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Articles in press have been peer-reviewed and accepted, which are not yet assigned to volumes/issues, but are citable by Digital Object Identifier (DOI).
Safety and Considerations of the COVID-19 Vaccine Massive Deployment
Junwei Li, Mingyue Song, Deyin Guo, Yongxiang Yi
doi: 10.1007/10.1007/s12250-021-00408-5
Received: 20 February 2021 Accepted: 26 April 2021 Published: 01 June 2021
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Cholesterol-25-Hydroxylase Suppresses Seneca Valley Virus Infection via Producing 25-Hydroxycholesterol to Block Adsorption Procedure
Hui Li, Zekai Zhao, Xiangmin Li, Liuxing Qin, Wei Wen, Huanchun Chen, Ping Qian
doi: 10.1007/s12250-021-00377-9
Received: 03 June 2020 Accepted: 22 September 2020 Published: 01 June 2021
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Cholesterol-25-hydroxylase (CH25H) is a membrane protein associated with endoplasmic reticulum, and it is an interferon-stimulated factor regulated by interferon. CH25H catalyzes cholesterol to produce 25-hydroxycholesterol (25HC) by adding a second hydroxyl to the 25th carbon atom of cholesterol. Recent studies have shown that both CH25H and 25HC could inhibit the replication of many viruses. In this study, we found that ectopic expression of CH25H in HEK-293T and BHK-21 cell lines could inhibit the replication of Seneca Valley virus (SVV) and that there was no species difference. On the other hand, the knockdown of CH25H could enhance the replication of SVV in HEK-293T and BHK-21 cells, indicating the importance of CH25H. To some extent, the CH25H mutant without hydroxylase activity also lost its ability to inhibit SVV amplification. Further studies demonstrated that 25HC was involved in the entire life cycle of SVV, especially in repressing its adsorption process. This study reveals that CH25H exerts the advantage of innate immunity mainly by producing 25HC to block virion adsorption.

Modulation of Antiviral Immunity and Therapeutic Efficacy by 25-Hydroxycholesterol in Chronically SIV-Infected, ART-Treated Rhesus Macaques
Chunxiu Wu, Jin Zhao, Ruiting Li, Fengling Feng, Yizi He, Yanjun Li, Runhan Huang, Guangye Li, Heng Yang, Genhong Cheng, Ling Chen, Feng Ma, Pingchao Li, Caijun Sun
doi: 10.1007/s12250-021-00407-6
Received: 29 January 2021 Accepted: 19 April 2021 Published: 31 May 2021
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Cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) exert broadly antiviral activity including inhibiting HIV-1 infection. However, their antiviral immunity and therapeutic efficacy in a nonhuman primate model are unknown. Here, we report that the regimen of 25HC combined with antiretroviral therapy (ART), provides profound immunological modulation towards inhibiting viral replication in chronically SIVmac239-infected rhesus macaques (RMs). Compared to the ART alone, this regimen more effectively controlled SIV replication, enhanced SIV-specific cellular immune responses, restored the ratio of CD4/CD8 cells, reversed the hyperactivation state of CD4+ T cells, and inhibited the secretion of proinflammatory cytokines by CD4+ and CD8+ T lymphocytes in chronically SIV-infected RMs. Furthermore, the in vivo safety and the preliminary pharmacokinetics of the 25HC compound were assessed in this RM model. Taken together, these assessments help explain the profound relationship between cholesterol metabolism, immune modulation, and antiviral activities by 25HC. These results provide insight for developing novel therapeutic drug candidates against HIV-1 infection and other related diseases.

The Functional Characterization of Bat and Human P[3] Rotavirus VP8*s
Dandi Li, Mengxuan Wang, Tongyao Mao, Mingwen Wang, Qing Zhang, Hong Wang, Lili Pang, Xiaoman Sun, Zhaojun Duan
doi: 10.1007/s12250-021-00400-z
Received: 06 January 2021 Accepted: 12 April 2021 Published: 31 May 2021
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P[3] rotavirus (RV) has been identified in many species, including human, simian, dog, and bat. Several glycans, including sialic acid, histo-blood group antigens (HBGAs) are reported as RV attachment factors. The glycan binding specificity of different P[3] RV VP8*s were investigated in this study. Human HCR3A and dog P[3] RV VP8*s recognized glycans with terminal sialic acid and hemagglutinated the red blood cells, while bat P[3] VP8* showed neither binding to glycans nor hemagglutination. However, the bat P[3] VP8* mutant of C189Y obtained the ability to hemagglutinate the red blood cells, while human P[3] HCR3A/M2-102 mutants of Y189C lost the ability. Sequence alignment and structural analysis indicated that residue 189 played an important role in the ligand recognition and may contribute to the cross-species transmission. Structural superimposition exhibited that bat P[3] VP8* model was quite different from the simian P[3] Rhesus rotavirus (RRV) P[3] VP8*, indicating that bat P[3] RV was relatively distinct and partially contributed to the no binding to tested glycans. These results promote our understanding of P[3] VP8*/glycans interactions and the potential transmission of bat/human P[3] RVs, offering more insight into the RV infection and prevalence.

Re-isolation of Wuxiang Virus from Wild Sandflies Collected from Yangquan County, China
Qinyan Wang, Shihong Fu, Jingxia Cheng, Xiuyan Xu, Jing Wang, Bin Wu, Xiaodong Tian, Yan Li, Ying He, Fan Li, Kai Nie, Songtao Xu, Bin Wang, Huanyu Wang, Xiaoqing Lu, Guodong Liang
doi: 10.1007/s12250-021-00398-4
Received: 17 November 2020 Accepted: 25 March 2021 Published: 31 May 2021
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We previously isolated a new species of the genus Phlebovirus from wild sandflies collected from Wuxiang County in central China, which named the Wuxiang virus (WUXV). In this study, we re-isolated the WUXV from wild sandflies collected from two villages in Yangquan County, China in 2019. Four virus isolates that caused cytopathic effects in BHK-21 cells were successfully isolated from sandfly specimens collected from chicken pens and sheep pens. Phylogenetic analyses of the L, M and S gene segments of the viruses revealed that the four virus strains represented the previously isolated WUXV. The minimum infection rate (MIR) of the virus isolated from the sheep pen was 3.21, and the MIR of the virus isolated from the chicken pen was 3.45. The positive rates of Wuxiang virus neutralizing antibodies in serum samples of local healthy people and domestic chickens were 8.7% (4/46) and 100% (4/4), respectively, suggesting that Wuxiang virus can infect human and animal. In view of the fact that Wuxiang virus is infectious to humans and animals and has a relatively wide geographical distribution in China, it is of great public health significance to strengthen the investigation and study on the infection status of Wuxiang virus in humans and animals.

Computational Viromics: Applications of the Computational Biology in Viromics Studies
Congyu Lu, Yousong Peng
doi: 10.1007/s12250-021-00395-7
Received: 02 June 2020 Accepted: 14 April 2021 Published: 31 May 2021
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Retrospective Study of an Adenovirus Pneumonia Outbreak in Shenzhen in 2017
Zhiwei Lu, Heping Wang, Yanmin Bao, Zhichuan Li, Huan Zhang, Haixia Zhao, Yuejie Zheng
doi: 10.1007/s12250-021-00393-9
Received: 02 July 2020 Accepted: 21 April 2021 Published: 27 May 2021
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PML Suppresses Influenza Virus Replication by Promoting FBXW7 Expression
Hai-Yan Yan, Hui-Qiang Wang, Ming Zhong, Shuo Wu, Lu Yang, Ke Li, Yu-Huan Li
doi: 10.1007/s12250-021-00399-3
Received: 16 December 2020 Accepted: 29 March 2021 Published: 27 May 2021
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Influenza A viruses (IAV) are responsible for seasonal flu epidemics, which can lead to high morbidity and mortality each year. Like other viruses, influenza virus can hijack host cellular machinery for its replication. Host cells have evolved diverse cellular defense to resist the invasion of viruses. As the main components of promyelocytic leukemia protein nuclear bodies (PML-NBs), PML can inhibit the replication of many medically important viruses including IAV. However, the mechanism of PML against IAV is unclear. In the present study, we found PML was induced in response to IAV infection and ectopic expression of PML could inhibit IAV replication, whereas knockdown of endogenous PML expression could enhance IAV replication. Further studies showed that PML increased the expression of FBXW7 by inhibiting its K48-linked ubiquitination and enhanced the interaction between FBXW7 and SHP2, which negatively regulated IAV replication during infection. Moreover, PML stabilized RIG-I to promote the production of type Ⅰ IFN. Collectively, these data indicated that PML inhibited IAV replication by enhancing FBXW7 expression in the antiviral immunity against influenza virus and extended the mechanism of PML in antiviral immunity.

Cucurbit[7]uril as a Broad-Spectrum Antiviral Agent against Diverse RNA Viruses
Jia Quan, Xiangjun Zhang, Yuanfu Ding, Shengke Li, Yang Qiu, Ruibing Wang, Xi Zhou
doi: 10.1007/s12250-021-00404-9
Received: 25 March 2021 Accepted: 06 April 2021 Published: 26 May 2021
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The emergence and re-emergence of RNA virus outbreaks highlight the urgent need for the development of broad-spectrum antivirals. Polyamines are positively-charged small molecules required for the infectivity of a wide range of RNA viruses, therefore may become good antiviral targets. Cucurbit[7]uril (CB[7]), a synthetic macrocyclic molecule, which can bind with amine-based organic compounds with high affinity, has been shown to regulate bioactive molecules through competitive binding. In this study, we tested the antiviral activity of CB[7] against diverse RNA viruses, including a panel of enteroviruses (i.e. human enterovirus A71, coxsackievirus A16, coxsackievirus B3, and echovirus 11), some flaviviruses (i.e. dengue virus and Zika virus), and an alphavirus representative Semliki forest virus. CB[7] can inhibit virus replications in a variety of cell lines, and its mechanism of action is through the competitive binding with polyamines. Our findings not only for the first time provide evidence that CB[7] can be a promising broad-spectrum antiviral agent, but more importantly, offer a novel therapeutic strategy to fight against RNA viruses by supramolecular sequestration of polyamines.

Anti-SARS-CoV-2 IgY Isolated from Egg Yolks of Hens Immunized with Inactivated SARS-CoV-2 for Immunoprophylaxis of COVID-19
Haiyan Shen, Yanxing Cai, Huan Zhang, Jie Wu, Lin Ye, Penghui Yang, Xiaojun Lin, Shibo Jiang, Ming Liao
doi: 10.1007/s12250-021-00371-1
Received: 29 November 2020 Accepted: 08 February 2021 Published: 21 May 2021
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Epidemiological Characteristics of Influenza A and B in Macau, 2010–2018
HoiMan Ng, Teng Zhang, Guoliang Wang, SiMeng Kan, Guoyi Ma, Zhe Li, Chang Chen, Dandan Wang, MengIn Wong, ChioHang Wong, Jinliang Ni, Xiaohua Douglas Zhang
doi: 10.1007/s12250-021-00388-6
Received: 04 December 2020 Accepted: 04 March 2021 Published: 20 May 2021
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Influenza is one of the major respiratory diseases in humans. Macau is a tourist city with high density of population and special population mobility. The study on the epidemiological characteristics of influenza in Macau should bring great value for preventing influenza in tourist cities like Macau in the world. In this study, we collected a total of 104, 874 samples with influenza-like illness (ILI) in Macau from 2010 to 2018. Chi-square test and binary multivariable logistic regression were used to investigate the epidemiological characteristics of influenza A and B in Macau. Among these ILI samples, the overall positive rate is 17.17% for influenza A and 6.97% for influenza B. The epidemics of influenza in three years (i.e., 2012, 2017 and 2018) differ from the remaining years (i.e., normal years). In a normal year, influenza A occurs year-round whereas influenza B is seasonal. Our research shows significant differences in influenza infections between different age groups in normal years. Interestingly, our analysis shows no significant difference between locals and tourists in influenza A and B infection in a normal year, whereas the odds of influenza A in tourists were significantly higher than those in locals in July 2017 and the odds of influenza B in tourists were significantly higher than those in locals in January–February 2012 and January–February 2018. This is possibly attributed by the policy of free vaccination to everyone in Macau. These findings should be valuable for preventing influenza in not only Macau but also the world.

Transcriptome Analyses Implicate Endogenous Retroviruses Involved in the Host Antiviral Immune System through the Interferon Pathway
Miao Wang, Liying Wang, Haizhou Liu, Jianjun Chen, Di Liu
doi: 10.1007/s12250-021-00370-2
Received: 10 October 2020 Accepted: 08 February 2021 Published: 19 May 2021
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Human endogenous retroviruses (HERVs) are the remains of ancient retroviruses that invaded our ancestors' germline cell and were integrated into the genome. The expression of HERVs has always been a cause for concern because of its association with various cancers and diseases. However, few previous studies have focused on specific activation of HERVs by viral infections. Our previous study has shown that dengue virus type 2 (DENV-2) infection induces the transcription of a large number of abnormal HERVs loci; therefore, the purpose of this study was to explore the relationship between exogenous viral infection and HERV activation further. In this study, we retrieved and reanalyzed published data on 21 transcriptomes of human cells infected with various viruses. We found that infection with different viruses could induce transcriptional activation of HERV loci. Through the comparative analysis of all viral datasets, we identified 43 key HERV loci that were up-regulated by DENV-2, influenza A virus, influenza B virus, Zika virus, measles virus, and West Nile virus infections. Furthermore, the neighboring genes of these HERVs were simultaneously up-regulated, and almost all such neighboring genes were interferon-stimulated genes (ISGs), which are enriched in the host's antiviral immune response pathways. Our data supported the hypothesis that activation of HERVs, probably via an interferon-mediated mechanism, plays an important role in innate immunity against viral infections.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery
Yun-Yun Jin, Hanwen Lin, Liu Cao, Wei-Chen Wu, Yanxi Ji, Liubing Du, Yiling Jiang, Yanchun Xie, Kuijie Tong, Fan Xing, Fuxiang Zheng, Mang Shi, Ji-An Pan, Xiaoxue Peng, Deyin Guo
doi: 10.1007/s12250-021-00385-9
Received: 02 March 2021 Accepted: 11 March 2021 Published: 17 May 2021
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SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.

Enriched Opportunistic Pathogens Revealed by Metagenomic Sequencing Hint Potential Linkages between Pharyngeal Microbiota and COVID-19
Dongyan Xiong, Caroline Muema, Xiaoxu Zhang, Xinming Pan, Jin Xiong, Hang Yang, Junping Yu, Hongping Wei
doi: 10.1007/s12250-021-00391-x
Received: 15 December 2020 Accepted: 15 March 2021 Published: 12 May 2021
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As a respiratory tract virus, SARS-CoV-2 infected people through contacting with the upper respiratory tract first. Previous studies indicated that microbiota could modulate immune response against pathogen infection. In the present study, we performed metagenomic sequencing of pharyngeal swabs from eleven patients with COVID-19 and eleven Non-COVID-19 patients who had similar symptoms such as fever and cough. Through metagenomic analysis of the above two groups and a healthy group from the public data, there are 6502 species identified in the samples. Specifically, the Pielou index indicated a lower evenness of the microbiota in the COVID-19 group than that in the Non-COVID-19 group. Combined with the linear discriminant analysis (LDA) and the generalized linear model, eighty-one bacterial species were found with increased abundance in the COVID-19 group, where 51 species were enriched more than 8 folds. The top three enriched genera were Streptococcus, Prevotella and Campylobacter containing some opportunistic pathogens. More interestingly, through experiments, we found that two Streptococcus strains, S. suis and S. agalactiae, could stimulate the expression of ACE2 of Vero cells in vitro, which may promote SARS-CoV-2 infection. Therefore, these enriched pathogens in the pharynxes of COVID-19 patients may involve in the virus-host interactions to affect SARS-CoV-2 infection and cause potential secondary bacterial infections through changing the expression of the viral receptor ACE2 and/or modulate the host's immune system.

Accelerated Evolution of H7N9 Subtype Influenza Virus under Vaccination Pressure
Yifan Wu, Jingkai Hu, Xuanjiang Jin, Xiao Li, Jinfeng Wang, Mengmeng Zhang, Jianglin Chen, Shumin Xie, Wenbao Qi, Ming Liao, Weixin Jia
doi: 10.1007/s12250-021-00383-x
Received: 21 October 2020 Accepted: 17 March 2021 Published: 11 May 2021
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No avian H7N9 outbreaks have occurred since the introduction of H7N9 inactivated vaccine in the fall of 2017. However, H7N9 is still prevalent in poultry. To surveil the prevalence, genetic characteristics, and antigenic changes of H7N9, over 7000 oropharyngeal and cloaca swab specimens were collected from live poultry markets and farms in 15 provinces of China from 2017 to 2019. A total of 85 influenza virus subtype H7N9 strains were isolated and 20 representative strains were selected for genetic analysis and antigenicity evaluation. Results indicated the decreased prevalence of low-pathogenic H7N9 strains while highly-pathogenic H7N9 strains became dominated since the introduction of vaccine. Phylogenetic analysis showed that strains from 2019 formed an independent small branch and were genetically distant to strains isolated in 2013–2018. Analysis of key amino acid sites showed that the virus strains may adapt to the host environment evolutionally through mutation. Our analysis predicted additional potential glycosylation sites for HA and NA genes in the 2019 strains. Sequence analysis of HA gene in strains isolated from 2018 to 2019 showed that there were an increased nucleotide substitution rate and an increased mutation rate in the first and second nucleotides of coding codons within the open reading frame. The hemagglutination inhibition (HI) assay showed that H7-Re1 and H7-Re2 exhibited a lower HI titer for isolates from 2019, while H7-Re3 and rLN79 showed a high HI titer. The protective effect of the vaccine decreased after 15 months of use. Overall, under vaccination pressure, the evolution of influenza virus subtype H7N9 has accelerated.

Expansion of GARP-Expressing CD4+CD25-FoxP3+ T Cells and SATB1 Association with Activation and Coagulation in Immune Compromised HIV-1-Infected Individuals in South Africa
Eman Teer, Danzil E. Joseph, Leanne Dominick, Richard H. Glashoff, Essop M. Faadiel
doi: 10.1007/s12250-021-00386-8
Received: 17 September 2020 Accepted: 23 February 2021 Published: 11 May 2021
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Although antiretroviral treatment lowers the burden of human immunodeficiency virus (HIV)-related disease, it does not always result in immunological recovery. This manifests as persistent chronic inflammation, immune activation or exhaustion that can promote the onset of co-morbidities. As the exact function of regulatory T (Treg) cells in HIV remains unclear, this cross-sectional study investigated three expression markers (Forkhead box protein P3 [FOXP3], glycoprotein A repetitions predominant [GARP], special AT-rich sequence binding protein 1 [SATB1]) and compared their expansion between CD4+CD25- and CD4+CD25++ T cells. Age-matched study subjects were recruited (Western Cape, South Africa) and sub-divided: HIV-negative subjects (n = 12), HIV-positive naïve treated (n = 22), HIV-positive treated based on CD4 count cells/μL (CD4 > 500 and CD4 < 500) (n = 34) and HIV-treated based on viral load (VL) copies/mL (VL < 1000 and VL > 1000) (n = 34). Markers of immune activation (CD38) and coagulation (CD142) on T cells (CD8) were assessed by flow cytometry together with FOXP3, GARP and SATB1 expression on CD4+CD25- and CD4+CD25++ T cells. Plasma levels of interleukin-10 (IL-10; anti-inflammatory marker), IL-6 (inflammatory marker) and D-dimer (coagulation marker) were assessed. This study revealed three major findings in immuno-compromised patients with virological failure (CD4 < 500; VL > 1000): (1) the expansion of the unconventional Treg cell subset (CD4+CD25-FOXP3+) is linked with disease progression markers; (2) increased GARP expression in the CD4+CD25- and CD4+CD25++ subsets; and (3) the identification of a strong link between CD4+CD25-SATB1+ cells and markers of immune activation (CD8+CD38+) and coagulation (CD8+CD142+ and D-dimer).

Development of RNA Polymerase III-Driven Reverse Genetics System for the Rescue of a Plant Rhabdovirus
Xiaoyan Zhang, Kai Sun, Yan Liang, Chenglu Zhao, Zhenghe Li
doi: 10.1007/s12250-021-00390-y
Received: 23 November 2020 Accepted: 25 March 2021 Published: 03 May 2021
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Detection of SARS-CoV-2 RNA in Medical Wastewater in Wuhan During the COVID-19 Outbreak
Jun-Bo Zhou, Wen-Hua Kong, Sheng Wang, Yi-Bing Long, Lian-Hua Dong, Zhen-Yu He, Man-Qing Liu
doi: 10.1007/s12250-021-00373-z
Received: 28 July 2020 Accepted: 14 January 2021 Published: 03 May 2021
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Porcine Bocavirus: A 10-Year History since Its Discovery
Manita Aryal, Guangliang Liu
doi: 10.1007/s12250-021-00365-z
Received: 04 July 2020 Accepted: 11 December 2020 Published: 28 April 2021
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Porcine bocavirus (PBoV) is a single-stranded DNA virus, belongs to the genus Bocaparvovirus of family Parvoviridae. It was discovered along with porcine circovirus 2 (PCV 2) and torque tenovirus (TTV) in the lymph nodes of pigs suffering from postweaning multisystemic wasting syndrome (PMWS) in Sweden in 2009. PBoV has been reported throughout the world, mostly in weaning piglets, and has a broad range of tissue tropism. Since PBoV is prevalent in healthy as well as clinically infected pigs and is mostly associated with coinfection with other viruses, the pathogenic nature of PBoV is still unclear. Currently, there are no cell lines available for the study of PBoV, and animal model experiments have not been described. This review summarizes the current state of knowledge about PBoV, including the epidemiology, evolution analysis, detection methods, pathogenesis and public health concerns.

A Virulent PEDV Strain FJzz1 with Genomic Mutations and Deletions at the High Passage Level Was Attenuated in Piglets via Serial Passage In Vitro
Pengfei Chen, Xiongwei Zhao, Shuting Zhou, Tianxing Zhou, Xiangmei Tan, Xia Wu, Wu Tong, Fei Gao, Lingxue Yu, Yifeng Jiang, Hai Yu, Zhibiao Yang, Guangzhi Tong, Yanjun Zhou
doi: 10.1007/s12250-021-00368-w
Received: 28 September 2020 Accepted: 28 December 2020 Published: 28 April 2021
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Highly virulent porcine epidemic diarrhea virus (PEDV) strains re-emerged and circulated in China at the end of 2010, causing significant economic losses in the pork industry worldwide. To understand the genetic dynamics of PEDV during its passage in vitro,the PEDV G2 strain FJzz1 was serially propagated in Vero cells for up to 200 passages. The susceptibility and adaptability of the FJzz1 strain increased gradually as it was serially passaged in vitro. Sequence analysis revealed that amino acid (aa) changes were mainly concentrated in the S glycoprotein,which accounted for 72.22%–85.71% of all aa changes. A continuous aa deletion (55I56G57E→55K56Δ57Δ) occurred in the N-terminal domain of S1 (S1-NTD). To examine how the aa changes affected its virulence,FJzz1-F20 and FJzz1-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets. All the piglets in the FJzz1-F20-infected group showed typical diarrhea at 24 h postinfection,and the piglets died successively by 48 h postinfection. However,the clinical signs of the piglets in the FJzz1-F200-infected group were significantly weaker,and no deaths occurred. The FJzz1-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues,and no obvious histopathological lesions. Type I and III interferon were induced in the FJzz1-F200 infection group,together with pro-inflammatory cytokines,such as TNF-α,IL-1β and IL-8. These results indicate that the identified genetic changes may contribute to the attenuation of FJzz1 strain,and the attenuated FJzz1-F200 may have the potential for developing PEDV live-attenuated vaccines.

Human Endogenous Retroviruses as Biomedicine Markers
Yuhe Song, Xiang Li, Xiaoman Wei, Jie Cui
doi: 10.1007/s12250-021-00387-7
Received: 22 August 2020 Accepted: 15 March 2021 Published: 27 April 2021
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Human endogenous retroviruses (HERVs) were formed via ancient integration of exogenous retroviruses into the human genome and are considered to be viral "fossils". The human genome is embedded with a considerable amount of HERVs, witnessing the long-term evolutionary history of the viruses and the host. Most HERVs have lost coding capability during selection but still function in terms of HERV-mediated regulation of host gene expression. In this review, we summarize the roles of HERV activation in response to viral infections and diseases, and emphasize the potential use of HERVs as biomedicine markers in the early diagnosis of diseases such as cancer, which provides a new perspective for the clinical application of HERVs.

Genetic Mutation of SARS-CoV-2 during Consecutive Passages in Permissive Cells
Ying Chen, Mei-Qin Liu, Yun Luo, Ren-Di Jiang, Hao-Rui Si, Yan Zhu, Bei Li, Xu-Rui Shen, Hao-Feng Lin, Kai Zhao, Ben Hu, Zheng-Li Shi, Xing-Lou Yang
doi: 10.1007/s12250-021-00384-w
Received: 08 December 2020 Accepted: 15 March 2021 Published: 26 April 2021
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Beagle Dogs Have Low Susceptibility to Florida Clade 2 H3N8 Equine Avian Influenza
Pei Zhou, Xiangyu Xiao, Xinkai Hu, Jie Dong, Haoyao Zhang, Yanchao Li, Shoujun Li
doi: 10.1007/s12250-021-00366-y
Received: 03 August 2020 Accepted: 25 January 2021 Published: 15 April 2021
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Rapid Acquisition of High-Quality SARS-CoV-2 Genome via AmpliconOxford Nanopore Sequencing
Yi Yan, Ke Wu, Jun Chen, Haizhou Liu, Yi Huang, Yong Zhang, Jin Xiong, Weipeng Quan, Xin Wu, Yu Liang, Kunlun He, Zhilong Jia, Depeng Wang, Di Liu, Hongping Wei, Jianjun Chen
doi: 10.1007/s12250-021-00378-8
Received: 28 October 2020 Accepted: 18 February 2021 Published: 13 April 2021
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Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing. While the rapid acquisition of SARS-CoV-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background. To address this issue, we modified and evaluated an approach by utilizing SARS-CoV-2-specific amplicon amplification and Oxford Nanopore PromethION platform. This workflow started with the throat swab of the COVID-19 patient, combined reverse transcript PCR, and multi-amplification in one-step to shorten the experiment time, then can quickly and steadily obtain high-quality SARS-CoV-2 genome within 24 h. A comprehensive evaluation of the method was conducted in 42 samples: the sequencing quality of the method was correlated well with the viral load of the samples; high-quality SARS-CoV-2 genome could be obtained stably in the samples with Ct value up to 39.14; data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20; variation analysis indicated that the method can detect the existing and emerging genomic mutations as well; Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing, making it as low as 0.4/10, 000 bp. In summary, high-quality SARS-CoV-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARS-CoV-2.

Recombinant GII.4[P31] Was Predominant Norovirus Circulating in Beijing Area, China, 2018–2020
Junhong Ai, Meng Zhang, Fang Jin, Zhengde Xie
doi: 10.1007/s12250-021-00381-z
Received: 28 November 2020 Accepted: 20 February 2021 Published: 09 April 2021
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Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infection in Mice and Non-Human Primates
Yan-Feng Yao, Ze-Jun Wang, Ren-Di Jiang, Xue Hu, Hua-Jun Zhang, Yi-Wu Zhou, Ge Gao, Ying Chen, Yun Peng, Mei-Qin Liu, Ya-Nan Zhang, Juan Min, Jia Lu, Xiao-Xiao Gao, Jing Guo, Cheng Peng, Xu-Rui Shen, Qian Li, Kai Zhao, Lian Yang, Xin Wan, Bo Zhang, Wen-Hui Wang, Jia Wu, Peng Zhou, Xing-Lou Yang, Shuo Shen, Chao Shan, Zhi-Ming Yuan, Zheng-Li Shi
doi: 10.1007/s12250-021-00376-w
Received: 23 January 2021 Accepted: 08 February 2021 Published: 09 April 2021
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The ongoing coronavirus disease 2019 (COVID-19) pandemic caused more than 96 million infections and over 2 million deaths worldwide so far. However, there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the disease causative agent. Vaccine is the most effective approach to eradicate a pathogen. The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations. Here we evaluated the safety, immunogenicity, and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates. Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates, and subsequently provided partial (in low dose) or full (in high dose) protection of challenge in the tested animals. In addition, passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice. These results warranted positive outcomes in future clinical trials in humans.

Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation
Bei Wang, Chongyang Zhang, Xiaobo Lei, Lili Ren, He Huang, Jianwei Wang, Zhendong Zhao
doi: 10.1007/s12250-021-00369-9
Received: 07 December 2020 Accepted: 09 February 2021 Published: 09 April 2021
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating pandemic worldwide. Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic, and scientists all over the world have made great efforts to this end. However, manipulation of the SARS-CoV-2 should be performed in the biosafety level 3 laboratory. This makes experiments complicated and time-consuming. Therefore, a safer system for working with this virus is urgently needed. Here, we report the construction of plasmid-based, non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae. Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes. Using SARS-CoV-2 replicons, the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed, and the half-maximal effective concentration (EC50) value of remdesivir and E64-D was estimated by different quantification methods respectively, indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h, Inhibits the Host Larval Cathepsin and Chitinase Activities
Huan Yu, Yi-Yi Ou-Yang, Chang-Jin Yang, Ni Li, Madoka Nakai, Guo-Hua Huang
doi: 10.1007/s12250-021-00374-y
Received: 29 August 2020 Accepted: 16 November 2020 Published: 08 April 2021
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3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of HvAV-3h. In the study, 3h-31 was initially transcribed and expressed at 3 h post-infection (hpi) in the infected Spodoptera exigua fat body cells (SeFB). 3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate an infectious baculovirus (AcMNPV-31). In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31, and lucent tubular structures were found around the virogenic stroma in the AcMNPV-31-infected SeFB cells. In vivo, both LD50 and LD90 values of AcMNPV-31 were significantly higher than those of the wild-type AcMNPV (AcMNPV-wt) in third instar S. exigua larvae. An interesting finding was that the liquefaction of the larvae killed by the infection of AcMNPV-31 was delayed. Chitinase and cathepsin activities of AcMNPV-31-infected larvae were significantly lower than those of AcMNPV-wt-infected larvae. The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi, which showed that larval cathepsin activity was significantly upregulated, but chitinase activity was not significantly changed due to the RNAi of 3h-31. Based on the obtained results, we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities, so as to restrain the hosts in their larval stages.

Host Interferon-Stimulated Gene 20 Inhibits Pseudorabies Virus Proliferation
Xiaoyong Chen, Dage Sun, Sujie Dong, Huanjie Zhai, Ning Kong, Hao Zheng, Wu Tong, Guoxin Li, Tongling Shan, Guangzhi Tong
doi: 10.1007/s12250-021-00380-0
Received: 29 October 2020 Accepted: 23 February 2021 Published: 08 April 2021
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Host interferon-stimulated gene 20 (ISG20) exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling. Here, we examined the role of ISG20 during pseudorabies virus (PRV) proliferation. We found that ISG20 modulates PRV replication by enhancing IFN signaling. Further, ISG20 expression was upregulated following PRV infection and poly(I: C) treatment. Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells, whereas knockdown of ISG20 promoted PRV proliferation. In addition, ISG20 expression upregulated IFN-β expression and enhanced IFN downstream signaling during PRV infection. Notably, PRV UL24 suppressed the transcription of ISG20, thus antagonizing its antiviral effect. Further domain mapping analysis showed that the N terminus (amino acids 1-90) of UL24 was responsible for the inhibition of ISG20 transcription. Collectively, these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay.

Stability of SARS-CoV-2 on the Surfaces of Three Meats in the Setting That Simulates the Cold Chain Transportation
Xiao-Li Feng, Bei Li, Hao-Feng Lin, Hong-Yi Zheng, Ren-Rong Tian, Rong-Hua Luo, Mei-Qin Liu, Ren-Di Jiang, Yong-Tang Zheng, Zheng-Li Shi, Yu-Hai Bi, Xing-Lou Yang
doi: 10.1007/s12250-021-00367-x
Received: 09 October 2020 Accepted: 01 February 2021 Published: 08 April 2021
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Ozone Water Is an Effective Disinfectant for SARS-CoV-2
Xiao Hu, Zhen Chen, Zhengyuan Su, Fei Deng, Xinwen Chen, Qi Yang, Pan Li, Quanjiao Chen, Jun Ma, Wuxiang Guan, Rongjuan Pei, Yun Wang
doi: 10.1007/s12250-021-00379-7
Received: 13 January 2021 Accepted: 23 February 2021 Published: 31 March 2021
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Human Endogenous Retrovirus Type W Envelope from Multiple Sclerosis Demyelinating Lesions Shows Unique Solubility and Antigenic Characteristics
Benjamin Charvet, Justine Pierquin, Joanna Brunel, Rianne Gorter, Christophe Quétard, Branka Horvat, Sandra Amor, Jacques Portoukalian, Hervé Perron
doi: 10.1007/s12250-021-00372-0
Received: 02 February 2021 Accepted: 08 February 2021 Published: 26 March 2021
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In multiple sclerosis (MS), human endogenous retrovirus W family (HERV-W) envelope protein, pHERV-W ENV, limits remyelination and induces microglia-mediated neurodegeneration. To better understand its role, we examined the soluble pHERV-W antigen from MS brain lesions detected by specific antibodies. Physico-chemical and antigenic characteristics confirmed differences between pHERV-W ENV and syncytin-1. pHERV-W ENV monomers and trimers remained associated with membranes, while hexamers self-assembled from monomers into a soluble macrostructure involving sulfatides in MS brain. Extracellular hexamers are stabilized by internal hydrophobic bonds and external hydrophilic moieties. HERV-W studies in MS also suggest that this diffusible antigen may correspond to a previously described high-molecular-weight neurotoxic factor secreted by MS B-cells and thus represents a major agonist in MS pathogenesis. Adapted methods are now needed to identify encoding HERV provirus(es) in affected cells DNA. The properties and origin of MS brain pHERV-W ENV soluble antigen will allow a better understanding of the role of HERVs in MS pathogenesis. The present results anyhow pave the way to an accurate detection of the different forms of pHERV-W ENV antigen with appropriate conditions that remained unseen until now.

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus
Zhongshun Liu, Baoqin Xuan, Shubing Tang, Zhikang Qian
doi: 10.1007/s12250-021-00382-y
Received: 10 July 2020 Accepted: 11 March 2021 Published: 24 March 2021
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Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic cutaneous T-cell lymphomas. In addition to its anticancer activity, SAHA has significant effects on the growth of many viruses. The effect of SAHA on replication of human cytomegalovirus (HCMV) has not, however, been investigated. Here, we showed that replication of HCMV was significantly suppressed by treatment with SAHA at concentrations that did not show appreciable cytotoxicity. SAHA reduced transcription and protein levels of HCMV immediate early genes, showing that SAHA acts at an early stage in the viral life-cycle. RNA-sequencing data mining showed that numerous pathways and molecules were affected by SAHA. Interferon -mediated immunity was one of the most relevant pathways in the RNA-sequencing data, and we confirmed that SAHA inhibits HCMV-induced IFN-mediated immune responses using quantitative Real-time PCR (qRT-PCR). Fatty acid-binding protein 4 (FABP4), which plays a role in lipid metabolism, was identified by RNA-sequencing. We found that FABP4 expression was reduced by HCMV infection but increased by treatment with SAHA. We then showed that knockdown of FABP4 partially rescued the effect of SAHA on HCMV replication. Our data suggest that FABP4 contributes to the inhibitory effect of SAHA on HCMV replication.
Role of Intracellular Distribution of Feline and Bovine SAMHD1 Proteins in Lentiviral Restriction
Chu Wang, Lina Meng, Jialin Wang, Kaikai Zhang, Sizhu Duan, Pengyu Ren, Yingzhe Wei, Xinyu Fu, Bin Yu, Jiaxin Wu, Xianghui Yu
doi: 10.1007/s12250-021-00351-5
Received: 03 August 2020 Accepted: 28 December 2020 Published: 22 March 2021
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Human SAMHD1 (hSAM) restricts lentiviruses at the reverse transcription step through its dNTP triphosphohydrolase (dNTPase) activity. Besides humans, several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1. However, the intracellular distribution of feline and bovine SAMHD1 (fSAM and bSAM) and its significance in their lentiviral restriction function is not known. Here, we demonstrated that fSAM and bSAM were both predominantly localized to the nucleus and nuclear localization signal (11KRPR14)-deleted fSAM and bSAM relocalized to the cytoplasm. Both cytoplasmic fSAM and bSAM retained the antiviral function against different lentiviruses and cytoplasmic fSAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type (WT) protein as cytoplasmic hSAM. Further investigation revealed that cytoplasmic fSAM was resistant to Vpx-induced degradation like cytoplasmic hSAM, while cytoplasmic bSAM was not, but they all demonstrated the same in vitro dNTPase activity and all could interact with Vpx as their WT proteins, indicating that cytoplasmic hSAM and fSAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation. Our results suggested that fSAM- and bSAM-mediated lentiviral restriction does not require their nuclear localization and that fSAM shares more common features with hSAM. These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo.

Significant Inhibition of Porcine Epidemic Diarrhea Virus In Vitro by Remdesivir, Its Parent Nucleoside and β-D-N4-hydroxycytidine
Yuanchao Xie, Xiaozhen Guo, Tianwen Hu, Daibao Wei, Xiuli Ma, Jiaqiang Wu, Bing Huang, Jingshan Shen
doi: 10.1007/s12250-021-00362-2
Received: 15 October 2020 Accepted: 22 January 2021 Published: 22 March 2021
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Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is widespread in the world. In recent years, the increased virulence of the virus due to viral variations, has caused great economic losses to the pig industry in many countries. It is always worthy to find effective therapeutic methods for PED. As an important class of antivirals, nucleoside drugs which target viral polymerases have been applied in treating human viral infections for half a century. Herein, we evaluated the anti-PEDV potential of three broad-spectrum antiviral nucleoside analogs, remdesivir (RDV), its parent nucleoside (RDV-N) and β-D-N4-hydroxycytidine (NHC). Among them, RDV-N was the most active agent in Vero E6 cells with EC50 of 0.31 μmol/L, and more potent than RDV (EC50 = 0.74 μmol/L) and NHC (EC50 = 1.17 μmol/L). The activity of RDV-N was further confirmed using an indirect immuno-fluorescence assay. Moreover, RDV-N exhibited a good safety profile in cells and in mice. The high sequence similarity of the polymerase functional domains of PEDV with other five porcine coronaviruses indicated a broader antiviral spectrum for the three compounds. Generally, RDV-N is a promising broad-spectrum antiviral nucleoside, and it would be worthy to make some structural modifications to increase its oral bioavailability.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates
Xiangqing Ding, Kangli Cao, Jing Wang, Yanmin Wan, Qinyun Chen, Yanqin Ren, Yongtang Zheng, Mingzhao Zhu, Renrong Tian, Wenjun Wang, Chen Zhao, Xiaoyan Zhang, Jianqing Xu
doi: 10.1007/s12250-021-00361-3
Received: 10 November 2020 Accepted: 13 January 2021 Published: 15 March 2021
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Raising a heterologous tier 2 neutralizing antibody (nAb) response remains a daunting task for HIV vaccine development. In this study, we explored the utility of diverse HIV-1 envelope (Env) immunogens in a sequential immunization scheme as a solution to this task. This exploration stemmed from the rationale that gp145, a membrane-bound truncation form of HIV Env, may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode. We first showed that gp140 DNA prime-gp145 Tiantan vaccinia (TV) boost likely represents a general format for inducing potent nAb response in mice. However, when examined in rhesus macaque, this modality showed little effectiveness. To improve the efficacy, we extended the original modality by adding a strong protein boost, namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle (NP), which was generated by a newly developed click approach. The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule. Importantly, the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge. Collectively, our studies highlighted that diversification of Env immunogens, in both types and formulations, under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.
The CREB Regulated Transcription Coactivator 2 Suppresses HIV-1 Transcription by Preventing RNA Pol II from Binding to HIV-1 LTR
Ling Ma, Shumin Chen, Zhen Wang, Saisai Guo, Jianyuan Zhao, Dongrong Yi, Quanjie Li, Zhenlong Liu, Fei Guo, Xiaoyu Li, Pingping Jia, Jiwei Ding, Chen Liang, Shan Cen
doi: 10.1007/s12250-021-00363-1
Received: 25 September 2020 Accepted: 09 December 2020 Published: 15 March 2021
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The CREB-regulated transcriptional co-activators (CRTCs), including CRTC1, CRTC2 and CRTC3, enhance transcription of CREB-targeted genes. In addition to regulating host gene expression in response to cAMP, CRTCs also increase the infection of several viruses. While human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter harbors a cAMP response element and activation of the cAMP pathway promotes HIV-1 transcription, it remains unknown whether CRTCs have any effect on HIV-1 transcription and HIV-1 infection. Here, we reported that CRTC2 expression was induced by HIV-1 infection, but CRTC2 suppressed HIV-1 infection and diminished viral RNA expression. Mechanistic studies revealed that CRTC2 inhibited transcription from HIV-1 LTR and diminished RNA Pol II occupancy at the LTR independent of its association with CREB. Importantly, CRTC2 inhibits the activation of latent HIV-1. Together, these data suggest that in response to HIV-1 infection, cells increase the expression of CRTC2 which inhibits HIV-1 gene expression and may play a role in driving HIV-1 into latency.
Porcine Picornavirus 3C Protease Degrades PRDX6 to Impair PRDX6-mediated Antiviral Function
Congcong Wang, Huanhuan Feng, Xiangle Zhang, Kangli Li, Fan Yang, Weijun Cao, Huisheng Liu, Lili Gao, Zhaoning Xue, Xiangtao Liu, Zixiang Zhu, Haixue Zheng
doi: 10.1007/s12250-021-00352-4
Received: 30 September 2020 Accepted: 17 December 2020 Published: 15 March 2021
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Peroxiredoxin-6 (PRDX6) is an antioxidant enzyme with both the activities of peroxidase and phospholipase A2 (PLA2), which is involved in regulation of many cellular reactions. However, the function of PRDX6 during virus infection remains unknown. In this study, we found that the abundance of PRDX6 protein was dramatically decreased in foot-and-mouth disease virus (FMDV) infected cells. Overexpression of PRDX6 inhibited FMDV replication. In contrast, knockdown of PRDX6 expression promoted FMDV replication, suggesting an antiviral role of PRDX6. To explore whether the activity of peroxidase and PLA2 was associated with PRDX6-mediated antiviral function, a specific inhibitor of PLA2 (MJ33) and a specific inhibitor of peroxidase activity (mercaptosuccinate) were used to treat the cells before FMDV infection. The results showed that incubation of MJ33 but not mercaptosuccinate promoted FMDV replication. Meanwhile, overexpression of PRDX6 slightly enhanced type I interferon signaling. We further determined that the viral 3Cpro was responsible for degradation of PRDX6, and 3Cpro-induced reduction of PRDX6 was independent of the proteasome, lysosome, and caspase pathways. The protease activity of 3Cpro was required for induction of PRDX6 reduction. Besides, PRDX6 suppressed the replication of another porcine picornavirus Senecavirus A (SVA), and the 3Cpro of SVA induced the reduction of PRDX6 through its proteolytic activity as well. Together, our results suggested that PRDX6 plays an important antiviral role during porcine picornavirus infection, and the viral 3Cpro induces the degradation of PRDX6 to overcome PRDX6-mediated antiviral function.
Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids
Xingang Chen, Xiaoqin Yang, Chengfeng Lei, Fujun Qin, Xiulian Sun, Jia Hu
doi: 10.1007/s12250-021-00353-3
Received: 20 October 2020 Accepted: 18 December 2020 Published: 15 March 2021
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Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.
Porcine Coronaviruses: Overview of the State of the Art
Hanna Turlewicz-Podbielska, Małgorzata Pomorska-Mól
doi: 10.1007/s12250-021-00364-0
Received: 26 May 2020 Accepted: 19 November 2020 Published: 15 March 2021
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Like RNA viruses in general, coronaviruses (CoV) exhibit high mutation rates which, in combination with their strong tendency to recombine, enable them to overcome the host species barrier and adapt to new hosts. It is currently known that six CoV are able to infect pigs. Four of them belong to the genus Alphacoronavirus [transmissible gastroenteritis coronavirus (TEGV), porcine respiratory coronavirus (PRCV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV)], one of them to the genus Betacoronavirus [porcine hemagglutinating encephalomyelitis virus (PHEV)] and the last one to the genus Deltacoronavirus (PDCoV). PHEV was one of the first identified swine CoV and is still widespread, causing subclinical infections in pigs in several countries. PRCV, a spike deletion mutant of TGEV associated with respiratory tract infection, appeared in the 1980s. PRCV is considered non-pathogenic since its infection course is mild or subclinical. Since its appearance, pig populations have become immune to both PRCV and TGEV, leading to a significant reduction in the clinical and economic importance of TGEV. TGEV, PEDV and PDCoV are enteropathogenic CoV and cause clinically indistinguishable acute gastroenteritis in all age groups of pigs. PDCoV and SADS-CoV have emerged in 2014 (US) and in 2017 (China), respectively. Rapid diagnosis is crucial for controlling CoV infections and preventing them from spreading. Since vaccines are available only for some porcine CoV, prevention should focus mainly on a high level of biosecurity. In view of the diversity of CoV and the potential risk factors associated with zoonotic emergence, updating the knowledge concerning this area is essential.
Establishment of a Reverse Genetic System of Severe Fever with Thrombocytopenia Syndrome Virus Based on a C4 Strain
Mingyue Xu, Bo Wang, Fei Deng, Hualin Wang, Manli Wang, Zhihong Hu, Jia Liu
doi: 10.1007/s12250-021-00359-x
Received: 18 December 2020 Accepted: 21 January 2021 Published: 15 March 2021
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Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease (SFTS) in humans with a case fatality rate up to 30%. To date, the molecular biology involved in SFTSV infection remains obscure. There are seven major genotypes of SFTSV (C1–C4 and J1–J3) and previously a reverse genetic system was established on a C3 strain of SFTSV. Here, we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain. First, we obtained the 5′- and 3′-terminal untranslated region (UTR) sequences of the Large (L), Medium (M) and Small (S) segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis, and developed functional T7 polymerase-based L-, M- and S-segment minigenome assays. Then, full-length cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells. Viral infectivity, growth kinetics, and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus. Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus. However, one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus. Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture. The results help us to understand the molecular biology of SFTSV, and provide a useful tool for developing vaccines and antivirals against SFTS.
Surveillance of Class I Newcastle Disease Virus at Live Bird Markets and Commercial Poultry Farms in Eastern China Reveals the Epidemic Characteristics
Xiaolong Lu, Xiaoquan Wang, Tiansong Zhan, Yifan Sun, Xin Wang, Naiqing Xu, Tianxing Liao, Yu Chen, Min Gu, Shunlin Hu, Xiaowen Liu, Xiufan Liu
doi: 10.1007/s12250-021-00357-z
Received: 25 October 2020 Accepted: 21 January 2021 Published: 15 March 2021
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Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a highly contagious and economically devastating viral disease of birds (Habib et al. 2018). NDV, also termed as avian paramyxovirus type 1 (APMV-1), belongs to the genus Orthoavulavirus in the family Paramyxoviridae according to the International Committee on Taxonomy of Viruses (ICTV) (Amarasinghe et al. 2019). According to the latest phylogenetic classification system, NDVs can be further divided into two groups: class I and class II. There are three sub-genotypes in a single class I genotype 1, including sub-genotype 1.1.1, 1.1.2, and 1.2 (Dimitrov et al. 2019). Class I NDV, with the genome size of 15,198 nucleotides, is distributed globally and isolated frequently from wild birds and domestic poultry (Miller et al. 2010). Waterfowl can harbor lentogenic NDV strains and act as a natural reservoir for NDV (Kim et al. 2007; Wang et al. 2016), and class I NDVs have been reported to transfer from waterfowls to chickens and circulated in chicken flocks extensively in China (Zhu et al. 2014; Chen et al. 2020). Although class I NDVs are generally avirulent, they are likely to increase their virulence. For instance, class I NDVs can enhance virulence through several passages in chicken due to the mutations at the F cleavage site (Yu et al. 2002), and they can also evolve into a virulent virus through only a few point mutations (Collins et al. 1998). Furthermore, the 1990 Ireland ND outbreak was caused by a virulent class I NDV (Alexander et al. 1992). However, owing to its avirulence, class I NDVs are often under-reported or neglected within other surveillance efforts. To better evaluate the prevalence of class I NDVs, we performed the continuous surveillance of class I NDVs and revealed the epidemic characteristics of class I NDVs at live bird markets (LBMs) and commercial poultry farms in eastern China.
Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage, Infectivity, and Immune Response
Xueqin Zhu, Yang Liu, Jiao Guo, Junyuan Cao, Zonglin Wang, Gengfu Xiao, Wei Wang
doi: 10.1007/s12250-021-00358-y
Received: 02 November 2020 Accepted: 13 January 2021 Published: 10 March 2021
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Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein complex (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies (asparagine to glutamine, asparagine to alanine, and serine/tyrosine to alanine mutants) to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd (N89), 5th (N119), or 8th (N365) glycosylation motif. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd (N89Q) or 8th (N365Q) glycan completely inhibited the transduction efficiency of pseudotyped particles. We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st (N79Q), 3rd (N99Q), 5th (N119Q), or 6th (N167Q) glycan significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st (N79Q), 2nd (N89Q), 3rd (N99Q), 4th (N109Q), 5th (N119Q), 6th (N167Q), or 9th (N373Q) glycan enhanced the proportion of CD8+ effector T cells. Deletion of specific glycan improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycandeficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine.
Systematic Analysis of 42 Autographa Californica Multiple Nucleopolyhedrovirus Genes Identifies An Additional Six Genes Involved in the Production of Infectious Budded Virus
Tong Chen, Xiaoyan Duan, Hengrui Hu, Yu Shang, Yangbo Hu, Fei Deng, Hualin Wang, Manli Wang, Zhihong Hu
doi: 10.1007/s12250-021-00355-1
Received: 30 July 2020 Accepted: 29 December 2020 Published: 08 March 2021
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Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.
Artesunate and Dihydroartemisinin Inhibit Rabies Virus Replication
Jun Luo, Yue Zhang, Yang Wang, Qing Liu, Jiesen Li, Hongling He, Yongwen Luo, Shile Huang, Xiaofeng Guo
doi: 10.1007/s12250-021-00349-z
Received: 05 August 2020 Accepted: 16 November 2020 Published: 05 March 2021
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Rabies is caused by infection of rabies virus (RABV) and remains a serious threat to the global public health. Except for the requirement for cold chain and high cost of human rabies immune globulin, no small molecule drugs are currently available for clinical treatment of rabies. So, it is of great importance to identify novel compounds that can effectively inhibit RABV infection. Artesunate (ART) and dihydroartemisinin (DHA), two derivatives of artemisinin, are widely used for treatment of malaria in adults and children, showing high safety. In this study, we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration (0.1 μmol/L). The antiviral effects of ART and DHA were independent of viral strains and cell lines. Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells, implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells. Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription. Treatment with ART or DHA (5 mg/kg) by intramuscular injection improved, to some extent, the survival rate of RABV-challenged mice. Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice. The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.
Effects of Overwintering on the Survival and Vector Competence of Aedes albopictus in the Urban Life Cycle of Dengue Virus in Guangzhou, China
Yue Chen, Ronghua Chen, Jianrong Gao, Chunyuan Li, Jun Liu, Zhijian Zhou, Ruiwen Ren
doi: 10.1007/s12250-021-00356-0
Received: 27 September 2020 Accepted: 21 December 2020 Published: 05 March 2021
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The Pearl River Delta, where Aedes albopictus (Ae. albopictus) is the only vector for dengue transmission, has exhibited one of the highest dengue burdens in southern China in recent decades. However, whether dengue virus (DENV) can overwinter in Ae. albopictus in the Pearl River Delta has not been determined to date. In this study, 300 field-derived Ae. albopictus mosquitoes from Guangzhou that were infected with the predominant endemic DENV-1 strain were investigated under simulated urban balcony environment from October 16, 2016, to June 16, 2017. The vertical transmission of DENV in the infected overwintering Ae. albopictus was analyzed. The DENV infected overwintering mosquitoes were evaluated for viral load at nine-time points using reverse transcription-quantitative PCR. The vector competence of the infected overwintering Ae. albopictus was also investigated by using suckling mice. Adult mosquitoes and larvae were found during the observation period. The vertical transmission of DENV-1 was documented. The DENV-1-positive rates between overwintering males and females had no difference. The proportion of DENV-1-positive overwintering mosquitoes decreased over time and had no difference beyond three months after the experiment. Overwintering mosquitoes can spread DENV-1 to hosts. No engorged mosquitoes at an ambient temperature below 15 ℃ were observed. The ratio of engorged mosquitoes was positively correlated with the ambient temperature ranging from 15 to 30 ℃. Our results demonstrated that DENV can overwinter in Ae. albopictus in the Pearl River Delta, Ae. albopictus is the competent vector for DENV, and maintain autochthonous dengue outbreaks in the Pearl River Delta through vertical transmission.
Sustainability of SARS-CoV-2 Induced Humoral Immune Responses in COVID-19 Patients from Hospitalization to Convalescence Over Six Months
Yang Zheng, Qing Zhang, Ashaq Ali, Ke Li, Nan Shao, Xiaoli Zhou, Zhiqin Ye, Xiaomin Chen, Shanshan Cao, Jing Cui, Juan Zhou, Dianbing Wang, Baidong Hou, Min Li, Mengmeng Cui, Lihua Deng, Xinyi Sun, Qian Zhang, Qinfang Yang, Yong li, Hui Wang, Yake Lei, Bo Yu, Yegang Cheng, Xiaolin Tong, Dong Men, Xian-En Zhang
doi: 10.1007/s12250-021-00360-4
Received: 24 November 2020 Accepted: 13 January 2021 Published: 04 March 2021
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Understanding the persistence of antibody in convalescent COVID-19 patients may help to answer the current major concerns such as the risk of reinfection, the protection period of vaccination and the possibility of building an active herd immunity. This retrospective cohort study included 172 COVID-19 patients who were hospitalized in Wuhan. A total of 404 serum samples were obtained over six months from hospitalization to convalescence. Antibodies in the specimens were quantitatively analyzed by the capture chemiluminescence immunoassays (CLIA). All patients were positive for the anti-SARS-CoV-2 IgM/IgG at the onset of COVID-19 symptoms, and the IgG antibody persisted in all the patients during the convalescence. However, only approximately 25% of patients can detect the IgM antibodies, IgM against N protein (N-IgM) and receptor binding domain of S protein (RBD-IgM) at the 27th week. The titers of IgM, N-IgM and RBD-IgM reduced to 16.7%, 17.6% and 15.2% of their peak values respectively. In contrast, the titers of IgG, N-IgG and RBD-IgG peaked at 4–5th week and reduced to 85.9%, 62.6% and 87.2% of their peak values respectively at the end of observation. Dynamic behavior of antibodies and their correlation in age, gender and severity groups were investigated. In general, the COVID-19 antibody was sustained at high levels for over six months in most of the convalescent patients. Only a few patients with antibody reducing to an undetectable level which needs further attention. The humoral immune response against SARS-CoV-2 infection in COVID-19 patients exhibits a typical dynamic of acquired immunity.
A Novel 2-dimensional Multiplex qPCR Assay for Single-Tube Detection of Nine Human Herpesviruses
Yingxue Li, Zhenzhou Wan, Lulu Zuo, Shenwei Li, Honglian Liu, Yingying Ma, Lianqun Zhou, Xia Jin, Yuye Li, Chiyu Zhang
doi: 10.1007/s12250-021-00354-2
Received: 26 September 2020 Accepted: 28 December 2020 Published: 26 February 2021
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Human herpesviruses are double-stranded DNA viruses that are classified into nine species. More than 90% of adults are ever infected with one or more herpesviruses. The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas. Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment. In this study, we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control. The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25 µL single-tube reaction, and does not cross-react with 20 other human viruses, including DNA and RNA viruses. The robustness of the novel assay was evaluated using 170 clinical samples. The novel assay showed a high consistency (100%) with the single qPCR assay for HHVs detection. The features of simple, rapid, high sensitivity, specificity, and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.
HRV16 Infection Induces Changes in the Expression of Multiple piRNAs
Jie Li, Xinling Wang, Yanhai Wang, Juan Song, Qinqin Song, Yanbin Wang, Jun Han
doi: 10.1007/s12250-021-00344-4
Received: 20 May 2020 Accepted: 30 October 2020 Published: 22 February 2021
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Human rhinovirus (HRV) is one of the most important cold-causing pathogens in humans. Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. However, the profile of human/host piRNA during HRV infection is largely unknown. Here we performed high-throughput sequencing of piRNAs from H1-HeLa cells infected with HRV16 at 12 h, 24 h, and 36 h. The results showed that 22,151,664, 24,362,486 and 22,726,546 piRNAs displayed differential expression after HRV16 infection for three time points. A significant differential expression of 21 piRNAs was found in all time points and further verified by RT-qPCR, including 7 known piRNAs and 14 newly found piRNAs. In addition, piRNA prediction was performed on Piano using the SVM algorithm and transposon information. It found that novel_pir78110, novel_pir78107, novel_pir78097, novel_pir78094 and novel_pir76584 are associated with the DNA/hobo of Drosophila, Ac of maize and Tam3 of snapdragon (hAT)-Charlie transposon. The novel_pir97924, novel_pir105705 and novel_pir105700 recognize long interspersed nuclear elements 1 (LINE-1). The novel_pir33182 and novel_pir46604 are related to the long terminal repeat (LTR)/(Endogenous Retrovirus1) ERV1 repetitive element. The novel_pir73855 is related to the LTR/ERVK repetitive element. Both novel_pir70108 and novel_pir70106 are associated with the LTR/ERVL-MaLR repetitive element. The novel_pir15900 is associated with the DNA/hAT-Tip100 repetitive element. Overall, our results indicated that rhinovirus infection could reduce the expression of some piRNAs to facilitate upregulation of LINE-1 transcription or retrotransposons' expression, which is helpful to further explore the mechanism of rhinovirus infection.
Current Status and Challenge of Pseudorabies Virus Infection in China
Lei Tan, Jun Yao, Yadi Yang, Wei Luo, Xiaomin Yuan, Lingchen Yang, Aibing Wang
doi: 10.1007/s12250-020-00340-0
Received: 20 July 2020 Accepted: 17 September 2020 Published: 22 February 2021
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Pseudorabies (PR), also called Aujeszky’s disease, is a highly infectious disease caused by pseudorabies virus (PRV). Without specific host tropism, PRV can infect a wide variety of mammals, including pig, sheep, cattle, etc., thereby causing severe clinical symptoms and acute death. PRV was firstly reported in China in 1950s, while outbreaks of emerging PRV variants have been documented in partial regions since 2011, leading to significant economic losses in swine industry. Although scientists have been devoting to the design of diagnostic approaches and the development of vaccines during the past years, PR remains a vital infectious disease widely prevalent in Chinese pig industry. Especially, its potential threat to human health has also attracted the worldwide attention. In this review, we will provide a summary of current understanding of PRV in China, mainly focusing on PRV history, the existing diagnosis methods, PRV prevalence in pig population and other susceptible mammals, molecular characteristics, and the available vaccines against its infection. Additionally, promising agents including traditional Chinese herbal medicines and novel inhibitors that may be employed to treat this viral infection, are also discussed.
Development of A MERS-CoV Replicon Cell Line for Antiviral Screening
Jing Chen, Bing-Jie Hu, Kai Zhao, Yun Luo, Hao-Feng Lin, Zheng-Li Shi
doi: 10.1007/s12250-020-00341-z
Received: 17 October 2020 Accepted: 23 November 2020 Published: 22 February 2021
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Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of  ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.
Alterations in Phenotypes and Responses of T Cells Within 6 Months of Recovery from COVID-19: A Cohort Study
Bali Zhao, Maohua Zhong, Qingyu Yang, Ke Hong, Jianbo Xia, Xia Li, Ying Liu, Yao-Qing Chen, Jingyi Yang, Chaolin Huang, Huimin Yan
doi: 10.1007/s12250-021-00348-0
Received: 11 October 2020 Accepted: 30 November 2020 Published: 09 February 2021
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The COVID-19 pandemic, caused by the SARS-CoV-2 infection, is a global health crisis. While many patients have clinically recovered, little is known about long-term alterations in T cell responses of COVID-19 convalescents. In this study, T cell responses in peripheral blood mononuclear cells of a long-time COVID-19 clinically recovered (20–26 weeks) cohort (LCR) were measured via flow cytometry and ELISpot. The T cell responses of LCR were comparatively analyzed against an age and sex matched short-time clinically recovered (4–9 weeks) cohort (SCR) and a healthy donor cohort (HD). All volunteers were recruited from Wuhan Jinyintan Hospital, China. Phenotypic analysis showed that activation marker PD-1 expressing on CD4+ T cells of LCR was still significantly lower than that of HD. Functional analysis indicated that frequencies of Tc2, Th2 and Th17 in LCR were comparable to those of HD, but Tc17 was higher than that of HD. In LCR, compared to the HD, there were fewer IFN-γ producing T cells but more IL-2 secreting T cells. In addition, the circulating Tfh cells in LCR were still slightly lower compared to HD, though the subsets composition had recovered. Remarkably, SARS-CoV-2 specific T cell responses in LCR were comparable to that of SCR. Collectively, T cell responses experienced long-term alterations in phenotype and functional potential of LCR cohort. However, after clinical recovery, SARS-CoV-2 specific T cell responses could be sustained at least for six months, which may be helpful in resisting re-infection.
Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates
Li-Nan Wang, Xiang-Lei Peng, Min Xu, Yuan-Bo Zheng, Yue-Ying Jiao, Jie-Mei Yu, Yuan-Hui Fu, Yan-Peng Zheng, Wu-Yang Zhu, Zhong-Jun Dong, Jin-Sheng He
doi: 10.1007/s12250-021-00345-3
Received: 28 February 2020 Accepted: 18 September 2020 Published: 09 February 2021
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Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.
Human PRV Infection in China: An Alarm to Accelerate Eradication of PRV in Domestic Pigs
Zhenhua Guo, Xin-Xin Chen, Gaiping Zhang
doi: 10.1007/s12250-021-00347-1
Received: 12 October 2020 Accepted: 21 December 2020 Published: 04 February 2021
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Pseudorabies, also known as Aujeszky’s disease, is one of the most economically important viral diseases in pigs and is lethal to other susceptible animals (Ren et al. 2020). The causative agent, pseudorabies virus (PRV), is an enveloped virus with a large double-stranded DNA genome encoding at least 70 proteins (Wong et al. 2019). PRV belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus and infects multiple animals, such as pigs, dogs, cats, rabbits, cattle, sheep, goats, minks, foxes, wolves, lynxes, etc. (He et al. 2019; Laval and Enquist 2020). Pigs are recognized as the natural hosts for the virus, and the PRV infection causes severe neurological symptoms in piglets with almost 100% mortality, respiratory/neurological signs in the nursery, respiratory signs in adult pigs, and reproductive disorders in sows (He et al. 2019; Ren et al. 2020). Most of the other infected non-natural animal hosts die within 24–48 h of disease onset, which is usually characterized by severe pruritus in the head and neck, accompanied by self-mutilation (Laval and Enquist 2020).
ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins
Jun Zhang, Tianhang Zheng, Xiaolei Zhou, Hong Wang, Zhaolong Li, Chen Huan, Baisong Zheng, Wenyan Zhang
doi: 10.1007/s12250-021-00346-2
Received: 06 August 2020 Accepted: 23 November 2020 Published: 03 February 2021
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Our recent study reported that ATP1B3 inhibits hepatitis B virus (HBV) replication via inducing NF-κB activation. However, ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication, suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression. Here, we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs, MHBs and SHBs, but had no effect on intracellular HBV DNA, RNA levels as well as HBV promoter activities. Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation, demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins. Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination. Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together. Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice
Zhao-Yang Wang, Zi-Da Zhen, Dong-Ying Fan, Pei-Gang Wang, Jing An
doi: 10.1007/s12250-020-00342-y
Received: 06 July 2020 Accepted: 07 December 2020 Published: 03 February 2021
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Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis. Axl, a receptor tyrosine kinase from TAM family, plays crucial role in many inflammatory diseases. We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection, which we speculate is due to the anti-inflammatory effect of Axl during JE. Currently, the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet. In this study, by using Axl deficient and heterozygous control mice, we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death, extended infiltration of inflammatory cells, and enhanced production of pro-inflammatory cytokines, in comparison to control mice. Additionally, consistent with our previous report, Axl deficiency had no impact on the infection and target cell tropism of JEV in brain. Taken together, our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.

Rearrangement of Actin Cytoskeleton by Zika Virus Infection Facilitates Blood–Testis Barrier Hyperpermeability
Yiwen Nie, Lixia Hui, Moujian Guo, Wei Yang, Rui Huang, Junsen Chen, Xinyue Wen, Meng Zhao, Ying Wu
doi: 10.1007/s12250-020-00343-x
Received: 27 August 2020 Accepted: 24 November 2020 Published: 03 February 2021
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In recent years, various serious diseases caused by Zika virus (ZIKV) have made it impossible to be ignored. Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier (BTB), or Sertoli cell barrier (SCB). However, little is known about the underlying mechanism. In this study, interaction between actin, an important component of the SCB, and ZIKV envelope (E) protein domain Ⅲ (EDⅢ) was inferred from coimmunoprecipitation (Co-IP) liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Confocal microscopy confirmed the role of actin filaments (F-actin) in ZIKV infection, during which part of the stress fibers, the bundles that constituted by paralleled actin filaments, were disrupted and presented in the cell periphery. Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement. Perturbation of F-actin by cytochalasin D (CytoD) or Jasplakinolide (Jas) enhanced the infection of ZIKV. More importantly, the transepithelial electrical resistance (TEER) of an in vitro mouse SCB (mSCB) model declined with the progression of ZIKV infection or overexpression of E protein. Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression, highlighting the role of E protein in ZIKV-induced disruption of the BTB. We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network, thereby compromising BTB integrity.

Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells
Jia Dan, Lin Nie, Xudong Jia, Cuiying Xu, Jing Cai, Yuan Lin, Jun Hu, Wenbo Zhu, Yinyin Li, Dong Chen, Ying Liu, Cheng Hu, Guangmei Yan, Jiankai Liang, Qinfen Zhang
doi: 10.1007/s12250-020-00339-7
Received: 11 July 2020 Accepted: 26 October 2020 Published: 22 January 2021
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Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells, but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear. Here, we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy, specifically examining viral entry, assembly, maturation and release. We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization, a typical indicator of apoptosis. Specifically, our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids. In the early and late stage of infection, three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication, maturation and release of the virus. Taken together, our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System
Yongsen Cao, Huanbin Zhou, Xueping Zhou, Fangfang Li
doi: 10.1007/s12250-020-00338-8
Received: 19 August 2020 Accepted: 21 November 2020 Published: 18 January 2021
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Laboratory-based Surveillance and Clinical Profile of Sporadic HEV Infection in Shanghai, China
Jie Lu, Qing Li, Jiayuan Jiang, Ziqiang Li, Peiyun Wang, Zike Sheng, Rongtao Lai, Huijuan Zhou, Wei Cai, Hui Wang, Qing Guo, Honglian Gui, Qing Xie
doi: 10.1007/s12250-020-00336-w
Received: 17 September 2020 Accepted: 30 October 2020 Published: 12 January 2021
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The study aimed to describe the epidemiological, virological and clinical features of sporadic HEV infection in eastern China. A total of 6112 patient sera were tested for anti-HEV IgG or anti-HEV IgM during one consecutive year (between August 2018 and July 2019). HEV RNA presence was evaluated by RT-PCR and HEV sequences were phylogenetically analyzed. Clinical features of confirmed HEV-infected patients were delineated. The sero-positivity rate of anti-HEV IgG maintained stable around 40%, while an obvious winter spike of anti-HEV IgM prevalence was observed. A total of 111 patients were confirmed of HEV viremia by molecular diagnosis. Subtype 4d was predominant. Phylogenetic analyses suggest that certain strains circulate across species and around the country. Subjects with confirmed current HEV infection had a high median age (58 years) and males were predominant (62.2%). Most patients presented with jaundice (75.7%) and anorexia (68.0%). Significantly elevated levels of liver enzymes and bilirubin were observed. Remarkably, the baseline bilirubin level was positively correlated with illness severity. Pre-existing HBV carriage may deteriorate illness. The clinical burden caused by locally acquired HEV infection is increasing. Surveillance should be enforced especially during the transition period from winter to spring. Patients with higher level of bilirubin at disease onset had slower recovery from HEV infection.

Genetic Analysis of Human Adenovirus Type 7 Strains Circulating in Different Parts of China
Yali Duan, Changchong Li, Li Deng, Shuhua An, Yun Zhu, Wei Wang, Meng Zhang, Li liXu, Baoping Xu, Xiangpeng Chen, Zhengde Xie
doi: 10.1007/s12250-020-00334-y
Received: 24 April 2020 Accepted: 10 October 2020 Published: 05 January 2021
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To investigate the molecular epidemiology and genetic variation of human adenovirus type 7 (HAdV-7) in children with acute respiratory infections (ARI) in China. HAdV-7-positive respiratory samples collected from children with ARI in Beijing, Shijiazhuang, Wenzhou and Guangzhou from 2014–2018 were selected for gene amplification and sequence analysis. Fifty-seven HAdV-7 clinical strains with hexon, penton base and fiber gene sequences were obtained. Meanwhile 17 strains were selected randomly from different cities for whole genome sequencing. Phylogenetic and variation analyses were performed based on the obtained sequences, HAdV-7 prototype strain Gomen (AY594255), vaccine strains (AY495969 and AY594256) and representative sequences of strains. The phylogenetic trees constructed based on whole genome sequences, major capsid protein genes (hexon, penton base and fiber) and the early genes (E1, E2, E3 and E4) were not completely consistent. The HAdV-7 strains obtained in this study always clustered with most of the circulating strains worldwide from the 1980s to the present. Compared with the HAdV-7 prototype strain Gomen (AY594255), some amino acid mutations in loop1 and loop2 of hexon and the RGD loop region of the penton base gene were observed. Recombination analysis showed that partial regions of 55 kDa protein and 100 kDa hexon-assembly associated protein genes among all HAdV-7 strains in this study were from HAdV-16 and HAdV-3, respectively. Our study demonstrated the molecular evolution characteristics of HAdV-7 strains circulating in China and provided basic reference data for the prevention, control and vaccine development of HAdV-7.
Foot-and-Mouth Disease Virus Inhibits RIP2 Protein Expression to Promote Viral Replication
Huisheng Liu, Qiao Xue, Zixiang Zhu, Fan Yang, Weijun Cao, Xiangtao Liu, Haixue Zheng
doi: 10.1007/s12250-020-00322-2
Received: 10 July 2020 Accepted: 17 September 2020 Published: 05 January 2021
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Receptors interaction protein 2 (RIP2) is a specific adaptor molecule in the downstream of NOD2. The role of RIP2 during foot-and-mouth disease virus (FMDV) infection remains unknown. Here, our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-β and NF-κB signal pathways during FMDV infection. FMDV infection triggered RIP2 transcription, while it reduced the expression of RIP2 protein. Detailed analysis showed that FMDV 2B, 2C, 3Cpro, and Lpro proteins were responsible for inducing the reduction of RIP2 protein. 3Cpro and Lpro are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis. The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2. Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2. The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1 (PABPC1). The interaction between RIP2 and 2C was observed in the context of viral infection, and the residues 1-61 were required for the interaction. These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.
Cell Cycle Arrest Protein CDKN2C Is Not an HBV Host Factor
Guiwen Guan, Liwei Zheng, Jingyuan Xi, Xingwen Yang, Xiangmei Chen, Fengmin Lu
doi: 10.1007/s12250-020-00337-9
Received: 27 July 2020 Accepted: 16 November 2020 Published: 05 January 2021
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Phylogenetic Analysis of the Dengue Virus Strains Causing the 2019 Dengue Fever Outbreak in Hainan, China
Jiang Du, Liyuan Zhang, Xiaoyuan Hu, Ruoyan Peng, Gaoyu Wang, Yi Huang, Wenqi Wang, Kunliang Wu, Qiang Wang, Haoxiang Su, Fan Yang, Yun Zhang, Chuanning Tang, Xiuji Cui, Lina Niu, Gang Lu, Meifang Xiao, Yongguo Du, Feifei Yin
doi: 10.1007/s12250-020-00335-x
Received: 16 August 2020 Accepted: 03 December 2020 Published: 05 January 2021
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Dengue virus is an arthropod-borne pathogen that is transmitted to humans primarily by Aedes spp. mosquitos, causing the acute infectious disease, dengue fever (DF). Until 2019, no dengue outbreak had been reported in Hainan Province for over 20 years. However, in early September of 2019, an increasing number of infected cases appeared and the DF outbreak lasted for over one month in Haikou City, Hainan Province. In our study, we collected 97 plasma samples from DF patients at three hospitals, as well as 1585 mosquito larvae samples from puddles in different areas of Haikou. There were 49 (50.5%) plasma samples found to be strongly positive and 9 (9.3%) plasma samples were weakly positive against the NS1 antigen. We discovered DENV both in the patient's plasma samples and mosquito larvae samples, and isolated the virus from C6/36 cells inoculated with the acute phase serum of patients. Phylogenetic analysis revealed that the new strains were the most closely related to the epidemic strain in the southern regions of China, belonging to lineage Ⅳ, genotype Ⅰ, DENV-1. Compared to the seven closest strains from neighboring countries and provinces, a total of 18 amino acid mutations occurred in the coding sequences (CDS) of the new isolated strain, DENV1 HMU-HKU-2. Our data shows that dengue virus is re-emerged in Hainan, and pose new threats for public health. Thus regular molecular epidemiological surveillance is necessary for control and prevention of DENV transmission.
Metagenomic Profiling of Viruses Associated with Rhipicephalus microplus Ticks in Yunnan Province, China
Junming Shi, Shu Shen, Hui Wu, Yunzhi Zhang, Fei Deng
doi: 10.1007/s12250-020-00319-x
Received: 22 August 2020 Accepted: 10 October 2020 Published: 05 January 2021
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Ticks are well known as vectors of many viruses which usually do great harm to human and animal health. Yunnan Province, widely covered by flourishing vegetation and mainly relying on farming husbandry, is abundant with Rhipicephalus microplus ticks. Therefore, it is of great significance to characterize the viral profile present in R. microplus parasitizing on cattle in Yunnan Province. In this study, a total of 7387 R. microplus ticks were collected from cattle and buffalo in the northwest and southeast areas of Yunnan Province from 2015 to 2017. We investigated the virome of R. microplus using next-generation sequencing (NGS) and the prevalence of important identified viruses among tick groups by RT-PCR. It revealed the presence of diverse virus concerning chu-, rhabdo-, phlebo-, flavi- and parvo- viruses in Yunnan. These viruses consist of single-stranded, circular and segmented sense RNAs, showing a greatly diversity in genomic organization. Furthermore, continuous epidemiological survey among ticks reveals broad prevalence of three viruses (Yunnan mivirus 1, Wuhan tick vrius 1 and YN tick-associated phlebovirus 1) and two possible prevalent viruses including a flavivirus-like segmented virus (Jingmen tick virus) and a bovine hokovirus 2 in Yunnan. Serological investigation among cattle indicates that these identified viruses may be infectious to cattle and can elicit corresponding antibody. Our findings on R. microplus-associated viral community will contribute to the prevention of viral disease and tracking the viral evolution. Further analysis is needed to better elucidate the pathogenicity and natural circulation of these viruses.
Herpesviruses and the Type Ⅲ Interferon System
Yue Yin, Herman W. Favoreel
doi: 10.1007/s12250-020-00330-2
Received: 09 September 2020 Accepted: 27 October 2020 Published: 05 January 2021
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Type Ⅲ interferons (IFNs) represent the most recently discovered group of IFNs. Together with type Ⅰ IFNs (e.g. IFN-α/β), type Ⅲ IFNs (IFN-λ) are produced as part of the innate immune response to virus infection, and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs). It was initially thought that type Ⅰ IFNs and type Ⅲ IFNs perform largely redundant functions. However, it has become evident that type Ⅲ IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers, thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world, versus the generally more broad, potent and systemic antiviral effects of type Ⅰ IFNs. Herpesviruseses are large DNA viruses, which enter their host via mucosal surfaces and establish lifelong, latent infections. Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses, our current knowledge on the interaction of herpesviruses with type Ⅲ IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV). This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.
Monocyte/Macrophage-Mediated Innate Immunity in HIV-1 Infection: From Early Response to Late Dysregulation and Links to Cardiovascular Diseases Onset
Eman Teer, Danzil E. Joseph, Richard H. Glashoff, M.Faadiel Essop
doi: 10.1007/s12250-020-00332-0
Received: 29 July 2020 Accepted: 26 October 2020 Published: 05 January 2021
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Although monocytes and macrophages are key mediators of the innate immune system, the focus has largely been on the role of the adaptive immune system in the context of human immunodeficiency virus (HIV) infection. Thus more attention and research work regarding the innate immune system—especially the role of monocytes and macrophages during early HIV-1 infection—is required. Blood monocytes and tissue macrophages are both susceptible targets of HIV-1 infection, and the early host response can determine whether the nature of the infection becomes pathogenic or not. For example, monocytes and macrophages can contribute to the HIV reservoir and viral persistence, and influence the initiation/extension of immune activation and chronic inflammation. Here the expansion of monocyte subsets (classical, intermediate and non-classical) provide an increased understanding of the crucial role they play in terms of chronic inflammation and also by increasing the risk of coagulation during HIV-1 infection. This review discusses the role of monocytes and macrophages during HIV-1 pathogenesis, starting from the early response to late dysregulation that occurs as a result of persistent immune activation and chronic inflammation. Such changes are also linked to downstream targets such as increased coagulation and the onset of cardiovascular diseases.
Development of Improved Mumps Vaccine Candidates by Mutating Viral mRNA Cap Methyltransferase Sites in the Large Polymerase Protein
Xiaoqiang Hao, Yilong Wang, Mengying Zhu, Dongming Zhou, Rongxian Liu, Bin Wang, Yao-Wei Huang, Zhengyan Zhao
doi: 10.1007/s12250-020-00326-y
Received: 26 July 2020 Accepted: 20 October 2020 Published: 07 December 2020
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Although a live attenuated vaccine is available for controlling mumps virus (MuV), mumps still outbreaks frequently worldwide. The attenuated MuV vaccine strain S79 is widely used in mumps vaccination in China, but still with many shortcomings, among which the most prominent are the side effects and decreased immunity. Therefore, there is a need to further improve the safety and efficacy of the current MuV vaccine. In the present study, we further attenuated MuV S79 vaccine strain by inhibiting viral mRNA methyltransferase (MTase). We generated a panel of eight recombinant MuVs (rMuVs) carrying mutations in the MTase catalytic site or S-adenosylmethionine (SAM) binding site in the large (L) polymerase protein. These rMuVs are genetically stable and seven rMuVs are more attenuated in replication in cell culture and five rMuVs are more attenuated in replication in lungs of cotton rats compared with the parental vaccine strain S79. Importantly, cotton rats vaccinated with these seven rMuV mutants produced high levels of serum neutralizing antibodies and were completely protected against challenge with a wild-type MuV strain (genotype F). Therefore, our results demonstrate that alteration in the MTase catalytic site or SAM binding site in MuV L protein improves the safety or the immunogenicity of the MuV vaccine and thus mRNA cap MTase may be an effective target for the development of new vaccine candidates for MuV.
Beta- and Novel Delta-Coronaviruses Are Identified from Wild Animals in the Qinghai-Tibetan Plateau, China
Wentao Zhu, Jing Yang, Shan Lu, Ruiting Lan, Dong Jin, Xue-lian Luo, Ji Pu, Shusheng Wu, Jianguo Xu
doi: 10.1007/s12250-020-00325-z
Received: 03 August 2020 Accepted: 27 September 2020 Published: 01 December 2020
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Outbreaks of severe virus infections with the potential to cause global pandemics are increasingly concerning. One type of those commonly emerging and re-emerging pathogens are coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2). Wild animals are hosts of different coronaviruses with the potential risk of cross-species transmission. However, little is known about the reservoir and host of coronaviruses in wild animals in Qinghai Province, where has the greatest biodiversity among the world's high-altitude regions. Here, from the next-generation sequencing data, we obtained a known beta-coronavirus (beta-CoV) genome and a novel delta-coronavirus (delta-CoV) genome from faecal samples of 29 marmots, 50 rats and 25 birds in Yushu Tibetan Autonomous Prefecture, Qinghai Province, China in July 2019. According to the phylogenetic analysis, the beta-CoV shared high nucleotide identity with Coronavirus HKU24. Although the novel delta-CoV (MtCoV) was closely related to Sparrow deltacoronavirus ISU42824, the protein spike of the novel delta-CoV showed highest amino acid identity to Sparrow coronavirus HKU17 (73.1%). Interestingly, our results identified a novel host (Montifringilla taczanowskii) for the novel delta-CoV and the potential cross-species transmission. The most recent common ancestor (tMRCA) of MtCoVs along with other closest members of the species of Coronavirus HKU15 was estimated to be 289 years ago. Thus, this study increases our understanding of the genetic diversity of beta-CoVs and delta-CoVs, and also provides a new perspective of the coronavirus hosts.
Tumor Necrosis Factor α Reduces SNAP29 Dependent Autolysosome Formation to Increase Prion Protein Level and Promote Tumor Cell Migration
Huan Li, Ren Wang, Ze Yu, Run Shi, Jie Zhang, Shanshan Gao, Ming Shao, Shuzhong Cui, Zhenxing Gao, Jiang Xu, Man-Sun Sy, Chaoyang Li
doi: 10.1007/s12250-020-00320-4
Received: 12 August 2020 Accepted: 10 October 2020 Published: 25 November 2020
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Tumor Necrosis Factor α (TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein (PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration; this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B (NF-кB) signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3 (FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29 (SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated, and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-кB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration.
SOX5 Regulates Cell Proliferation, Apoptosis, Migration and Invasion in KSHV-Infected Cells
Wu-Mei Yuan, Ya-Ge Fan, Meng Cui, Ting Luo, Ya-E Wang, Zhan-Jun Shu, Juan Zhao, Jun Zheng, Yan Zeng
doi: 10.1007/s12250-020-00313-3
Received: 01 July 2020 Accepted: 16 September 2020 Published: 24 November 2020
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Kaposi's sarcoma (KS) originates from vascular endothelial cells, with KS-associated herpesvirus (KSHV) as the etiological agent. SRY-box transcription factor 5 (SOX5) plays different roles in various types of cancer, although its role in KS remains poorly understood. In this study, we identified the role of SOX5 in KS tissues and KSHV-infected cells and elucidated the molecular mechanism. Thirty-two KS patients were enrolled in this study. Measurement of SOX5 mRNA and protein levels in human KS tissues and adjacent control tissues revealed lower levels in KS tissues, with KS patients having higher SOX5 level in the early stages of the disease compared to the later stages. And SOX5 mRNA and protein was also lower in KSHV-infected cells (iSLK-219 and iSLK-BAC) than normal cells (iSLK-Puro). Additionally, SOX5 overexpression inhibited cell proliferation and promoted apoptosis and decreased KSHV-infected cell migration and invasion. Moreover, we found that SOX5 overexpression suppressed the epithelial-to-mesenchymal transition of KSHV-infected cells. These results suggest SOX5 is a suppressor factor during KS development and a potential target for KS treatment.
Generation of A Stable GFP-reporter Zika Virus System for High-throughput Screening of Zika Virus Inhibitors
Jing-Wei Zhang, Han Wang, Jing Liu, Le Ma, Rong-Hong Hua, Zhi-Gao Bu
doi: 10.1007/s12250-020-00316-0
Received: 31 July 2020 Accepted: 18 September 2020 Published: 24 November 2020
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Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549. The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.
Broad Cell Tropism of SADS-CoV In Vitro Implies Its Potential Cross-Species Infection Risk
Yun Luo, Ying Chen, Rong Geng, Bei Li, Jing Chen, Kai Zhao, Xiao-Shuang Zheng, Wei Zhang, Peng Zhou, Xing-Lou Yang, Zheng-Li Shi
doi: 10.1007/s12250-020-00321-3
Received: 15 July 2020 Accepted: 26 October 2020 Published: 17 November 2020
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Sumoylation of Human Parainfluenza Virus Type 3 Phosphoprotein Correlates with A Reduction in Viral Replication
Qi Cheng, Wenjing Huai, Xiaoyan Wu, Mingzhou Chen
doi: 10.1007/s12250-020-00314-2
Received: 11 September 2020 Accepted: 29 September 2020 Published: 16 November 2020
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Human parainfluenza virus type 3 (HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein (P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein (L). P connects nucleocapsid protein (N) with L to initiate genome transcription and replication. Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine (PK492R/K532R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492R/K532R had no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies. Finally, we found that incorporation of K492R/K532R into a recombinant HPIV3 (rHPIV3-PK492R/K532R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.
Piperlongumine Inhibits Zika Virus Replication In vitro and Promotes Up-Regulation of HO-1 Expression, Suggesting An Implication of Oxidative Stress
Weizhi Lu, Linjuan Shi, Jing Gao, Huimin Zhu, Ying Hua, Jintai Cai, Xianbo Wu, Chengsong Wan, Wei Zhao, Zhang Bao
doi: 10.1007/s12250-020-00310-6
Received: 16 April 2020 Accepted: 25 August 2020 Published: 13 November 2020
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Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new infectious diseases, Zika virus has become a global public health concern. Recent studies have found that Zika virus can continuously infect human brain microvascular endothelial cells. These cells are the primary components of the bloodɃbrain barrier of the cerebral cortex, and further infection of brain tissue may cause severe damage such as encephalitis and fetal pituitary disease. The present study found that a biologically active base, piperlongumine (PL), inhibited Zika virus replication in human brain microvascular endothelial cells, Vero cells, and human umbilical vein endothelial cells. PL also significantly increased heme oxygenase-1 (HO-1) gene expression, while silencing HO-1 expression and using the reactive oxygen species scavenger, N-acetylcysteine, attenuated the inhibitory effect of PL on Zika virus replication. These results suggest that PL induces oxidative stress in cells by increasing reactive oxygen species. This, in turn, induces an increase in HO-1 expression, thereby inhibiting Zika virus replication. These findings provide novel clues for drug research on the prevention and treatment of Zika virus.
Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase
Zuopeng Zhang, Sen Yuan, Shuting Xu, Deyin Guo, Lang Chen, Wei Hou, Min Wang
doi: 10.1007/s12250-020-00311-5
Received: 14 March 2020 Accepted: 14 September 2020 Published: 13 November 2020
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Human immunodeficiency virus (HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome (AIDS). Treatment with combination antiretroviral therapy (cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway (UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases (E3s) based on known human E3s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation. Herein, a series of prototypical chimeric E3s have been designed and constructed, and original substrate-binding domains of these E3s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146LI was then further optimized to generate 146LIS (146LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair (HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.
Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou, China
Xingui Tian, Ye Fan, Changbing Wang, Zhenwei Liu, Wenkuan Liu, Yun Xu, Chuncong Mo, Aiping You, Xiao Li, Xia Rong, Rong Zhou
doi: 10.1007/s12250-020-00307-1
Received: 29 June 2020 Accepted: 31 August 2020 Published: 09 November 2020
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Human adenoviruses (HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3, -7, -4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population. Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies (NAb) against HAdV-3, -4, -7, -14, -55, and -11 in total 278 healthy populations between 0 months and 49 years of age (228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at 12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1–2, 3–5, and 6–17 years, respectively. The seroprevalence was very low (0%~8.1%) for all other four types. In adults aged between 18 and 49 years, HAdV-3, -4, and -7 (>50.00%) were the most common types, followed by HAdV-14 (38.00%), -55 (34.00%), and -11 (24.00%). Adults tended to have high NAb titers against HAdV-4 and -55. HAdV-55-seropositive donors tended to be HAdV-11- and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14, -55, -11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.
Repurposing of Antazoline Hydrochloride as an Inhibitor of Hepatitis B Virus DNA Secretion
Jing Li, Yangyang Hu, Yifei Yuan, Yinan Zhao, Qiqi Han, Canyu Liu, Xue Hu, Yuan Zhou, Yun Wang, Yu Guo, Chunchen Wu, Xinwen Chen, Rongjuan Pei
doi: 10.1007/s12250-020-00306-2
Received: 26 March 2020 Accepted: 16 September 2020 Published: 09 November 2020
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Hepatitis B virus (HBV) belongs to Hepadnaviridae family and mainly infects hepatocytes, which can cause acute or chronic hepatitis. Currently, two types of antiviral drugs are approved for chronic infection clinically: interferons and nucleos(t)ide analogues. However, the clinical cure for chronic infection is still rare, and it is a huge challenge for all researchers to develop high-efficiency, safe, non-tolerant, and low-toxicity anti-HBV drugs. Antazoline hydrochloride is a first-generation antihistamine with anticholinergic properties, and it is commonly used to relieve nasal congestion and in eye drops. Recently, an in vitro high-throughput evaluation system was constructed to screen nearly 800 compounds from the Food and Drug Administration (FDA)-approved Drug Library. We found that arbidol hydrochloride and antazoline hydrochloride can effectively reduce HBV DNA in the extracellular supernatant in a dose-dependent manner, with EC50 of 4.321 μmol/L and 2.910 μmol/L in HepAD38 cells, respectively. Moreover, the antiviral effects and potential mechanism of action of antazoline hydrochloride were studied in different HBV replication systems. The results indicate that antazoline hydrochloride also has a significant inhibitory effect on HBV DNA in the extracellular supernatant of Huh7 cells, with an EC50 of 2.349 μmol/L. These findings provide new ideas for screening and research related to HBV agents.
Hantavirus Infection during Pregnancy
Deng-Hui Lu, Hong Jiang, Jian-Qi Lian
doi: 10.1007/s12250-020-00300-8
Received: 25 January 2020 Accepted: 07 August 2020 Published: 19 October 2020
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Hantavirus infection is a global health challenge, causing widespread public concern. In recent years, cases of hantavirus infection in pregnant women have been reported in many countries. The infected pregnant women and their fetuses appear to have more severe clinical symptoms and worse clinical outcomes. Hence, to study the prevalence of hantavirus infection in pregnant women, this study will focus on the epidemiological distribution of the virus, different virus species penetrating the placental barrier, and factors affecting the incidence and clinical outcome of the infection in pregnant women and their fetuses. In addition, this review will also discuss the diagnostic tools and treatments for pregnant patients and provide an overview of the relevant future research.
PTEN Lipid Phosphatase Activity Enhances Dengue Virus Production through Akt/FoxO1/Maf1 Signaling
Bin Liu, Ting-Ting Gao, Xiao-Yu Fu, Zhen-Hao Xu, Hao Ren, Ping Zhao, Zhong-Tian Qi, Zhao-Ling Qin
doi: 10.1007/s12250-020-00291-6
Received: 10 January 2020 Accepted: 31 July 2020 Published: 12 October 2020
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Dengue virus (DENV) is an arthropod-borne viral pathogen and a global health burden. Knowledge of the DENV-host interactions that mediate virus pathogenicity remains limited. Host lipid metabolism is hijacked by DENV for virus replication in which lipid droplets (LDs) play a key role during the virus lifecycle. In this study, we reveal a novel role for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in LDs-mediated DENV infection. We demonstrate that PTEN expression is downregulated upon DENV infection through post-transcriptional regulation and, in turn, PTEN overexpression enhances DENV replication. PTEN lipid phosphatase activity was found to decrease cellular LDs area and number through Akt/FoxO1/Maf1 signaling, which, together with autophagy, enhanced DENV replication and virus production. We therefore provide mechanistic insight into the interaction between lipid metabolism and the DENV replication cycle.
Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo
Mengwei Li, Yuxu Wang, Jing Jin, Jie Dou, Qinglong Guo, Xue Ke, Changlin Zhou, Min Guo
doi: 10.1007/s12250-020-00302-6
Received: 19 July 2019 Accepted: 11 September 2020 Published: 12 October 2020
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Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain to be elucidated. Here, four honeysuckle extracts, including acids extract, flavonoids extract, total extract and acids-flavonoids mixture, were prepared to clarify the main active antiviral components. The cytopathic effect reduction assay showed that all the four extracts inhibited the replication of influenza viruses H1N1, H3N2 and the oseltamivir-resistant mutant strain H1N1-H275Y. The acids-flavonoids mixture had the strongest inhibitory effects in vitro with EC50 values of 3.8, 4.1, and >20 μg/mL against H1N1, H3N2 and H1N1-H275Y, respectively, showing competitive antiviral activity with oseltamivir and ribavirin. Honeysuckle acids extract also showed the most significant antiviral activity in vivo. Oral administration of the acids extract at a dosage of 600 mg/kg/d effectively alleviated viral pneumonia, maintained body weight and improved the survival rate to 30% of the mice infected with a lethal dose of H1N1. The results of time-of-drug addition experiment and neuraminidase (NA) inhibition assay showed that honeysuckle extracts had a broad-spectrum inhibitory effect against influenza virus NAs. The flavonoid extract showed the strongest inhibitory effect on the NA of influenza virus H7N9 with an IC50 of 24.7 μg/mL. These results suggested that these extracts might exert their antiviral activity by suppressing the release of influenza viruses. Briefly, our findings demonstrate that acids and flavonoids extracts of honeysuckle are the major antiviral active components, and the acids extract has the potential to be developed into an antiviral agent against influenza virus, especially for oseltamivir-resistant viruses.
Application of Human Adenovirus Genotyping by Phylogenetic Analysis in an Outbreak to Identify Nosocomial Infection
Chuanyu Yang, Chunmei Zhu, Yuan Qian, Jie Deng, Baoyuan Zhang, Runan Zhu, Fang Wang, Yu Sun, Dongmei Chen, Qi Guo, Yutong Zhou, Lei Yu, Ling Cao, Linqing Zhao
doi: 10.1007/s12250-020-00299-y
Received: 14 May 2020 Accepted: 05 August 2020 Published: 01 October 2020
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Nosocomial infections are common in pediatric patients and can be fatal in infants and immunocompromised patients. In September 2018, a high positive rate of human adenovirus HAdV was occurred among hospitalized children in the Children's Hospital Affiliated to the Capital Institute of Paediatrics in Beijing. To investigate whether this outbreak of HAdV was related to nosocomial infections or the result of community infections, we collected respiratory specimens from patients with acute respiratory infections in a respiratory ward during June to December 2018, and screened for respiratory viruses. Among 1, 840 cases included, 95 (5.2%, 95/1840) were positive for HAdV and 81 were genotyped based on phylogenetic analysis, including seven as HAdV-1 (8.6%), 30 HAdV-3 (37.0%), two HAdV-6 (2.5%), and 42 HAdV-7 (51.9%). More HAdV-positive samples were collected in August (4.7%, 12/255), September (15.0%, 41/274) and October (6.9%, 17/247), with a peak in September 2018. By combining the results of HAdV phylogenetic analysis with clinical data of patients, there were 77 cases (4.2%, 77/1840; 81.1%, 77/95) excluded from nosocomial infections, eight cases representing possible infections transmitted by visitors or attending parents, three cases without sequences that might have been due to infection transmitted by roommates positive for HAdV, one case of a roommate without an HAdV sequence, and six cases that shared highly homologous sequences with those of their roommates, for which nosocomial infections might be considered. In conclusion, genotyping of HAdVs based on phylogenetic analysis combined with clinical information provides a powerful method to distinguish nosocomial infections from community acquired infection, especially when tracing the origins of nosocomial infections.
A Sensitive and High-Throughput Flow Cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus Type 3
Zhenwei Liu, Xingui Tian, Wenkuan Liu, Yuting Xian, Weilue Chen, Rong Zhou
doi: 10.1007/s12250-020-00295-2
Received: 18 January 2020 Accepted: 24 August 2020 Published: 29 September 2020
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The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAdV-3) are the microneu-tralization (MN) assay, which has insufficient sensitivity, and the plaque reduction neutralization test (PRNT), which is not suitable for high-throughput screening. Herein, we describe the development of a flow cytometry-based neutralization (FCN) assay for measuring the neutralization of sera, cell culture supernatants, and chimeric antibodies against HAdV-3 on the basis of a recombinant HAdV-3 (rHAdV-3) construct expressing the enhanced green fluorescent protein (EGFP). For flow cytometry-based assays, the optimal cell confluence was determined as 90%, and the virus was titrated using the assay. The established FCN assay follows the percentage law and an optimal MOI of not less than 5×10-4 was determined by using a purified chimeric antibody. In addition, comparison of the anti-HAdV-3 NAb titers of 72 human serum samples by the MN and FCN assays, showed that both assays correlated strongly with each other. Our FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and robotically. Importantly, the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture supernatants. Thus, this sensitive and high-throughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAdV-3 and for screening anti-HAdV-3 NAbs in cell culture supernatants.
Molecular Characterization of Human Respiratory Adenoviruses Infection in Xining City, China In 2018
Juan Yu, Shengcang Zhao, Huaxiang Rao
doi: 10.1007/s12250-020-00282-7
Received: 18 March 2020 Accepted: 05 August 2020 Published: 14 September 2020
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Freeze-Drying Formulations Increased the Adenovirus and Poxvirus Vaccine Storage Times and Antigen Stabilities
Ye Chen, Qibin Liao, Tianyue Chen, Yuchao Zhang, Weien Yuan, Jianqing Xu, Xiaoyan Zhang
doi: 10.1007/s12250-020-00250-1
Received: 19 April 2020 Published: 21 July 2020
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Successful vaccines induce specific immune responses and protect against various viral and bacterial infections. Noninactivated vaccines, especially viral vector vaccines such as adenovirus and poxvirus vaccines, dominate the vaccine market because their viral particles are able to replicate and proliferate in vivo and produce lasting immunity in a manner similar to natural infection. One challenge of human and livestock vaccination is vaccine stability related to the antigenicity and infectivity. Freeze-drying is the typical method to maintain virus vaccine stability, while cold chain transportation is required for temperatures about 2 ℃–8 ℃. The financial and technological resource requirements hinder vaccine distribution in underdeveloped areas. In this study, we developed a freeze-drying formula consisting of bovine serum albumin (BSA), L-glutamic acid (L-Glu), polyethylene glycol (PEG), and dextran (DEX) to improve the thermal stability and activity of viral vaccines, including vaccinia recombinant vaccine (rTTV-OVA) and adenovirus vaccine (Ad5-ENV). We compared a panel of five different formulations (PEG: DEX: BSA: L-GLU=50:9:0:0(#1), 50:5:4:0(#2), 50:10:9:0(#3), 50:0:0:9(#4), and 50:1:0:8(#5), respectively) and optimized the freeze-drying formula for rTTV-OVA and Ad5-ENV. We found that the freeze-drying formulations #2 and #3 could maintain rTTV-OVA infectivity at temperatures of 4 ℃ and 25 ℃ and that rTTV-OVA immunogenicity was retained during lyophilization. However, formulations #4 and #5 maintained Ad5-ENV infectivity under the same conditions, and Ad5-ENV immunogenicity had maximum retention with freeze-drying formulation #4. In summary, we developed new freeze-drying formulations that increased virus vaccine storage times and retained immunogenicity at an ambient temperature.
Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: "Adenovirus Vaccine Within an Adenovirus Vector"
Yuqian Yan, Shuping Jing, Liqiang Feng, Jing Zhang, Zhiwei Zeng, Min Li, Shan Zhao, Junxian Ou, Wendong Lan, Wenyi Guan, Xiaowei Wu, Jianguo Wu, Donald Seto, Qiwei Zhang
doi: 10.1007/s12250-020-00234-1
Received: 22 February 2020 Accepted: 13 April 2020 Published: 26 May 2020
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Human adenoviruses (HAdVs) are highly contagious and result in large number of acute respiratory disease (ARD) cases with severe morbidity and mortality. Human adenovirus type 3 (HAdV-3) is the most common type that causes ARD outbreaks in Asia, Europe, and the Americas. However, there is currently no vaccine approved for its general use. The hexon protein contains the main neutralizing epitopes, provoking strong and lasting immunogenicity. In this study, a novel recombinant and attenuated adenovirus vaccine candidate against HAdV-3 was constructed based on a commercially-available replication-defective HAdV-5 gene therapy and vaccine vector. The entire HAdV-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system. The resultant recombinants expressing the HAdV-3 hexon protein were rescued in AD293 cells, identified and characterized by RT-PCR, Western blots, indirect immunofluorescence, and electron microscopy. This potential vaccine candidate had a similar replicative efficacy as the wild-type HAdV-3 strain. However, and importantly, the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twenty-generation passages in AD293 cells. This represents a significant safety feature. The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAdV-3. Therefore, this recombinant, attenuated, and safe adenovirus vaccine is a promising HAdV-3 vaccine candidate. The strategy of using a clinically approved and replication-defective HAdV-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.


From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease (HFMD)
Xiangchuan He, Miaomiao Zhang, Chen Zhao, Peiyong Zheng, Xiaoyan Zhang, Jianqing Xu
2021, 36(2): 167-175.  doi: 10.1007/s12250-020-00294-3
Received: 22 April 2020 Accepted: 25 August 2020 Published: 30 September 2020
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Hand, foot, and mouth disease (HFMD) recently emerged as a global public threat. The licensure of inactivated enterovirus A71 (EV-A71) vaccine was the first step in using a vaccine to control HFMD. New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage. The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines. The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes. The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a cross-protective antibody response.

Research Article
African Swine Fever Virus MGF360-12L Inhibits Type I Interferon Production by Blocking the Interaction of Importin α and NF-кB Signaling Pathway
Yisha Zhuo, Zeheng Guo, Tongtong Ba, Cheng Zhang, Lihua He, Cuiping Zeng, Hanchuan Dai
2021, 36(2): 176-186.  doi: 10.1007/s12250-020-00304-4
Received: 26 February 2020 Accepted: 13 July 2020 Published: 03 November 2020
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African swine fever (ASF) is an infectious transboundary disease of domestic pigs and wild boar and spreading throughout Eurasia. There is no vaccine and treatment available. Complex immune escape strategies of African swine fever virus (ASFV) are crucial factors affecting immune prevention and vaccine development. MGF360 genes have been implicated in the modulation of the IFN-I response. The molecular mechanisms contributing to innate immunity are poorly understood. In this study, we demonstrated that ASFV MGF360-12L (MGF360 families 12L protein) significantly inhibited the mRNA transcription and promoter activity of IFN-β and NF-кB, accompanied by decreases of IRF3, STING, TBK1, ISG54, ISG56 and AP-1 mRNA transcription. Also, MGF360-12L might suppress the nuclear localization of p50 and p65 mediated by classical nuclear localization signal (NLS). Additionally, MGF360-12L could interact with KPNA2, KPNA3, and KPNA4, which interrupted the interaction between p65 and KPNA2, KPNA3, KPNA4. We further found that MGF360-12L could interfere with the NF-кB nuclear translocation by competitively inhibiting the interaction between NF-кB and nuclear transport proteins. These findings suggested that MGF360-12L could inhibit the IFN-I production by blocking the interaction of importin a and NF-кB signaling pathway, which might reveal a novel strategy for ASFV to escape the host innate immune response.

African Swine Fever Virus MGF-110-9L-deficient Mutant Has Attenuated Virulence in Pigs
Dan Li, Yinguang Liu, Xiaolan Qi, Yuan Wen, Pan Li, Zhao Ma, Yongjie Liu, Haixue Zheng, Zhijie Liu
2021, 36(2): 187-195.  doi: 10.1007/s12250-021-00350-6
Received: 18 July 2020 Accepted: 17 December 2020 Published: 10 March 2021
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African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), an often lethal disease in domestic and wild pigs. ASF represents a major threat to the swine industry worldwide. Currently, no commercial vaccine is available because of the complexity of ASFV or biosecurity concerns. Live attenuated viruses that are naturally isolated or genetically manipulated have demonstrated reliable protection against homologous ASFV strain challenge. In the present study, a mutant ASFV strain with the deletion of ASFV MGF-110-9L (ASFV-Δ9L) was generated from a highly virulent ASFV CN/GS/2018 parental strain, a genotype II ASFV. Relative to the parental ASFV isolate, deletion of the MGF-110-9L gene significantly decreased the ability of ASFV-Δ9L to replicate in vitro in primary swine macrophage cell cultures. The majority of animals inoculated intramuscularly with a low dose of ASFV-Δ9L (10 HAD50) remained clinically normal during the 21-day observational period. Three of five ASFV-Δ9L-infected animals displayed low viremia titers and low virus shedding and developed a strong virus-specific antibody response, indicating partial attenuation of the ASFV-Δ9L strain in pigs. The findings imply the potential usefulness of the ASFV-Δ9L strain for further development of ASF control measures.

African Swine Fever Virus Protein E199L Promotes Cell Autophagy through the Interaction of PYCR2
Sheng Chen, Xinheng Zhang, Yu Nie, Hongxin Li, Weiguo Chen, Wencheng Lin, Feng Chen, Qingmei Xie
2021, 36(2): 196-206.  doi: 10.1007/s12250-021-00375-x
Received: 23 November 2020 Accepted: 01 February 2021 Published: 08 April 2021
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African swine fever virus (ASFV), as a member of the large DNA viruses, may regulate autophagy and apoptosis by inhibiting programmed cell death. However, the function of ASFV proteins has not been fully elucidated, especially the role of autophagy in ASFV infection. One of three Pyrroline-5-carboxylate reductases (PYCR), is primarily involved in conversion of glutamate to proline. Previous studies have shown that depletion of PYCR2 was related to the induction of autophagy. In the present study, we found for the first time that ASFV E199L protein induced a complete autophagy process in Vero and HEK-293T cells. Through co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) analysis, we firstly identified that E199L interact with PYCR2 in vitro. Importantly, our work provides evidence that E199L down-regulated the expression of PYCR2, resulting in autophagy activation. Overall, our results demonstrate that ASFV E199L protein induces complete autophagy through interaction with PYCR2 and down-regulate the expression level of PYCR2, which provide a valuable reference for the role of autophagy during ASFV infection and contribute to the functional clues of PYCR2.

Porcine Immunoglobulin Fc Fused P30/P54 Protein of African Swine Fever Virus Displaying on Surface of S. cerevisiae Elicit Strong Antibody Production in Swine
Chen Chen, Deping Hua, Jingxuan Shi, Zheng Tan, Min Zhu, Kun Tan, Lilin Zhang, Jinhai Huang
2021, 36(2): 207-219.  doi: 10.1007/s12250-020-00278-3
Received: 06 December 2019 Accepted: 13 July 2020 Published: 11 September 2020
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African swine fever virus (ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae (S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcγ and P54-Fcα fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcγ and P54-Fcα fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay. Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage (PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques
Meishen Ren, Hong Mei, Ming Zhou, Zhen F. Fu, Heyou Han, Dingren Bi, Fuhu Peng, Ling Zhao
2021, 36(2): 220-230.  doi: 10.1007/s12250-020-00323-1
Received: 12 May 2020 Accepted: 20 October 2020 Published: 07 January 2021
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African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.

SRP54 Negatively Regulates IFN-Beta Production and Antiviral Response by Targeting RIG-I and MDA5
Dong-Peng Wang, Hong-Yan Zhang, Bo-Wei Liao, Zhen Tong, Zhi-Sheng Xu, Yan-Yi Wang, Yan Yang
2021, 36(2): 231-240.  doi: 10.1007/s12250-020-00267-6
Received: 11 April 2020 Accepted: 08 June 2020 Published: 07 August 2020
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During virus infection, RIG-I-like receptors (RLRs) recognize viral RNAs and recruit the adaptor protein VISA to activate downstream signaling, leading to activation of transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs). IFNs further induce expression of hundreds of IFN-stimulated genes (ISGs) that suppress viral replication and facilitate the adaptive immune response. Dysregulated production of IFNs is implicated in various immune diseases. Here we identified Signal Recognition Particle 54 (SRP54) as a negative regulator of RLRs-induced antiviral signaling. Overexpression of SRP54 inhibited RNA virus-triggered induction of IFN-β and increased viral replication, whereas knockdown of SRP54 had opposite effects. Mechanistically, SRP54 interacted with both RIG-I and MDA5 and impaired their association with VISA. Our findings demonstrate that SRP54 acts as a negative regulator of RLRs-mediated innate immune response by disrupting the recruitment of VISA to RIG-I/MDA5.

Human Cytomegalovirus Influences Host circRNA Transcriptions during Productive Infection
Jingui Deng, Yujing Huang, Qing Wang, Jianming Li, Yanping Ma, Ying Qi, Zhongyang Liu, Yibo Li, Qiang Ruan
2021, 36(2): 241-253.  doi: 10.1007/s12250-020-00275-6
Received: 26 April 2020 Accepted: 08 July 2020 Published: 05 August 2020
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Human cytomegalovirus (HCMV) is a double-strand DNA virus widely infected in human. Circular RNAs (circRNAs) are non-coding RNAs with most functions of which keep unknown, and the effects of HCMV productive infection on host circRNA transcriptions remain unclear. In this study, we profiled 283 host circRNAs that significantly altered by HCMV productive infection in human embryonic lung fibroblasts (HELF) by RNA deep sequencing and bioinformatics analysis. Among these, circSP100, circMAP3K1, circPLEKHM1, and circTRIO were validated for their transcriptions and sequences. Furthermore, characteristics of circSP100 were investigated by RT-qPCR and northern blot. It was implied that circSP100 was produced from the sense strand of the SP100 gene containing six exons. Kinetics of circSP100 and SP100 mRNA were significantly different after infection: circSP100 levels increased gradually along with infection, whereas SP100 mRNA levels increased in the beginning and dropped at 24 h post-infection (hpi). Meanwhile, a total number of 257 proteins, including 10 HCMV encoding proteins, were identified potentially binding to cytoplasmic circSP100 by RNA antisense purification (RAP) and mass spectrometry. Enrichment analysis showed these proteins were mainly involved in the spliceosome, protein processing, ribosome, and phagosome pathways, suggesting multiple functions of circSP100 during HCMV infection.

Identification of Aristolactam Derivatives That Act as Inhibitors of Human Immunodeficiency Virus Type 1 Infection and Replication by Targeting Tat-Mediated Viral Transcription
YoungHyun Shin, Chul Min Park, Hong Gi Kim, Dong-Eun Kim, Min Suk Choi, Jeong-ah Kim, Byeong-Sun Choi, Cheol-Hee Yoon
2021, 36(2): 254-263.  doi: 10.1007/s12250-020-00274-7
Received: 15 May 2020 Published: 10 August 2020
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Despite the success of antiretroviral therapy (ART), efforts to develop new classes of antiviral agents have been hampered by the emergence of drug resistance. Dibenzo-indole-bearing aristolactams are compounds that have been isolated from various plants species and which show several clinically relevant effects, including anti-inflammatory, antiplatelet, and anti-mycobacterial actions. However, the effect of these compounds on human immunodeficiency virus type 1 (HIV-1) infection has not yet been studied. In this study, we discovered an aristolactam derivative bearing dibenzo[cd, f]indol-4(5H)-one that had a potent anti-HIV-1 effect. A structure-activity relationship (SAR) study using nine synthetic derivatives of aristolactam identified the differing effects of residue substitutions on the inhibition of HIV-1 infection and cell viability. Among the compounds tested,1,2,8,9-tetramethoxy-5-(2-(piperidin-1-yl)ethyl)-dibenzo[cd, f]indol-4(5H)-one (Compound 2) exhibited the most potent activity by inhibiting HIV-1 infection with a half-maximal inhibitory concentration (IC50) of 1.03 μmol/L and a half-maximal cytotoxic concentration (CC50) of 16.91 μmol/L (selectivity index,16.45). The inhibitory effect of the compounds on HIV-1 infection was linked to inhibition of the viral replication cycle. Mode-of-action studies showed that the aristolactam derivatives did not affect reverse transcription or integration; instead, they specifically inhibited Tat-mediated viral transcription. Taken together, these findings show that several aristolactam derivatives impaired HIV-1 infection by inhibiting the activity of Tat-mediated viral transcription, and suggest that these derivatives could be antiviral drug candidates.

Termination of Transcription of LAT Increases the Amounts of ICP0 mRNA but Does Not Alter the Course of HSV-1 Infection in Latently Infected Murine Ganglia
Haifang Jiang, Jiaming Wu, Xianjie Liu, Ruitao Lu, Manling Zhou, Meiling Chen, Yonghong Liu, Grace Guoying Zhou, Wenmin Fu
2021, 36(2): 264-272.  doi: 10.1007/s12250-020-00287-2
Received: 20 May 2020 Accepted: 31 July 2020 Published: 07 September 2020
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