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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).
A single nonsynonymous mutation on ZIKV E protein-coding sequences leads to markedly increased neurovirulence in vivo
Zhihua Liu, Yawei Zhang, Mengli Cheng, Ningning Ge, Jiayi Shu, Zhiheng Xu, Xiao Su, Zhihua Kou, Yigang Tong, Chengfeng Qin, Xia Jin
Received: 09 September 2021 Accepted: 20 October 2021 Published: 17 November 2021
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Zika virus (ZIKV) can infect a wide range of tissues including the developmental brain of human fetus. Whether specific viral genetic variants are linked to neuropathology is incompletely understood. To address this, we have intracranially serially passaged a clinical ZIKV isolate (SW01) in neonatal mice and discovered variants that exhibit markedly increased virulence and neurotropism. Deep sequencing analysis combining with molecular virology studies revealed that a single 67D (Aspartic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism in vivo. Notably, virus clones with D67N mutation had higher viral production and caused more severe cytopathic effect (CPE) in human neural astrocytes U251 cells in vitro, indicating its potential neurological toxicity to human brain. These findings revealed that a single mutation D67N on ZIKV envelope may lead to severe neuro lesion that may help to explain the neurovirulence of ZIKV and suggest monitoring the occurrence of this mutation during nature infection maybe important.
Updates on CRISPR-based gene editing in HIV-1/AIDS therapy
Zhihao Zhang, Wei Hou, Shuliang Chen
Received: 11 August 2021 Accepted: 10 November 2021 Published: 15 November 2021
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Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss- the caveats and problems that should be addressed before the clinical use of these versatile CRISPR- based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.
Epidemiological Evidence of Mosquito-Borne Viruses among Persons and Vectors in Iran: A Study from North to South
Abbas Ahmadi Vasmehjani, Farhad Rezaei, Mohammad Farahmand, Talat Mokhtari-Azad, Mohammad Reza Yaghoobi-Ershadi, Mohsen Keshavarz, Hamid Reza.Baseri, Morteza Zaim, Mahmood Iranpour, Habibollah Turki, Mohammad esmaeilpour-bandboni
Received: 12 July 2020 Accepted: 09 August 2021 Published: 22 October 2021
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Nucleolin Interacts with the Rabbit Hemorrhagic Disease Virus Replicase RdRp, Nonstructural Proteins p16 and p23, Playing a Role in Virus Replication
Jie Zhu, Qiuhong Miao, Hongyuan Guo, Aoxing Tang, Dandan Dong, Jingyu Tang, Fang Wang, Guangzhi Tong, Guangqing Liu
Received: 23 June 2021 Accepted: 15 September 2021 Published: 19 October 2021
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Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using twostep affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

An Integrated Rapid Nucleic Acid Detection Assay Based on Recombinant Polymerase Amplification for SARS-CoV-2
Ying Tang, Yiqin Wang, Yuchang Li, Huai Zhao, Sen Zhang, Ying Zhang, Jing Li, Yuehong Chen, Xiaoyan Wu, Chengfeng Qin, Tao Jiang, Xiaoping Kang
Received: 03 June 2020 Accepted: 16 June 2020 Published: 19 October 2021
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Metagenomic Analysis of Viral Community in the Yangtze River Expands Known Eukaryotic and Prokaryotic Virus Diversity in Freshwater
Juan Lu, Shixing Yang, Xiaodan Zhang, Xiangming Tang, Ju Zhang, Xiaochun Wang, Hao Wang, Quan Shen, Wen Zhang
Received: 21 July 2021 Accepted: 03 September 2021 Published: 14 October 2021
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Viruses in aquatic ecosystems are characterized by extraordinary abundance and diversity. Thus far, there have been limited studies focused on viral communities in river water systems. Here, we investigated the virome of the Yangtze River Delta using viral metagenomic analysis. The compositions of viral communities from six sampling sites were analyzed and compared. By using library construction and next generation sequencing, contigs and singlet reads similar to viral sequences were classified into 17 viral families, including nine dsDNA viral families, four ssDNA viral families and four RNA viral families. Statistical analysis using Friedman test suggested that there was no significant difference among the six sampling sites (P > 0.05). The viromes in this study were all dominated by the order Caudovirales, and a group of Freshwater phage uvFW species were particularly prevalent among all the samples. The virome from Nanjing presented a unique pattern of viral community composition with a relatively high abundance of family Parvoviridae. Phylogenetic analyses based on virus hallmark genes showed that the Caudovirales order and CRESS-DNA viruses presented high genetic diversity, while viruses in the Microviridae and Parvoviridae families and the Riboviria realm were relatively conservative. Our study provided the first insight into viral community composition in large river ecosystem, revealing the diversity and stability of river water virome, contributing to the proper utilization of freshwater resource.

Immunogenicity of a Recombinant VSV-Vectored SARS-CoV Vaccine Induced Robust Immunity in Rhesus Monkeys after Single-Dose Immunization
Dan Shan, Xiaoyan Tang, Renqiang Liu, Dan Pan, Xijun Wang, Jinying Ge, Zhiyuan Wen, Zhigao Bu
Received: 23 February 2021 Accepted: 04 August 2021 Published: 08 October 2021
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Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31th 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVƤG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVƤG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVƤG-SARS is safe and effective in mice at a dose of 1106 TCID50. More importantly, only a single-dose immunization of 2107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVƤG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.

Seroepidemiologic Study on Convalescent Sera from Dengue Fever Patients in Jinghong, Yunnan
Yingshuo Ma, Man Li, Lyu Xie, Na Gao, Dongying Fan, Kaihao Feng, Yao Yao, Yong Zhou, Ziyang Sheng, Hongning Zhou, Hui Chen, Jing An
Received: 29 April 2021 Accepted: 28 August 2021 Published: 29 September 2021
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After dengue virus (DENV) infection, antibody-dependent enhancement (ADE) is easy to occur when the neutralizing antibody (NAb) gradually decreases to a sub-neutralizing concentration. In this cohort surveillance, we utilized sera samples collected from dengue fever patients at different convalescent phases in Jinghong City, to investigate the dynamic change rule of DENV-specific antibodies, and to analyze the risk of ADE caused by secondary infection with heterologous serotypes DENVs. For baseline serosurvey, 191 four-year and 99 six-year sera samples during convalescence were collected in 2017 and 2019, respectively. The positive rate of DENV-specific immunoglobulin G was 98.4% in 2017, which significantly decreased to 82.8% in 2019. The geometric mean titer (GMT) of NAb decreased from 1:155.35 to 1:46.66. Among 290 overall samples, 73 paired consecutive samples were used for follow-up serosurvey. In four-year sera, the GMTs of NAb against DENV3 and cross-reactive antibodies against DENV-1, DENV-2 and DENV-4 were 1:167.70, 1:13.80, 1:18.54 and 1:45.26, respectively, which decreased to 1:53.18, 1:10.30, 1:14.60 and 1:8.17 in six-year sera. In age-stratified analysis, due to the increasing number of ADE positive samples from 2017 to 2019 in 31–40 and 51–60 years groups, the risk of ADE in DENV-4 infection was positively associated with the extension of convalescent phase, and the odd ratio was higher than other groups. With the recovery period lengthened, the risk of secondary infection with DENV-1 and DENV-2 was reduced. Our results offer essential experimental data for risk prediction of severe dengue in hyper-endemic dengue areas, and provide crucial scientific insight for the development of effective dengue vaccines.

Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL
Shengda Xie, Zhenjie Liang, Xingmiao Yang, Junhui Pan, Du Yu, Tongtong Li, Ruibing Cao
doi: 10.1007/s12250-021-00442-3
Received: 14 May 2021 Accepted: 12 July 2021 Published: 06 September 2021
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Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a transmembrane protein, can promote the replication of various flaviviruses, such as dengue (DENV), Zika (ZIKV), and West Nile (WNV) viruses. However, the effect of AXL on JEV infection has not yet been determined. In the present study, we demonstrate that AXL is down-regulated after JEV infection in the late stage. JEV NS2B-3 protein specifically interacted with AXL, and promoted AXL degradation through the ubiquitin–proteasome pathway. AXL-degradation increased cell apoptosis by disrupting phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction. In addition, the degradation of AXL promoted JEV release to supernatant, whereas the virus in the cell lysates decreased. The supplementation of AXL ligand Gas6 inhibited the JEV-mediated degradation of AXL. Altogether, we discover a new function of NS2B-3 during the process of JEV replication, and provide a new insight into the interactions between JEV and cell hosts.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture
Quanmei Tu, Weixu Feng, Zhuo Chen, Qijia Li, Yu Zhao, Jun Chen, Pengfei Jiang, Xiangyang Xue, Lifang Zhang, Kong-Nan Zhao
doi: 10.1007/s12250-021-00439-y
Received: 22 February 2021 Accepted: 21 May 2021 Published: 30 August 2021
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We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae (budding yeast) cultures (Zhao and Frazer 2002, Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication (day 3–16), middle weak replication (day 23–34/45) and late stable replication (day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

Three Novel Avastroviruses Identified in Dead Wild Crows
Chunge Zhang, Yongchun Yang, Tao Hu, Hong Zhou, Cheng Zhang, Jian Cao, Juan Li, Peihan Wang, Gary Wong, Xiaodu Wang, Houhui Song, George F. Gao, Weifeng Shi, Yuhai Bi
doi: 10.1007/s12250-021-00416-5
Received: 22 February 2021 Accepted: 27 April 2021 Published: 30 August 2021
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The Establishment and Spatiotemporal History of A Novel HIV-1 CRF01_AE Lineage in Shenyang City, Northeastern China in 2002–2019
Minghui An, Wei Song, Bin Zhao, Xue Dong, Lin Wang, Wen Tian, Xin Li, Lu Wang, Zhenxing Chu, Junjue Xu, Haibo Ding, Xiaoxu Han, Hong Shang
doi: 10.1007/s12250-021-00435-2
Received: 24 May 2021 Accepted: 28 June 2021 Published: 23 August 2021
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Generation and Characterization of a Nanobody Against SARS-CoV
Jiang-Fan Li, Lei He, Yong-Qiang Deng, Shu-Hui Qi, Yue-Hong Chen, Xiao-Lu Zhang, Shi-Xiong Hu, Rui-Wen Fan, Guang-Yu Zhao, Cheng-Feng Qin
doi: 10.1007/s12250-021-00436-1
Received: 28 March 2021 Accepted: 06 May 2021 Published: 17 August 2021
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The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Structure and Function of N-Terminal Zinc Finger Domain of SARS-CoV-2 NSP2
Jun Ma, Yiyun Chen, Wei Wu, Zhongzhou Chen
doi: 10.1007/s12250-021-00431-6
Received: 14 June 2021 Accepted: 15 July 2021 Published: 16 August 2021
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SARS-CoV-2 has become a global pandemic threatening human health and safety. It is urgent to find effective therapeutic agents and targets with the continuous emergence of novel mutant strains. The knowledge of the molecular basis and pathogenesis of SARS-CoV-2 in host cells requires to be understood comprehensively. The unknown structure and function of nsp2 have hindered our understanding of its role in SARS-CoV-2 infection. Here, we report the crystal structure of the N-terminal of SARS-CoV-2 nsp2 to a high resolution of 1.96?. This novel structure contains three zinc fingers, belonging to the C2H2, C4, and C2HC types, respectively. Structure analysis suggests that nsp2 may be involved in binding nucleic acids and regulating intracellular signaling pathways. The binding to single or double-stranded nucleic acids was mainly through the large positively charged region on the surface of nsp2, and K111, K112, K113 were key residues. Our findings lay the foundation for a better understanding of the relationship between structure and function for nsp2. It is helpful to make full use of nsp2 as further research and development of antiviral targets and drug design.

A multi-center study on Molecular Epidemiology of Human Respiratory Syncytial Virus from Children with Acute Lower Respiratory Tract Infections in the Mainland of China between 2015 and 2019
Xiangpeng Chen, Yun Zhu, Wei Wang, Changchong Li, Shuhua An, Gen Lu, Rong Jin, Baoping Xu, Yunlian Zhou, Aihuan Chen, Lei Li, Meng Zhang, Zhengde Xie
doi: 10.1007/s12250-021-00430-7
Received: 29 January 2021 Accepted: 18 May 2021 Published: 16 August 2021
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Human respiratory syncytial virus (RSV) is a major pathogen of acute lower respiratory tract infection among young children. To investigate the prevalence and genetic characteristics of RSV in China, we performed a molecular epidemiological study during 2015-2019. A total of 964 RSV-positive specimens were identified from 5529 enrolled patients during a multi-center study. RSV subgroup A (RSV-A) was the predominant subgroup during this research period except in 2016. Totally, 535 sequences of the second hypervariable region (HVR-2) of the G gene were obtained. Combined with 182 Chinese sequences from GenBank, phylogenetic trees showed that 521 RSV-A sequences fell in genotypes ON1 (512), NA1 (6) and GA5 (3), respectively; while 196 RSV-B sequences fell in BA9 (193) and SAB4 (3). ON1 and BA9 were the only genotypes after December 2015. Genotypes ON1 and BA9 can be separated into 10 and 7 lineages, respectively. The HVR-2 of genotype ON1 had six amino acid changes with a frequency more than 10%, while two substitutions H258Q and H266L were co-occurrences. The HVR-2 of genotype BA9 had nine amino acid substitutions with a frequency more than 10%, while the sequences with T290I and T312I were all from 2018 to 2019. One N-glycosylation site at 237 was identified among ON1 sequences, while two N-glycosylation sites (296 and 310) were identified in the 60-nucleotide duplication region of BA9. To conclusion, ON1 and BA9 were the predominant genotypes in China during 2015-2019. For the genotypes ON1 and BA9, the G gene exhibited relatively high diversity and evolved continuously.

SARS-CoV-2 Genomic Sequencing Revealed N501Y and L452R Mutants of S/A Lineage in Tianjin Municipality, China
Xiaoyan Li, Xin Gao, Ming Zou, Zhichao Zhuang, Zhaolin Tan, Baolu Zheng, Aiping Yu, Xu Su
doi: 10.1007/s12250-021-00432-5
Received: 31 March 2021 Accepted: 26 May 2021 Published: 11 August 2021
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In Vitro Inhibition of Alphaviruses by Lycorine
Na Li, Zhen Wang, Rui Wang, Zhe-Rui Zhang, Ya-Nan Zhang, Cheng-Lin Deng, Bo Zhang, Lu-Qing Shang, Han-Qing Ye
doi: 10.1007/s12250-021-00438-z
Received: 08 May 2021 Accepted: 08 June 2021 Published: 10 August 2021
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Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. As an emerging virus, CHIKV imposes a threat to public health. Currently, there are no vaccines or antivirals available for the prevention of CHIKV infection. Lycorine, an alkaloid from Amaryllidaceae plants, has antiviral activity against a number of viruses such as coronavirus, flavivirus and enterovirus. In this study, we found that lycorine could inhibit CHIKV in cell culture at a concentration of 10 μmol/L without apparent cytotoxicity. In addition, it exhibited broad-spectrum anti-alphavirus activity, including Sindbis virus (SINV), Semliki Forest virus (SFV), and Venezuelan equine encephalomyelitis virus (VEEV). The time of addition studies indicated that lycorine functions at an early post-entry stage of CHIKV life cycle. The results based on two different CHIKV replicons provided further evidence that lycorine exerts its antiviral activity mainly by inhibiting CHIKV translation. Overall, our study extends the antiviral spectrum of lycorine.

Inhibition of the Neddylation Pathway Suppresses Enterovirus Replication
Zhe Zhang, Haoran Guo, Jing Wang, Yan Li, Yanhang Gao, Quan Liu, Junqi Niu, Wei Wei
doi: 10.1007/s12250-021-00427-2
Received: 04 February 2021 Accepted: 26 May 2021 Published: 05 August 2021
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Genomic Characterization of a New Coronavirus from Migratory Birds in Jiangxi Province of China
Wentao Zhu, Wentao Song, Guoyin Fan, Jing Yang, Shan Lu, Dong Jin, Xue-lian Luo, Ji Pu, Haiying Chen, Jianguo Xu
doi: 10.1007/s12250-021-00402-x
Received: 25 January 2021 Accepted: 29 March 2021 Published: 08 July 2021
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Consecutive Monitoring of Interleukin-6 Is Needed for COVID-19 Patients
Xiaohua Chen, Juan Zhou, Chen Chen, Baidong Hou Hou, Ashaq Ali, Feng Li, Zhaolin Hua, Yingtao Wu, Qin Yang, Min Chen, Rong Zhang, Qianchuan Huang, Jinya Ding, Xian-En Zhang, Dong Men
doi: 10.1007/s12250-021-00425-4
Received: 26 January 2021 Accepted: 21 May 2021 Published: 07 July 2021
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Two Inhibitors Against the 3C-Like Proteases of Swine Coronavirus and Feline Coronavirus
Mengxin Zhou, Yutong Han, Mengxia Li, Gang Ye, Guiqing Peng
doi: 10.1007/s12250-021-00415-6
Received: 19 February 2021 Accepted: 06 May 2021 Published: 06 July 2021
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Coronaviruses (CoVs) are important human and animal pathogens that cause respiratory and gastrointestinal diseases. Porcine epidemic diarrhoea (PED), characterized by severe diarrhoea and vomiting in pigs, is a highly lethal disease caused by porcine epidemic diarrhoea virus (PEDV) and causes substantial losses in the swine industry worldwide. However, currently available commercial drugs have not shown great therapeutic effects. In this study, a fluorescence resonance energy transfer (FRET)-based assay was applied to screen a library containing 1, 590 compounds and identified two compounds, 3-(aminocarbonyl)-1-phenylpyridinium and 2, 3-dichloronaphthoquinone, that target the 3C-like protease (3CLpro) of PEDV. These compounds are of low molecular weight (MW) and greatly inhibited the activity of this enzyme (IC50 values were obtained in this study). Furthermore, these compounds exhibited antiviral capacity against another member of the CoV family, feline infectious peritonitis virus (FIPV). Here, the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified (with EC50 values) and cell viability assays were performed. The results of putative molecular docking models indicate that these compounds, labeled compound 1 and compound 2, contact the conserved active sites (Cys144, Glu165, Gln191) of 3CLpro via hydrogen bonds. These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.

Non-Structural Protein 5 of Zika Virus Interacts with p53 in Human Neural Progenitor Cells and Induces p53-Mediated Apoptosis
Ping Li, Hualian Jiang, Hong Peng, Weijie Zeng, Yongheng Zhong, Miao He, Luyang Xie, Junhai Chen, Deyin Guo, Junyu Wu, Chun-Mei Li
doi: 10.1007/s12250-021-00422-7
Received: 05 April 2021 Accepted: 08 June 2021 Published: 05 July 2021
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Zika virus (ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells (NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection,yet the detailed mechanism is not well understood. In the present study,we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that,among the ten viral proteins,the nonstructural protein 5 (NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach,we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition,the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore,lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of hNPCs. Taken together,these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs,which may contribute to the ZIKV-caused abnormal neurodevelopment.

Desmoglein 2 (DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia
Jing Zhang, Kui Ma, Xiangyu Wang, Yinbo Jiang, Shan Zhao, Junxian Ou, Wendong Lan, Wenyi Guan, Xiaowei Wu, Heping Zheng, Bin Yang, Chengsong Wan, Wei Zhao, Jianguo Wu, Qiwei Zhang
doi: 10.1007/s12250-021-00414-7
Received: 12 February 2021 Accepted: 06 May 2021 Published: 05 July 2021
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Human adenovirus type 55 (HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult community-acquired pneumonia (CAP). Previous studies have shown that the receptor of HAdV-B14,which genome is highly similar with HAdV-B55,is human Desmoglein 2 (DSG2). However,whether the receptor of HAdV-B55 is DSG2 is undetermined because there are three amino acid mutations in the fiber gene between HAdV-B14 and HAdV-B55. Here,firstly we found the 3T3 cells,a mouse embryo fibroblast rodent cell line which does not express human DSG2,were able to be infected by HAdV-B55 after transfected with pcDNA3.1-DSG2,while normal 3T3 cells were still unsusceptible to HAdV-B55 infection. Next,A549 cells with hDSG2 knock-down by siRNA were hard to be infected by HAdV-B3/-B14/-B55,while the control siRNA group was still able to be infected by all these types of HAdVs. Finally,immunofluorescence confocal microscopy indicated visually that Cy3-conjugated HAdV-B55 viruses entered A549 cells by binding to DSG2 protein. Therefore,DSG2 is a major receptor of HAdV-B55 causing adult CAP. Our finding is important for better understanding of interactions between adenoviruses and host cells and may shed light on the development of new drugs that can interfere with these processes as well as for the development of potent prophylactic vaccines.

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants
Yuanyuan Qu, Xueyan Zhang, Meiyu Wang, Lina Sun, Yongzhong Jiang, Cheng Li, Wei Wu, Zhen Chen, Qiangling Yin, Xiaolin Jiang, Yang Liu, Chuan Li, Jiandong Li, Tianlei Ying, Dexin Li, Faxian Zhan, Youchun Wang, Wuxiang Guan, Shiwen Wang, Mifang Liang
doi: 10.1007/s12250-021-00409-4
Received: 16 March 2021 Accepted: 06 May 2021 Published: 05 July 2021
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study,12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them,two RBD-binding antibodies (F61 and H121) showed high-affinity neutralization against SARS-CoV-2,whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis,F61 identified a linear epitope located in residues G446–S494,which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites,while H121 recognized a conformational epitope located on the side face of RBD,outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly,these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351,and reacted with mutations of N501Y,E484K,and L452R,indicated that it may also neutralize the recent India endemic strain B.1.617. The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants,which mitigated the risk of viral escape. Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-CoV-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-CoV-2 variants.

CRISPR/Cas12a Technology Combined with RT-ERA for Rapid and Portable SARS-CoV-2 Detection
Sihua Liu, Mengqian Huang, Yanan Xu, Jun Kang, Sheng Ye, Si Liu, Zhiyun Wang, Hongyun Liu, Jibin Yu, Kongxin Hu, Tao Wang
doi: 10.1007/s12250-021-00406-7
Received: 24 November 2020 Accepted: 28 March 2021 Published: 02 July 2021
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ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles
Qiuhan Zhang, Siliang Li, Ping Lei, Zixian Li, Feifei Chen, Qi Chen, Yulu Wang, Jiami Gong, Qi Tang, Xinjin Liu, Ke Lan, Shuwen Wu
doi: 10.1007/s12250-021-00417-4
Received: 14 March 2021 Accepted: 27 April 2021 Published: 01 July 2021
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Similar to that of other enteroviruses, the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase Ⅲ (PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate (PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase (RdRp) to modulate RdRp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2 (ANXA2) can interact with 3D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membrane-binding capacity, mediates the interaction of ANXA2 with EV71 3D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.

Profiles of SARS-CoV-2 RNA and Antibodies in Inpatients with COVID-19 not Related with Clinical Manifestation: A Single Centre Study
Li Zhao, Ruqin Gao, Roujian Lu, Huijuan Wang, Yao Deng, Peihua Niu, Fachun Jiang, Baoying Huang, Jiwei Liang, Jing Jia, Feng Zhang, Wenling Wang, Guizhen Wu, Wenjie Tan
doi: 10.1007/s12250-021-00411-w
Received: 13 August 2020 Accepted: 30 April 2021 Published: 01 July 2021
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Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone
Penghui Li, Chen Yao, Ting Wang, Tong Wu, Wenfu Yi, Yue Zheng, Yuanjiu Miao, Jianhong Sun, Zhongyuan Tan, Yan Liu, Xiaowei Zhang, Hanzhong Wang, Zhenhua Zheng
doi: 10.1007/s12250-021-00396-6
Received: 17 July 2020 Accepted: 15 March 2021 Published: 30 June 2021
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Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

New Simian Enterovirus 19 (EV-A122) Strains in China Reveal Large-Scale Inter-Serotype Recombination between Simian EV-As
Zhenzhi Han, Jinbo Xiao, Yang Song, Shuangli Zhu, Dongyan Wang, Huanhuan Lu, Tianjiao Ji, Dongmei Yan, Wenbo Xu, Yong Zhang
doi: 10.1007/s12250-021-00412-9
Received: 06 December 2020 Accepted: 13 April 2021 Published: 29 June 2021
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Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro
Huimin Xiao, Jingliang Li, Xu Yang, Zhaolong Li, Ying Wang, Yajuan Rui, Bin Liu, Wenyan Zhang
doi: 10.1007/s12250-021-00410-x
Received: 31 January 2021 Accepted: 12 April 2021 Published: 25 June 2021
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Enteroviruses (EVs) 3C proteins suppress type Ⅰ interferon (IFN) responses mediated by retinoid acid-inducible gene I (RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25 (TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3 (CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-β production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.

Clinical Characteristics of Human Adenovirus Plastic Bronchitis in 10 Pediatric Cases: A Retrospective Study of Seven Years
Lingjian Zeng, Jianhua Wei, Yuyi Tang, Enmei Liu, Qubei Li, Na Zang
doi: 10.1007/s12250-021-00394-8
Received: 02 November 2020 Accepted: 25 April 2021 Published: 22 June 2021
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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: 22 June 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 the 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.

Current Status of Human Papillomavirus-Related Head and Neck Cancer: From Viral Genome to Patient Care
Haoru Dong, Xinhua Shu, Qiang Xu, Chen Zhu, Andreas M. Kaufmann, Zhi-Ming Zheng, Andreas E. Albers, Xu Qian
doi: 10.1007/s12250-021-00413-8
Received: 21 January 2021 Accepted: 18 May 2021 Published: 21 June 2021
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Human papillomavirus (HPV) infection identified as a definitive human carcinogen is increasingly being recognized for its role in carcinogenesis of human cancers. Up to 38%–80% of head and neck squamous cell carcinoma (HNSCC) in oropharyngeal location (OPSCC) and nearly all cervical cancers contain the HPV genome which is implicated in causing cancer through its oncoproteins E6 and E7. Given by the biologically distinct HPV-related OPSCC and a more favorable prognosis compared to HPV-negative tumors, clinical trials on de-escalation treatment strategies for these patients have been studied. It is therefore raised the questions for the patient stratification if treatment de-escalation is feasible. Moreover, understanding the crosstalk of HPV-mediated malignancy and immunity with clinical insights from the proportional response rate to immune checkpoint blockade treatments in patients with HNSCC is of importance to substantially improve the treatment efficacy. This review discusses the biology of HPV-related HNSCC as well as successful clinically findings with promising candidates in the pipeline for future directions. With the advent of various sequencing technologies, further biomolecules associated with HPV-related HNSCC progression are currently being identified to be used as potential biomarkers or targets for clinical decisions throughout the continuum of cancer care.

The Application of a Safe Neutralization Assay for Ebola Virus Using Lentivirus-Based Pseudotyped Virus
Zengguo Cao, Hongli Jin, Gary Wong, Ying Zhang, Cuicui Jiao, Na Feng, Fangfang Wu, Shengnan Xu, Hang Chi, Yongkun Zhao, Tiecheng Wang, Weiyang Sun, Yuwei Gao, Songtao Yang, Xianzhu Xia, Hualei Wang
doi: 10.1007/s12250-021-00405-8
Received: 28 July 2020 Accepted: 19 April 2021 Published: 21 June 2021
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Dynamic Host Immune and Transcriptomic Responses to Respiratory Syncytial Virus Infection in a Vaccination-Challenge Mouse Model
Yu Zhao, Chen Ma, Jie Yang, Xiufen Zou, Zishu Pan
doi: 10.1007/s12250-021-00418-3
Received: 08 April 2021 Accepted: 20 April 2021 Published: 17 June 2021
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Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children. Inactivated RSV vaccine was developed in the late 1960's,but the vaccine-enhanced disease (VED) occurred to vaccinated infants upon subsequent natural RSV infection. The excessive inflammatory immunopathology in the lungs might be involved in the VED,but the underlying mechanisms remain not fully understood. In this study,we utilized UV-inactivated RSV in the prime/boost approach followed by RSV challenge in BALB/c mice to mimic RSV VED. The dynamic virus load,cytokines,histology and transcriptome profiles in lung tissues of mice were investigated from day 1 to day 6 post-infection. Compared to PBS-treated mice,UV-RSV vaccination leads to a Th2 type inflammatory response characterized by enhanced histopathology,reduced Treg cells and increased IL4+CD4 T cells in the lung. Enhanced production of several Th2 type cytokines (IL-4,IL-5,IL-10) and TGF-β,reduction of IL-6 and IL-17 were observed in UV-RSV vaccinated mice. A total of 5582 differentially expressed (DE) genes between PBS-treated or vaccinated mice and naïve mice were identified by RNA-Seq. Eleven conserved high-influential modules (HMs) were recognized,majorly grouped into regulatory networks related to cell cycle and cell metabolism,signal transduction,immune and inflammatory responses. At an early time post-infection,the vaccinated mice showed obvious decreased expression patterns of DE genes in 11 HMs compared to PBS-treated mice. The extracellular matrix (HM5) and immune responses (HM8) revealed tremendous differences in expression and regulation characteristics of transcripts between PBS-treated and vaccinated mice at both early and late time points. The highly connected genes in HM5 and HM8 networks were further validated by RT-qPCR. These findings reveal the relationship between RSV VED and immune responses,which could benefit the development of novel RSV vaccines.

The C/EBPb-Dependent Induction of TFDP2 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Proliferation
Min Zhu, Xiaoyang Li, Ruiqi Sun, Peidian Shi, Aiping Cao, Lilin Zhang, Yanyu Guo, Jinhai Huang
doi: 10.1007/s12250-021-00403-w
Received: 07 February 2021 Accepted: 28 April 2021 Published: 17 June 2021
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Antigenic Drift of the Hemagglutinin from an Influenza A (H1N1) pdm09 Clinical Isolate Increases its Pathogenicity In Vitro
Lei Xing, Yunbo Chen, Boqian Chen, Ling Bu, Ying Liu, Zhiqi Zeng, Wenda Guan, Qigao Chen, Yongping Lin, Kun Qin, Honglin Chen, Xilong Deng, Xinhua Wang, Wenjun Song
doi: 10.1007/s12250-021-00401-y
Received: 06 January 2021 Accepted: 12 April 2021 Published: 09 June 2021
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Development and Characterization of SYBR Green Ⅰ Based RT-PCR Assay for Detection of Omsk Hemorrhagic Fever Virus
Ya-Nan Zhang, Si-Qing Liu, Cheng-Lin Deng, Zhi-Ming Yuan, Bo Zhang, Xiao-Dan Li, Han-Qing Ye
doi: 10.1007/s12250-021-00389-5
Received: 10 January 2021 Accepted: 17 March 2021 Published: 02 June 2021
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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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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, M. Faadiel Essop
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.

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.

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.
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.
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.


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
2021, 36(4): 565-576.  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.
Herpesviruses and the Type Ⅲ Interferon System
Yue Yin, Herman W. Favoreel
2021, 36(4): 577-587.  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.
Current Status and Challenge of Pseudorabies Virus Infection in China
Lei Tan, Jun Yao, Yadi Yang, Wei Luo, Xiaomin Yuan, Lingchen Yang, Aibing Wang
2021, 36(4): 588-607.  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.
Research Article
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
2021, 36(4): 608-622.  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.
Metagenomic Profiling of Viruses Associated with Rhipicephalus microplus Ticks in Yunnan Province, China
Junming Shi, Shu Shen, Hui Wu, Yunzhi Zhang, Fei Deng
2021, 36(4): 623-635.  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.
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
2021, 36(4): 636-643.  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.
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
2021, 36(4): 644-654.  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.
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
2021, 36(4): 655-666.  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.
Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice
Zhao-Yang Wang, Zi-Da Zhen, Dong-Ying Fan, Pei-Gang Wang, Jing An
2021, 36(4): 667-677.  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.
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
2021, 36(4): 678-691.  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.
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
2021, 36(4): 692-705.  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.
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
2021, 36(4): 706-720.  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.
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
2021, 36(4): 721-729.  doi: 10.1007/s12250-021-00349-z
Received: 05 August 2020 Accepted: 16 November 2020 Published: 04 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.
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
2021, 36(4): 730-735.  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.
HRV16 Infection Induces Changes in the Expression of Multiple piRNAs
Jie Li, Xinling Wang, Yanhai Wang, Juan Song, Qinqin Song, Yanbin Wang, Jun Han
2021, 36(4): 736-745.  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.
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
2021, 36(4): 746-754.  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.
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
2021, 36(4): 755-761.  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.
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
2021, 36(4): 762-773.  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.
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
2021, 36(4): 774-783.  doi: 10.1007/s12250-021-00358-y
Received: 02 November 2020 Accepted: 13 January 2021 Published: 10 March 2021