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2020 Vol.35(2)

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of viral life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus (RABV). In this issue, Zhang et al. performed proteomics studies of the RABV virions with good purity and integrity to define 49 cellular proteins incorporated into mature virions, and 24 of them were likely involved in virus replication according to the functional annotation analysis. Furthermore, Zhang et al. used cryo-electron microscopy (Cryo-EM) to observe the purified RABV virions, generating high-resolution pictures of RABV particles with unprecedented structure details. The cover image clearly displays an intact bullet-shaped structure with four distinct electron-dense layers consisting of glycoprotein spike, viral envelope, matrix protein helix and RNP complex from outside to inside. Please see page 143–155 for details.

Review

Bacteriophages and Lysins in Biofilm Control

Marzanna Łusiak-Szelachowska, Beata Weber-Dąbrowska, Andrzej Górski

2020, 35(2): 125 doi: 10.1007/s12250-019-00192-3

Received: 30 May 2019 Accepted: 17 December 2019 Published: 03 March 2020
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To formulate the optimal strategy of combatting bacterial biofilms, in this review we update current knowledge on the growing problem of biofilm formation and its resistance to antibiotics which has spurred the search for new strategies to deal with this complication. Based on recent findings, the role of bacteriophages in the prevention and elimination of biofilm-related infections has been emphasized. In vitro, ex vivo and in vivo biofilm treatment models with single bacteriophages or phage cocktails have been compared. A combined use of bacteriophages with antibiotics in vitro or in vivo confirms earlier reports of the synergistic effect of these agents in improving biofilm removal. Furthermore, studies on the application of phage-derived lysins in vitro, ex vivo or in vivo against biofilm-related infections are encouraging. The strategy of combined use of phage and antibiotics seems to be different from using lysins and antibiotics. These findings suggest that phages and lysins alone or in combination with antibiotics may be an efficient weapon against biofilm formation in vivo and ex vivo, which could be useful in formulating novel strategies to combat bacterial infections. Those findings proved to be relevant in the prevention and destruction of biofilms occurring during urinary tract infections, orthopedic implant-related infections, periodontal and peri-implant infections. In conclusion, it appears that most efficient strategy of eliminating biofilms involves phages or lysins in combination with antibiotics, but the optimal scheme of their administration requires further studies.
Research Article

Genome Analysis of Dasineura jujubifolia Toursvirus 2, A Novel Ascovirus

Jun Wang, Minglu Yang, Haibing Xiao, Guo-Hua Huang, Fei Deng, Zhihong Hu

2020, 35(2): 134 doi: 10.1007/s12250-019-00177-2

Received: 28 May 2019 Accepted: 09 September 2019 Published: 29 November 2019
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So far, ascoviruses have only been identified from Lepidoptera host insects and their transmission vectors—endoparasitic wasps. Here, we reported the first finding of a complete novel ascovirus genome from a Diptera insect, Dasineura jujubifolia. Initially, sequence fragments with homology to ascoviruses were incidentally identified during metagenomic sequencing of the mitochondria of D. jujubifolia (Cecidomyiidae, Diptera) which is a major pest on Ziziphus jujuba. Then a full circular viral genome was assembled from the metagenomic data, which has an A+T percentage of 74% and contains 142, 600 bp with 141 open reading frames (ORFs). Among the 141 ORFs, 37 were conserved in all sequenced ascoviruses (core genes) including proteins predicted to participate in DNA replication, gene transcription, protein modification, virus assembly, lipid metabolism and apoptosis. Multi-gene families including those encode for baculovirus repeated open reading frames (BROs), myristylated membrane proteins, RING/U-box E3 ubiquitin ligases, and ATP-binding cassette (ABC) transporters were found in the virus genome. Phylogenetic analysis showed that the newly identified virus belongs to genus Toursvirus of Ascoviridae, and is therefore named as Dasineura jujubifolia toursvirus 2 (DjTV-2a). The virus becomes the second reported species of the genus after Diadromus pulchellus toursvirus 1 (DpTV-1a). The genome arrangement of DjTV-2a is quite different from that of DpTV-1a, suggesting these two viruses separated in an early time of evolution. The results suggest that the ascoviruses may infect a much broader range of hosts than our previous knowledge, and shed lights on the evolution of ascoviruses and particularly on that of the toursviruses.

Proteomic Profiling of Purified Rabies Virus Particles

Yan Zhang, Yuyang Wang, Ye Feng, Zhongzhong Tu, Zhiyong Lou, Changchun Tu

2020, 35(2): 143 doi: 10.1007/s12250-019-00157-6

Received: 11 May 2019 Accepted: 31 July 2019 Published: 19 August 2019
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While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus (RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC-MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.

Structural Basis of Glycan Recognition in Globally Predominant Human P[8] Rotavirus

Xiaoman Sun, Lei Dang, Dandi Li, Jianxun Qi, Mengxuan Wang, Wengang Chai, Qing Zhang, Hong Wang, Ruixia Bai, Ming Tan, Zhaojun Duan

2020, 35(2): 156 doi: 10.1007/s12250-019-00164-7

Received: 25 July 2019 Accepted: 21 August 2019 Published: 16 October 2019
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Rotavirus (RV) causes acute gastroenteritis in infants and children worldwide. Recent studies showed that glycans such as histo-blood group antigens (HBGAs) function as cell attachment factors affecting RV host susceptibility and prevalence. P[8] is the predominant RV genotype in humans, but the structural basis of how P[8] RVs interact with glycan ligands remains elusive. In this study, we characterized the interactions between P[8] VP8*s and glycans which showed that VP8*, the RV glycan binding domain, recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays. Importantly, we determined the structural basis of P[8] RV-glycans interaction from the crystal structures of a Rotateq P[8] VP8* in complex with core 2 and H type 1 glycans at 1.8? and 2.3?, respectively, revealing a common binding pocket and similar binding mode. Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[II] genogroup, while genotype-specific amino acid variations determined different glycan binding preference. Our data elucidated the detailed structural basis of the interactions between human P[8] RVs and different host glycan factors, shedding light on RV infection, epidemiology, and development of anti-viral agents.

Analysis of Expression Profiles of Long Noncoding RNAs and mRNAs in A549 Cells Infected with H3N2 Swine Influenza Virus by RNA Sequencing

Yina Zhang, Tianqi Yu, Yingnan Ding, Yahui Li, Jing Lei, Boli Hu, Jiyong Zhou

2020, 35(2): 171 doi: 10.1007/s12250-019-00170-9

Received: 23 May 2019 Accepted: 27 August 2019 Published: 27 November 2019
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Long noncoding RNAs (lncRNAs) participate in regulating many biological processes. However, their roles in influenza A virus (IAV) pathogenicity are largely unknown. Here, we analyzed the expression profiles of lncRNAs and mRNAs in H3N2-infected cells and mock-infected cells by high-throughput sequencing. The results showed that 6129 lncRNAs and 50, 031 mRNA transcripts in A549 cells displayed differential expression after H3N2 infection compared with mock infection. Among the differentially expressed lncRNAs, 4963 were upregulated, and 1166 were downregulated. Functional annotation and enrichment analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases (KEGG) suggested that target genes of the differentially expressed lncRNAs were enriched in some biological processes, such as cellular metabolism and autophagy. The up- or downregulated lncRNAs were selected and further verified by quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription PCR (RT-PCR). To the best of our knowledge, this is the first report of a comparative expression analysis of lncRNAs in A549 cells infected with H3N2. Our results support the need for further analyses of the functions of differentially expressed lncRNAs during H3N2 infection.

Premature Stop Codon at Residue 101 within HIV-1 Rev Does Not Influence Viral Replication of Clade BC but Severely Reduces Viral Fitness of Clade B

Zheng Wang, Xiaolin Ji, Yanling Hao, Kunxue Hong, Liying Ma, Dan Li, Yiming Shao

2020, 35(2): 181 doi: 10.1007/s12250-019-00179-0

Received: 02 March 2019 Accepted: 17 October 2019 Published: 11 December 2019
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HIV-1 Rev is an accessory protein that plays a key role in nuclear exportation, stabilization, and translation of the viral mRNAs. Rev of HIV-1 clade BC often shows a truncation of 16 AAs due to a premature stop codon at residue 101. This stop codon presents the highest frequency in clade BC and the lowest frequency in clade B. In order to discover the potential biological effect of this truncation on Rev activity and virus replication of clade BC, we constructed Rev expression vectors of clade BC with or without 16 AAs within C-terminal separately, and replaced the stop codon by Q in a CRF07_BC infectious clone. We found that 16 AAs truncation had no effect on expression and activity of Rev in clade BC. Also, the mutation from the stop codon to Q had no effect on virus replication of clade BC. Next, to investigate the effect of this truncation on Rev activity and replication capacity of clade B, Rev expression vectors of clade B carrying or lacking 16 AAs in C-terminal were constructed respectively, and residue Q at position 101 within Rev was substituted by the stop codon in a clade B infectious clone. It was found that 16 AAs truncation significantly down-regulated Rev expression and impaired clade B Rev activity. Furthermore, a Q-to-stop codon substitution within Rev significantly reduced viral replication fitness of clade B. These results indicate that the premature stop codon at residue 101 within Rev exerts diverse impact on viral replication among different HIV-1 clades.

Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein

Ke Zheng, Fang-Fang Jiang, Le Su, Xin Wang, Yu-Xin Chen, Huan-Chun Chen, Zheng-Fei Liu

2020, 35(2): 191 doi: 10.1007/s12250-019-00175-4

Received: 12 April 2019 Accepted: 09 September 2019 Published: 02 December 2019
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Viruses evolve rapidly and continuously threaten animal health and economy, posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine. We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase. We cloned pseudorabies virus genome into bacterial artificial chromosome, and used CRISPR-guided cytidine deaminase to directly convert cytidine (C) to uridine (U) to induce premature stop mutagenesis in viral genes. The editing efficiencies were 100%. Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus (PRV) genomes. Notably, in our study viral genome exists as a plasmid in E. coli, suggesting that this method is virus species-independent. This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Fuxian Zhang, Hong Guo, Qingxiu Chen, Zheng Ruan, Qin Fang

2020, 35(2): 200 doi: 10.1007/s12250-019-00178-1

Received: 16 April 2019 Accepted: 18 September 2019 Published: 19 December 2019
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Grass carp reovirus (GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus. To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots (QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl, chloroquine and bafilomycin A1 (Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection, hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether, our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

Ingo Jordan, Deborah Horn, Kristin Thiele, Lars Haag, Katharina Fiddeke, Volker Sandig

2020, 35(2): 212 doi: 10.1007/s12250-019-00176-3

Received: 06 June 2019 Accepted: 18 September 2019 Published: 12 December 2019
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Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

Monoclonal Antibody-Based Serological Detection of Rice Stripe Mosaic Virus Infection in Rice Plants or Leafhoppers

Liqian Guo, Jiayu Wu, Rui Chen, Jian Hong, Xueping Zhou, Jianxiang Wu

2020, 35(2): 227 doi: 10.1007/s12250-019-00186-1

Received: 17 August 2019 Accepted: 21 October 2019 Published: 19 December 2019
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Rice stripe mosaic virus (RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies (MAbs) (i.e., 1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20, 971, 520 (w/v, g/mL) through ACP-ELISA or diluted at 1:327, 680 (w/v, g/mL) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper (Recilia dorsalis) homogenate diluted at 1:307, 200 and 1:163, 840 (individual leafhopper/μL), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR (19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.
Letter

Screening and Identification of Marburg Virus Entry Inhibitors Using Approved Drugs

Li Zhang, Shan Lei, Hui Xie, Qianqian Li, Shuo Liu, Qiang Liu, Weijin Huang, Xinyue Xiao, Youchun Wang

2020, 35(2): 235 doi: 10.1007/s12250-019-00184-3

Received: 03 June 2019 Accepted: 21 October 2019 Published: 20 December 2019
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Meta-Transcriptome Profiling of Novel Invasive Pest Spodoptera frugiperda in Yunnan, China

Junming Shi, Weiwei Li, Yunyu Wang, Quanyan Chen, Fei Deng

2020, 35(2): 240 doi: 10.1007/s12250-019-00188-z

Received: 23 July 2019 Accepted: 01 November 2019 Published: 08 January 2020
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Identification and Characterization of the First Equine Parainfluenza Virus 5

Jinxin Xie, Panpan Tong, Aoyuntuya Zhang, Lei Zhang, Xiaozhen Song, Ling Kuang

2020, 35(2): 245 doi: 10.1007/s12250-019-00185-2

Received: 02 August 2019 Accepted: 18 November 2019 Published: 08 January 2020
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Meeting Report

WSV 2019: The First Committee Meeting of the World Society for Virology

Ahmed S. Abdel-Moneim, Matthew D. Moore, Mahmoud M. Naguib, Jesus L. Romalde, Maria Söderlund-Venermo

2020, 35(2): 248 doi: 10.1007/s12250-019-00189-y

Received: 26 November 2019 Accepted: 28 November 2019 Published: 19 December 2019
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The World Society for Virology (WSV) was founded and incorporated as a nonprofit organization in the United States in 2017. WSV seeks to strengthen and support both virological research and virologists who conduct research of viruses that affect humans, other animals, plants, and other organisms. One of the objectives of WSV is to connect virologists worldwide and support collaboration. Fulfilling this objective, virologists from fourteen countries in North America, Europe, Africa, Asia, and the Middle East met on 25–27th August 2019 in Stockholm, Sweden at the Karolinska University Hospital for the first Committee Meeting of WSV. This meeting included compelling keynote and honorary speeches and a series of 18 scientific talks were given encompassing a diverse array of subjects within virology. Followed by the scientific session, a business session was held where multiple aspects and next steps of the society were discussed and charted out.