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2019 Vol.34(5)

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. In this issue, Liu et al. used genome-wide CRISPR-based screening to identify the host cellular factors required for SFTSV infection, and the vesicular trafficking protein sorting nexin 11 (SNX11) was identified essential for the establishment of SFTSV infection, particularly for penetration from the endolysosome to the cytoplasm. The cover image shows endolysosomes significantly increased in SNX11-KO cells (bottom red panel) compared with the wild-type cells (upper blue panel). See page 508–520 for details.

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Review

Binding between Prion Protein and Aβ Oligomers Contributes to the Pathogenesis of Alzheimer's Disease

Chang Kong, Hao Xie, Zhenxing Gao, Ming Shao, Huan Li, Run Shi, Lili Cai, Shanshan Gao, Taolei Sun, Chaoyang Li

2019, 34(5): 475 doi: 10.1007/s12250-019-00124-1

Received: 23 February 2019 Accepted: 26 March 2019 Published: 15 May 2019
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A plethora of evidence suggests that protein misfolding and aggregation are underlying mechanisms of various neurodegenerative diseases, such as prion diseases and Alzheimer's disease (AD). Like prion diseases, AD has been considered as an infectious disease in the past decades as it shows strain specificity and transmission potential. Although it remains elusive how protein aggregation leads to AD, it is becoming clear that cellular prion protein (PrPC) plays an important role in AD pathogenesis. Here, we briefly reviewed AD pathogenesis and focused on recent progresses how PrPC contributed to AD development. In addition, we proposed a potential mechanism to explain why infectious agents, such as viruses, conduce AD pathogenesis. Microbe infections cause Aβ deposition and upregulation of PrPC, which lead to high affinity binding between Aβ oligomers and PrPC. The interaction between PrPC and Aβ oligomers in turn activates the Fyn signaling cascade, resulting in neuron death in the central nervous system (CNS). Thus, silencing PrPC expression may turn out be an effective treatment for PrPC dependent AD.
Research Article

DNA Methylation and SNPs in VCX are Correlated with Sex Differences in the Response to Chronic Hepatitis B

Xue-Qing Hu, Yuan Zhou, Jian Chen, Yu Zhao, Yi-Yu Lu, Qi-Long Chen, Yuanjia Hu, Yi-Yang Hu, Shi-Bing Su

2019, 34(5): 489 doi: 10.1007/s12250-019-00117-0

Received: 22 December 2018 Accepted: 25 March 2019 Published: 03 June 2019
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The study was conducted to explore the mechanisms of sex differences in the response to chronic hepatitis B (CHB) in terms of DNA methylation, SNP genotype, and gene expression. Genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) of CHB patients and healthy controls and evaluated using the Human Methylation 450 K Assay. The DNA methylation level at hg37 chromosome (CHR) X: 7810800 was further validated using pyrosequencing. SNP genotypes, VCX mRNA expression of PBMCs, and plasma VCX protein concentration were further examined using SNaPshot, RT-qPCR, and Western blot, respectively. Results showed that a total of 5529 CpG loci were differentially methylated between male and female CHB patients. DNA methylation level and CC + CT frequency at CHR X: 7810800, VCX mRNA expression of PBMCs, and plasma VCX protein concentration were higher in female than in male CHB patients. The CHR X: 7810800 locus was hypermethylated in CHB patients with CC + CT genotypes in comparison with those with the TT genotype. In cases of CC + CT genotypes, VCX mRNA expression was negatively correlated with the DNA methylation level. CHB patients with higher levels of HBV DNA, AST, and GGT or higher GPRI scores exhibited lower VCX expression. In conclusion, SNPs and DNA methylation at the CHR X: 7810800 locus cooperatively regulate VCX expression in CHB. The upregulated VCX expression in female CHB patients might represent a mechanism of protection from more severe liver dysfunction and extensive fibrosis, as observed in male CHB patients.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Phlebovirus in Domesticated Deer in South Korea

Min-Ah Yu, Kwang-Min Yu, Su-Jin Park, Young-Il Kim, Norbert John Robles, Young-Jae Si, Eun-Ha Kim, Hyeok-Il Kwon, Hye Won Jeong, Min-Suk Song, Seok-Yong Kim, Young Ki Choi

2019, 34(5): 501 doi: 10.1007/s12250-019-00137-w

Received: 08 January 2019 Accepted: 22 April 2019 Published: 25 June 2019
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Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) has a wide host range. Not only has it been found in humans, but also in many wild and domesticated animals. The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products, including raw blood. To investigate the prevalence of breeding domesticated deer, we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017. Of the 215 collected blood samples, 0.9% (2/215) were found to be positive for viral RNA by PCR, and sequence analysis showed the highest homology with the KADGH human isolate. Both SFTSV specific recombinant N and Gn protein-based ELISAs revealed that 14.0% (30/215) and 7.9% (17/215) of collected blood specimens were positive for SFTSV antibody. These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Tiezhu Liu, Jiajia Li, Yang Liu, Yuanyuan Qu, Aqian Li, Chuan Li, Quanfu Zhang, Wei Wu, Jiandong Li, Yan Liu, Dexin Li, Shiwen Wang, Mifang Liang

2019, 34(5): 508 doi: 10.1007/s12250-019-00141-0

Received: 22 March 2019 Accepted: 17 May 2019 Published: 18 June 2019
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Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11 (SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum (ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1 (LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall, these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.

Phosphatidylserine-Specific Phospholipase A1 is the Critical Bridge for Hepatitis C Virus Assembly

Qi Yang, Min Guo, Yuan Zhou, Xue Hu, Yun Wang, Chunchen Wu, Min Yang, Rongjuan Pei, Xinwen Chen, Jizheng Chen

2019, 34(5): 521 doi: 10.1007/s12250-019-00123-2

Received: 18 December 2018 Accepted: 07 March 2019 Published: 03 June 2019
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The phosphatidylserine-specific phospholipase A1 (PLA1A) is an essential host factor in hepatitis C virus (HCV) assembly. In this study, we mapped the E2, NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts, through which they form oligomeric protein complexes to participate in HCV assembly. Multiple regions of PLA1A were involved in their interaction and complex formation. Furthermore, the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A, and strongly suggest PLA1A-E2's physical interaction in cells. Meanwhile, we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1. Interestingly, these amino acids in the sequence are also essential for viral RNA replication. Further experiments revealed that these four proteins interact with each other. Moreover, PLA1A expression levels were elevated in livers from HCV-infected patients. In conclusion, we exposed the structural determinants of PLA1A, E2, NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.

PD1+CCR2+CD8+ T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection

Fang Zhang, Linlin Qi, Tong Li, Xiaojing Li, Dan Yang, Shengbo Cao, Jing Ye, Bin Wei

2019, 34(5): 538 doi: 10.1007/s12250-019-00134-z

Received: 25 February 2019 Accepted: 08 April 2019 Published: 18 June 2019
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Japanese encephalitis (JE) is a viral encephalitis disease caused by Japanese encephalitis virus (JEV) infection. Uncontrolled inflammatory responses in the central nervous system (CNS) are a hallmark of severe JE. Although the CCR2-CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8+ T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection (i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8+ T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8+ T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells (CD44+CD62L-). JEV infection dramatically enhanced the expression of programmed death 1 (PD-1) on infiltrating CD8+ T cells in the brain, as compared to that on peripheral CD8+ T cells in the spleen. This effect was more pronounced on infiltrating CCR2+CD8+ T cells than on CCR2-CD8+ T cells. In conclusion, we identified a new subset of CD8+ T cells (PD1+CCR2+CD8+ T cells) present in the CNS of mice during acute JEV infection. These CD8+ T cells might play a role in JE pathogenesis.

Proteomics Profiling of Host Cell Response via Protein Expression and Phosphorylation upon Dengue Virus Infection

Meng Miao, Fei Yu, Danya Wang, Yongjia Tong, Liuting Yang, Jiuyue Xu, Yang Qiu, Xi Zhou, Xiaolu Zhao

2019, 34(5): 549 doi: 10.1007/s12250-019-00131-2

Received: 07 March 2019 Accepted: 08 April 2019 Published: 27 May 2019
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Dengue virus (DENV) infection is a worldwide public health threat. To date, the knowledge about the pathogenesis and progression of DENV infection is still limited. Combining global profiling based on proteomic analysis together with functional verification analysis is a powerful strategy to investigate the interplay between the virus and host cells. In the present study, quantitative proteomics has been applied to evaluate host responses (as indicated by altered proteins and modifications) in human cells (using K562 cell line) upon DENV-2 infection, as DENV-2 spreads most widely among all DENV serotypes. Comparative analysis was performed to define differentially expressed proteins in the infected cells compared to the mock-control, and it revealed critical pathogen-induced changes covering a broad spectrum of host cellular compartments and processes. We also discovered more dramatic changes (>20%, 160 regulated phosphoproteins) in protein phosphorylation compared to protein expression (14%, 321 regulated proteins). Most of these proteins/phosphoproteins were involved in transcription regulation, RNA splicing and processing, immune system, cellular response to stimulus, and macromolecule biosynthesis. Western blot analysis was also performed to confirm the proteomic data. Potential roles of these altered proteins were discussed. The present study provides valuable large-scale protein-related information for elucidating the functional emphasis of host cell proteins and their post-translational modifications in virus infection, and also provides insight and protein evidence for understanding the general pathogenesis and pathology of DENV.

Development of a Specific CHIKV-E2 Monoclonal Antibody for Chikungunya Diagnosis

Jaemoo Kim, Jihyun Yang, Young Bong Kim, Hee-Jung Lee, Sehyun Kim, Haryoung Poo

2019, 34(5): 563 doi: 10.1007/s12250-019-00135-y

Received: 11 February 2019 Accepted: 26 April 2019 Published: 18 June 2019
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Chikungunya fever is a vector-borne viral disease transmitted to humans by chikungunya virus (CHIKV)-infected mosquitoes. There have been many outbreaks of CHIKV infection worldwide, and the virus poses ongoing risks to global health. To prevent and control CHIKV infection, it is important to improve the current CHIKV diagnostic approaches to allow for the detection of low CHIKV concentrations and to correctly distinguish CHIKV infections from those due to other mosquito-transmitted viruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Here, we produced monoclonal antibodies (mAbs) against the CHIKV envelope 2 protein (CHIKV-E2) and compared their sensitivity and specificity with commercially available mAbs using enzyme-linked immunosorbent assays (ELISA). Two anti-CHIKV-E2 mAbs, 19-1 and 21-1, showed higher binding affinities to CHIKV-E2 protein than the commercial mAbs did. In particular, the 19-1 mAb had the strongest binding affinity to inactivated CHIKV. Moreover, the 19-1 mAb had very little cross-reactivity with other mosquito-borne viruses, such as ZIKV, JEV, and DENV. These results suggest that the newly produced anti-CHIKV-E2 mAb, 19-1, could be used for CHIKV diagnostic approaches.

Proteasome Inhibitor PS-341 Effectively Blocks Infection by the Severe Fever with Thrombocytopenia Syndrome Virus

Sihua Liu, Hongyun Liu, Keke Zhang, Xueping Li, Yuqin Duan, Zhiyun Wang, Tao Wang

2019, 34(5): 572 doi: 10.1007/s12250-019-00162-9

Received: 24 March 2019 Accepted: 20 August 2019 Published: 21 October 2019
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Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptideboronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded nonstructural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Wenling Wang, Baoying Huang, Xiuping Wang, Wenjie Tan, Li Ruan

2019, 34(5): 583 doi: 10.1007/s12250-019-00138-9

Received: 01 February 2019 Accepted: 29 April 2019 Published: 25 June 2019
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Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein (NP) and extracellular domain of matrix protein 2 (M2e) genes from the influenza A virus A/Beijing/30/95 (H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34 (PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP- and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD50 of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.
Letter

Aminopeptidase N Knockout Pigs Are Not Resistant to Porcine Epidemic Diarrhea Virus Infection

Jian Zhang, Zhenfang Wu, Huaqiang Yang

2019, 34(5): 592 doi: 10.1007/s12250-019-00127-y

Received: 19 December 2018 Accepted: 15 April 2019 Published: 27 May 2019
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Porcine epidemic diarrhea virus (PEDV) is the etiologic agent of porcine epidemic diarrhea (PED), which is an acute, highly contagious, and devastating enteric viral disease in pigs. Surface expression of Aminopeptidase N (APN) is considered a functional receptor for infection by porcine epidemic diarrhea virus (PEDV). To test the role of APN in PEDV infection, we created APN knockout (KO) pigs by introducing frame shift mutations in the exon 2 or exon 3 of APN gene using CRIPSR/Cas9-mediated gene editing and somatic cell nuclear transfer. The resulting cloned pigs harbored homozygous or biallelic insertion/deletion in the target site of APN, and had no APN expression in the small intestines. After PEDV infection, both APN KO and wild type controls became infected and showed no differences in clinical signs, fecal viral shedding or antibody response. The results demonstrate that the expression of the APN gene is not necessary for infection of pigs with PEDV and that the absence of APN does not contribute to the resistance to the disease. This work indicates that an alternative receptor is needed to be identified for PEDV infection of pigs.

Prevalence and Molecular Phylogenetic Analysis of Severe Fever with Thrombocytopenia Syndrome Virus in Domestic Animals and Rodents in Hubei Province, China

Jing Cheng, Li Zhang, Bing Hu, Qin Wang, Ran Wu, Faxian Zhan, Shuang Rong, Jianbo Zhan

2019, 34(5): 596 doi: 10.1007/s12250-019-00119-y

Received: 08 January 2019 Accepted: 10 April 2019 Published: 03 June 2019
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To understand the role of domesticated animals and rodents in the circulation and transmission of SFTSV, a total of 544 serum samples were collected from several domestic animals and rodents from SFTSV epidemic areas (Suizhou, Macheng) and non-epidemic areas (Yicheng, Qianjiang, Xiantao) in Hubei Province, China, from July 2010 to November 2016. SFTSV-specific antibodies were detected in 80 (81.63%) of 98 sheep, 39 (67.24%) of 58 cattle, 12 (63.16%) of 19 dogs, and 4 (1.08%) of 369 rodents. SFTSV RNA was detected in serum samples from sheep (3.06%) and cattle (5.17%), but was not detected in rodent tissue samples. The virus isolated from sheep shared more than 97% sequence homology with local patients. Our findings suggest that sheep and cattle act as natural hosts and might play an important role in the transmission of SFTSV.