The roles of five conserved lentiviral RNA structures in HIV-1 replication Virology (IF 3.353) Pub Date : 2017-11-09 Yang Liu, Jianbo Chen, Olga A. Nikolaitchik, Belete A. Desimmie, Steven Busan, Vinay K. Pathak, Kevin M. Weeks, Wei-Shau Hu
The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.
Discovery of novel anelloviruses in small mammals expands the host range and diversity of the Anelloviridae Virology (IF 3.353) Pub Date : 2017-11-10 William Marciel de Souza, Marcílio Jorge Fumagalli, Jansen de Araujo, Gilberto Sabino-Santos Jr., Felipe Gonçalves Motta Maia, Marilia Farignoli Romeiro, Sejal Modha, Marcello Schiavo Nardi, Luzia Helena Queiroz, Edison Luiz Durigon, Márcio Roberto Teixeira Nunes, Pablo Ramiro Murcia, Luiz Tadeu Moraes Figueiredo
The Anelloviridae comprises single-stranded DNA viruses currently grouped in sixty-eight species classified in twelve genera. They have been found in many vertebrate hosts including primates. In this study, we describe the application of the high-throughput sequencing to examine the frequency and diversity of anelloviruses in rodents, bats and opossums captured in São Paulo State, Brazil. We report a total of twenty-six anelloviruses with sixteen nearly complete genomes and ten partial genomes, which include eleven potential novel species identified in rodents (Cricetidae), bats (Molossidae and Phyllostomidae), and opossums (Didelphidae). We also propose the inclusion of two potential new genera within the Anelloviridae family, provisionally named Omegatorquevirus and Sigmatorquevirus, including six and three novel species of anelloviruses, respectively. In summary, this study expands the diversity and the host range of the known anelloviruses.
Rab1A is required for assembly of classical swine fever virus particle Virology (IF 3.353) Pub Date : 2017-11-10 Jihui Lin, Chengbao Wang, Wulong Liang, Jing Zhang, Longxiang Zhang, Huifang Lv, Wang Dong, Yanming Zhang
Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein.
Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1 Virology (IF 3.353) Pub Date : 2017-11-10 Ahmed Atef Mesalam, Isabelle Desombere, Ali Farhoudi, Freya Van Houtte, Lieven Verhoye, Jonathan Ball, Jean Dubuisson, Steven K.H. Foung, Arvind H. Patel, Mats A.A. Persson, Geert Leroux-Roels, Philip Meuleman
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230–239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.
Wheat streak mosaic virus coat protein is a determinant for vector transmission by the wheat curl mite Virology (IF 3.353) Pub Date : 2017-11-10 Satyanarayana Tatineni, Anthony J. McMechan, Gary L. Hein
Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae), is transmitted by the wheat curl mite (Aceria tosichella Keifer). The requirement of coat protein (CP) for WSMV transmission by the wheat curl mite was examined using a series of viable deletion and point mutations. Mite transmission of WSMV was completely abolished with deletions comprising CP amino acids 58–100. In contrast, the amino-proximal (amino acids 6–27 and 36–57) and carboxy-terminal (14 amino acids) regions of CP were expendable for mite transmission. Mutation of aspartic acid residues at amino acid positions 289 or 326 (D289A or D326A) at the carboxy-proximal region of CP significantly reduced mite transmission. Remarkably, every wheat plant infected by mutants D289A or D326A through mite transmission but not with in vitro transcripts contained a second-site mutation of R131C and N275H, respectively. Collectively, these data demonstrate for the first time that CP is a determinant for an eriophyid-transmitted plant virus.
Integrase C-terminal residues determine the efficiency of feline foamy viral DNA integration Virology (IF 3.353) Pub Date : 2017-11-10 Jinsun Kim, Ga-Eun Lee, Martin Lochelt, Cha-Gyun Shin
Integrase (IN) is an essential enzyme in retroviral life cycle. It mediates viral cDNA integration into host cellular DNA. Feline foamy virus (FFV) is a member of the Spumavirus subfamily of Retroviridae. Recently, its life cycle has been proposed to be different from other retroviruses. Despite this important finding, FFV IN is not understood clearly. Here, we constructed point mutations in FFV IN C-terminal domain (CTD) to obtain a clear understanding of its integration mechanism. Mutation of the amino acid residues in FFV IN CTD interacting with target DNA reduced both IN enzymatic activities in vitro and viral productions in infected cells. Especially, the mutants, R307 and K340, made viral DNA integration less efficient and allowed accumulation of more unintegrated viral DNA, thereby suppressing viral replication. Therefore, we suggest that the CTD residues interacting with the target DNA play a significant role in viral DNA integration and replication.
Male Syrian hamsters are more susceptible to intravenous infection with species C human adenoviruses than are females Virology (IF 3.353) Pub Date : 2017-11-10 Baoling Ying, Jacqueline F. Spencer, Ann E. Tollefson, William S.M. Wold, Karoly Toth
Recently, increasing attention has been focused on the influence of sex on the course of infectious diseases. Thus far, the best-documented examples point toward an immune-mediated mechanism: the generally stronger immune response in females can result in a faster clearance of the pathogen or, conversely, a more severe immune-mediated pathology. Here, we report that human species C adenoviruses replicate more and cause more pathology in male Syrian hamsters than in females. We also show that this sex disparity is not caused by a stronger immune response to the infection by the female hamsters. Rather, the liver of male hamsters is more susceptible to adenovirus infection: after intravenous injection, more hepatocytes become infected in male animals than in females. We hypothesize that Kupffer cells (hepatic tissue macrophages) of female animals are more active in sequestering circulating virions, and thus protect hepatocytes more efficiently than those of males.
Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus Virology (IF 3.353) Pub Date : 2017-11-05 Phu-Tri Tran, Kristin Widyasari, Jang-Kyun Seo, Kook-Hyung Kim
Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV.
Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations Virology (IF 3.353) Pub Date : 2017-11-05 María Mercedes Elizalde, Ina Sevic, María Mora González López Ledesma, Rodolfo Héctor Campos, Luciana Barbini, Diego Martin Flichman
In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes.
Porcine reproductive and respiratory disease virus: Evolution and recombination yields distinct ORF5 RFLP 1-7-4 viruses with individual pathogenicity Virology (IF 3.353) Pub Date : 2017-11-05 Albert G.M. van Geelen, Tavis K. Anderson, Kelly M. Lager, Phani B. Das, Nicholas J. Otis, Nestor A. Montiel, Laura C. Miller, Vikas Kulshreshtha, Alexandra C. Buckley, Susan L. Brockmeier, Jianqiang Zhang, Phillip C. Gauger, Karen M. Harmon, Kay S. Faaberg
Recent cases of porcine reproductive and respiratory syndrome virus (PRRSV) infection in United States swine-herds have been associated with high mortality in piglets and severe morbidity in sows. Analysis of the ORF5 gene from such clinical cases revealed a unique restriction fragment polymorphism (RFLP) of 1-7-4. The genome diversity of seventeen of these viruses (81.4% to 99.8% identical; collected 2013–2015) and the pathogenicity of 4 representative viruses were compared to that of SDSU73, a known moderately virulent strain. Recombination analyses revealed genomic breakpoints in structural and nonstructural regions of the genomes with evidence for recombination events between lineages. Pathogenicity varied between the isolates and the patterns were not consistent. IA/2014/NADC34, IA/2013/ISU-1 and IN/2014/ISU-5 caused more severe disease, and IA/2014/ISU-2 did not cause pyrexia and had little effect on pig growth. ORF5 RFLP genotyping was ineffectual in providing insight into isolate pathogenicity and that other parameters of virulence remain to be identified.
MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs Virology (IF 3.353) Pub Date : 2017-11-05 Rodney P. Kincaid, Neena G. Panicker, Mary M. Lozano, Christopher S. Sullivan, Jaquelin P. Dudley, Farah Mustafa
Identification of the interaction and interaction domains of chicken anemia virus VP2 and VP3 proteins Virology (IF 3.353) Pub Date : 2017-11-05 Fenfen Sun, Wei Pan, Honglei Gao, Xiaole Qi, Liting Qin, Yongqiang Wang, Yulong Gao, Xiaomei Wang
Chicken anemia virus (CAV) is a small, single-stranded DNA virus of Anelloviridae family. Its genome segments encode three proteins, VP1, VP2, and VP3. This study identified an interaction between VP2 and VP3 and mapped the interaction domains. Through the yeast two-hybrid (Y2H) system, VP2 was found to interact with VP3. The presence of the VP2–VP3 complex in CAV-infected chicken cells was confirmed by co-immunoprecipitation. Confocal microscopy showed that VP2 and VP3 were expressed in the cytoplasm in cotransfected Vero cells. In the Y2H system, the interaction domains were identified as being within the N-terminal aa 1–30 and C-terminal aa 17–60 for VP2 and the N-terminal aa 46–60 and C-terminal aa 1–7 for VP3. This study showed the interaction between VP2 and VP3 of CAV and identified multiple independent interactive domains within the two proteins. This provides novel information for investigating the biological functions of these proteins.
Identification of the caveolae/raft-mediated endocytosis as the primary entry pathway for aquareovirus Virology (IF 3.353) Pub Date : 2017-11-05 Fuxian Zhang, Hong Guo, Jie Zhang, Qingxiu Chen, Qin Fang
Grass carp reovirus (GCRV), a member of the Aquareovirus genus in the Reoviridae family, is considered the most pathogenic aquareovirus. However, its productive viral entry pathways remain largely unclear. Using a combination of quantum dot (QD)-based live-virus tracking and biochemical assays, we found that extraction of cellular membrane cholesterol with methyl-β-cyclodextrin (MβCD) and nystatin strongly inhibited the internalization of GCRVs, and supplementation with cholesterol restored viral infection. In addition, the entry of the virus was restrained by genistein, an inhibitor known to block caveolar endocytosis. Subsequent real-time tracking experiments revealed that the QD-labeled GCRV particles were colocalized with caveolin-1, and transfection of cells with dominant-negative mutant (caveolin-1 Y14F) significantly reduced GCRV infection. In contrast, no effects on virus infection were detected when the clathrin-mediated endocytosis or the macropinocytosis inhibitors were used. Our results collectively suggest that aquareoviruses can use caveolae/raft-mediated endocytosis as the primary entry pathway to initiate productive infection.
Viral sequences in human cancer Virology (IF 3.353) Pub Date : 2017-11-05 Paul G. Cantalupo, Joshua P. Katz, James M. Pipas
We have developed a virus detection and discovery computational pipeline, Pickaxe, and applied it to NGS databases provided by The Cancer Genome Atlas (TCGA). We analyzed a collection of whole genome (WGS), exome (WXS), and RNA (RNA-Seq) sequencing libraries from 3052 participants across 22 different cancers. NGS data from nearly all tumor and normal tissues examined contained contaminating viral sequences. Intensive computational and manual efforts are required to remove these artifacts. We found that several different types of cancers harbored Herpesviruses including EBV, CMV, HHV1, HHV2, HHV6 and HHV7. In addition to the reported associations of Hepatitis B and C virus (HBV & HCV) with liver cancer, and Human papillomaviruses (HPV) with cervical cancer and a subset of head and neck cancers, we found additional cases of HPV integrated in a small number of bladder cancers. Gene expression and mutational profiles suggest that HPV drives tumorigenesis in these cases.
Hepatitis E virus subtype 3f strains isolated from Japanese hepatitis patients with no history of travel to endemic areas – The origin analyzed by molecular evolution Virology (IF 3.353) Pub Date : 2017-10-24 Tatsunori Nakano, Masaharu Takahashi, Kazuaki Takahashi, Shigeo Nagashima, Yusuke Suzuki, Yoichi Nishigaki, Eiichi Tomita, Hiroshi Okano, Yumi Oya, Katsuya Shiraki, Kojiro Takase, Kazushi Sugimoto, Junichi Koyama, Hitoshi Mizuo, Kazuto Ikezawa, Tatsuya Aikawa, Masahiro Arai, Hiroaki Okamoto
Hepatitis E virus subtype 3f (HEV-3f) strains are usually isolated in Europe and Thailand. Recently, HEV-3f strains were detected from six acute hepatitis E patients in Japan, none of whom had a history of travel to endemic areas. We inferred the origin and transmission route of the six HEV-3f strains. A time-scaled phylogenetic tree of the six strains with reference strains was constructed using a Bayesian statistical inference framework. The time-scaled tree indicated that the six strains independently derived from similar European strains between 2008 and 2014. The pattern suggested recent inflow of multiple HEV-3f strains from Europe to Japan. Japan imports a substantial amount of pork from European countries every year. The emergence of acute hepatitis cases caused by HEV-3f strains in Japan, in patients with no history of travel abroad, might be influenced by the increased opportunities to consume pork products imported from European countries.
Intramuscular delivery of replication-defective herpes simplex virus gives antigen expression in muscle syncytia and improved protection against pathogenic HSV-2 strains Virology (IF 3.353) Pub Date : 2017-10-22 Fernando Diaz, Sean Gregory, Hiroshi Nakashima, Mariano S. Viapiano, David M. Knipe
Herpes simplex virus 2 (HSV-2) is the leading cause of genital herpes and increases the risk of HIV infection, but there is no effective vaccine. A replication-defective HSV-2 mutant virus, dl5-29, is effective in animal models and has been in a phase I trial. Previous studies have shown that dl5-29 gives higher antibody responses and better protection when inoculated intramuscularly (IM) as compared with subcutaneously (SC). However, the basis for this effect has not been defined. We confirmed that IM inoculation of dl5-29 is more immunogenic and provides better protection than SC inoculation. IM inoculation of HSV-2 strains produced higher levels of a luciferase transgene than SC inoculation, as measured by intravital bioluminescence imaging. Intramuscular immunization also showed better protection against infection with a highly pathogenic African HSV-2, demonstrating that this single vaccine can be efficacious against HSV-2 strains from different geographic regions.
Inhibition of apoptosis in BHV-1-infected cells depends on Us3 serine/threonine kinase and its enzymatic activity Virology (IF 3.353) Pub Date : 2017-10-23 Agnieszka Brzozowska, Andrea D. Lipińska, Natalia Derewońko, Dorota Lesiak, Michał Rychłowski, Łukasz Rąbalski, Krystyna Bieńkowska-Szewczyk
Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers.To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors.
Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to “Megavirales” Virology (IF 3.353) Pub Date : 2017-10-21 Arcady Mushegian, Eli Levy Karin, Tal Pupko
The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes.
Shared ancestry of herpes simplex virus 1 strain Patton with recent clinical isolates from Asia and with strain KOS63 Virology (IF 3.353) Pub Date : 2017-10-20 Aldo Pourchet, Richard Copin, Matthew C. Mulvey, Bo Shopsin, Ian Mohr, Angus C. Wilson
Herpes simplex virus 1 (HSV-1) is a widespread pathogen that persists for life, replicating in surface tissues and establishing latency in peripheral ganglia. Increasingly, molecular studies of latency use cultured neuron models developed using recombinant viruses such as HSV-1 GFP-US11, a derivative of strain Patton expressing green fluorescent protein (GFP) fused to the viral US11 protein. Visible fluorescence follows viral DNA replication, providing a real time indicator of productive infection and reactivation. Patton was isolated in Houston, Texas, prior to 1973, and distributed to many laboratories. Although used extensively, the genomic structure and phylogenetic relationship to other strains is poorly known. We report that wild type Patton and the GFP-US11 recombinant contain the full complement of HSV-1 genes and differ within the unique regions at only eight nucleotides, changing only two amino acids. Although isolated in North America, Patton is most closely related to Asian viruses, including KOS63.
Evidence for the role of basic amino acids in the coat protein arm region of Cucumber necrosis virus in particle assembly and selective encapsidation of viral RNA Virology (IF 3.353) Pub Date : 2017-10-20 Syed Benazir Alam, Ron Reade, Jane Theilmann, D'Ann Rochon
Cucumber necrosis virus (CNV) is a T = 3 icosahedral virus with a (+)ssRNA genome. The N-terminal CNV coat protein arm contains a conserved, highly basic sequence (“KGRKPR”), which we postulate is involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV “KGRKPR” sequence; the four basic residues were mutated to alanine individually, in pairs, or in total. Virion accumulation and vRNA encapsidation were significantly reduced in mutants containing two or four substitutions and virion morphology was also affected, where both T = 1 and intermediate-sized particles were produced. Mutants with two or four substitutions encapsidated significantly greater levels of truncated RNA than that of WT, suggesting that basic residues in the “KGRKPR” sequence are important for encapsidation of full-length CNV RNA. Interestingly, “KGRKPR” mutants also encapsidated relatively higher levels of host RNA, suggesting that the “KGRKPR” sequence also contributes to selective encapsidation of CNV RNA.
Phosphorylation of transcriptional regulators in the retinoblastoma protein pathway by UL97, the viral cyclin-dependent kinase encoded by human cytomegalovirus Virology (IF 3.353) Pub Date : 2017-10-20 Satoko Iwahori, Robert F. Kalejta
Human cytomegalovirus (HCMV) encodes a viral cyclin-dependent kinase (v-CDK), the UL97 protein. UL97 phosphorylates Rb, p107 and p130, thereby inactivating all three retinoblastoma (Rb) family members. Rb proteins function through regulating the activity of transcription factors to which they bind. Therefore, we examined whether the UL97-mediated regulation of the Rb tumor suppressors also extended to their binding partners. We observed that UL97 phosphorylates LIN52, a component of p107- and p130-assembled transcriptionally repressive DREAM complexes that control transcription during the G0/G1 phases, and the Rb-associated E2F3 protein that activates transcription through G1 and S phases. Intriguingly, we also identified FoxM1B, a transcriptional regulator during the S and G2 phases, as a UL97 substrate. This survey extends the influence of UL97 beyond simply the Rb proteins themselves to their binding partners, as well as past the G1/S transition into later stages of the cell cycle.
The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis Virology (IF 3.353) Pub Date : 2017-10-20 Brigid Chiyoko Poling, Alexander M. Price, Micah A. Luftig, Bryan R. Cullen
The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (∆123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1–3 stem-loops are present in the 3’UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1–3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1–3 in the ∆123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the ∆123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1–3 also regulate EBNA-LP mRNA expression in cis.
Foot-and-mouth disease virus 5’-terminal S fragment is required for replication and modulation of the innate immune response in host cells Virology (IF 3.353) Pub Date : 2017-10-20 Anna Kloc, Fayna Diaz-San Segundo, Elizabeth A. Schafer, Devendra K. Rai, Mary Kenney, Teresa de los Santos, Elizabeth Rieder
The S fragment of the FMDV 5’ UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.
The down-regulation of casein kinase 1 alpha as a host defense response against infectious bursal disease virus infection Virology (IF 3.353) Pub Date : 2017-10-05 Lizhou Zhang, Hui Li, Yuming Chen, Xiang Gao, Zhen Lu, Li Gao, Yongqiang Wang, Yulong Gao, Honglei Gao, Changjun Liu, Hongyu Cui, Yanping Zhang, Qing Pan, Xiaole Qi, Xiaomei Wang
Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. Although the gene functions of IBDV have been well characterized, the host responses during IBDV infection remain much poor. In the present study, casein kinase 1 alpha (CK1α), a novel VP2-associated protein, was down-regulated during IBDV replication in DF1 cells. Further experiments showed that siRNA-mediated knockdown of CK1α inhibited IBDV replication, while overexpression of CK1α promoted IBDV growth. Finally, we revealed that the effects of CK1α expression level on IBDV replication were involved in the negative regulation of CK1α on type I interferon receptor (IFNAR1), because ubiquitination assay analyses demonstrated that CK1α could promote the ubiquitination of IFNAR1, thereby affecting the stability of this receptor. In conclusion, down-regulation of CK1α during IBDV infection as a host defense response increased abundance of IFNAR1, which in turn enhanced an inhibitory effect on IBDV replication.
SJL bone marrow-derived macrophages do not have IRF3 mutations and are highly susceptible to Theiler's virus infection Virology (IF 3.353) Pub Date : 2017-10-20 Kyung-No Son, Zhiguo Liang, Howard L. Lipton
It is well known that SJL mice are susceptible to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease while C57BL6 (B6) and B10 mice are resistant, and H-2s on a B10 background (B10.S) contributes modestly to susceptibility. A recent study linked two IRF3 non-conservative mutations in SJL compared to B10.S mice to resistance to TMEV infection of SJL peritoneal-derived macrophages, an observation of practical interest in light of the central role of IRF3 transcription factor in the type I interferon (IFN) response. However, we did not find these non-conservative mutations among SJL, B10.S, B6 and B10 mice in the IRF3 amino acid sequence, and show SJL bone marrow-derived macrophages infected with TMEV exhibit increased virus RNA replication and infectious virus yields as well as greater IL-6 production than C57Bl strain (including B10.S) cultures.
Inhibition of the lytic cycle of Kaposi's sarcoma-associated herpesvirus by cohesin factors following de novo infection Virology (IF 3.353) Pub Date : 2017-10-20 Zsolt Toth, Richard J. Smindak, Bernadett Papp
Establishment of Kaposi's sarcoma-associated herpesvirus (KSHV) latency following infection is a multistep process, during which polycomb proteins are recruited onto the KSHV genome, which is crucial for the genome-wide repression of lytic genes during latency. Strikingly, only a subset of lytic genes are expressed transiently in the early phase of infection prior to the binding of polycomb proteins onto the KSHV genome, which raises the question what restricts lytic gene expression in the first hours of infection. Here, we demonstrate that both CTCF and cohesin chromatin organizing factors are rapidly recruited to the viral genome prior to the binding of polycombs during de novo infection, but only cohesin is required for the genome-wide inhibition of lytic genes. We propose that cohesin is required for the establishment of KSHV latency by initiating the repression of lytic genes following infection, which is an essential step in persistent infection of humans.
Immunological and pathological consequences of coxsackievirus RNA persistence in the heart Virology (IF 3.353) Pub Date : 2017-10-20 Claudia T. Flynn, Taishi Kimura, Kwesi Frimpong-Boateng, Stephanie Harkins, J. Lindsay Whitton
Type B coxsackieviruses (CVB) can cause myocarditis and dilated cardiomyopathy (DCM), a potentially-fatal sequela that has been correlated to the persistence of viral RNA. Herein, we demonstrate that cardiac RNA persistence can be established even after an inapparent primary infection. Using an inducible Cre/lox mouse model, we ask: (i) Does persistent CVB3 RNA cause ongoing immune activation? (ii) If T1IFN signaling into cardiomyocytes is ablated after RNA persistence is established, is there any change in the abundance of persistent CVB3 RNA and/or does cytopathic infectious virus re-emerge? (iii) Does this loss of T1IFN responsiveness by cardiomyocytes lead to the recurrence/exacerbation of myocarditis? Our findings suggest that persistent enteroviral RNAs probably do not contribute to ongoing myocardial disease, and are more likely to be the fading remnants of a recent, possibly sub-clinical, primary infection which may have set in motion the process that ultimately ends in DCM.
Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entry Virology (IF 3.353) Pub Date : 2017-10-20 John Nahabedian, Amit Sharma, Maryska E. Kaczmarek, Greg K. Wilkerson, Sara L. Sawyer, Julie Overbaugh
Studying HIV-1 replication in the presence of functionally related proteins from different species has helped define host determinants of HIV-1 infection. Humans and owl monkeys, but not macaques, encode a CD4 receptor that permits entry of transmissible HIV-1 variants due to a single residue difference. However, little is known about whether divergent CCR5 receptor proteins act as determinants of host-range. Here we show that both owl monkey (Aotus vociferans) CD4 and CCR5 receptors are functional for the entry of transmitted HIV-1 when paired with human versions of the other receptor. By contrast, the owl monkey CD4/CCR5 pair is generally a suboptimal receptor combination, although there is virus-specific variation in infection with owl monkey receptors. Introduction of the human residues 15Y and 16T within a sulfation motif into owl monkey CCR5 resulted in a gain of function. These findings suggest there is cross-talk between CD4 and CCR5 involving the sulfation motif.
Determinant of receptor-preference switch in influenza hemagglutinin Virology (IF 3.353) Pub Date : 2017-10-18 Fengyun Ni, Elena Kondrashkina, Qinghua Wang
Influenza pandemic occurs when a new strain from other animal species overcomes the inter-species barriers and supports rapid human-to-human transmission. A critical prerequisite to this process is that hemagglutinin (HA) acquires a few key mutations to switch from avian receptors to human receptors. Previous studies suggest that H1 and H2/H3 HAs use different sets of mutations for the switch. This report shows that HA from the 1918 H1N1 pandemic virus (1918H1 HA) adopts the set of mutations used by H2/H3 HAs in receptor-preference switch when its 130-loop is made similar to those of H2/H3 HAs. Thus, the 130-loop appears to be the key determinant for the different mutations employed by pandemic H1 or H2/H3 HA. The correlation of the mutational routes and the 130-loop as unraveled in this study opens the door for efficient investigation of mutations required by other HA subtypes for inter-human airborne transmission.
Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses Virology (IF 3.353) Pub Date : 2017-10-19 Elissa J. Schwartz, Naveen K. Vaidya, Karin S. Dorman, Susan Carpenter, Robert H. Mealey
Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV.
Comparative transcriptome analysis reveals networks of genes activated in the whitefly, Bemisia tabaci when fed on tomato plants infected with Tomato yellow leaf curl virus Virology (IF 3.353) Pub Date : 2017-10-13 Daniel K. Hasegawa, Wenbo Chen, Yi Zheng, Navneet Kaur, William M. Wintermantel, Alvin M. Simmons, Zhangjun Fei, Kai-Shu Ling
Identification of N-linked glycosylation sites in the spike protein and their functional impact on the replication and infectivity of coronavirus infectious bronchitis virus in cell culture Virology (IF 3.353) Pub Date : 2017-10-13 Jie Zheng, Yoshiyuki Yamada, To Sing Fung, Mei Huang, Raymond Chia, Ding Xiang Liu
Spike (S) glycoprotein on the viral envelope is the main determinant of infectivity. The S protein of coronavirus infectious bronchitis virus (IBV) contains 29 putative asparagine(N)-linked glycosylation sites. These post-translational modifications may assist in protein folding and play important roles in the functionality of S protein. In this study, we used bioinformatics tools to predict N-linked glycosylation sites and to analyze their distribution in IBV strains and variants. Among these sites, 8 sites were confirmed in the S protein extracted from partially purified virus particles by proteomics approaches. N-D and N-Q substitutions at 13 predicted sites were introduced into an infectious clone system. The impact on S protein-mediated cell-cell fusion, viral recovery and infectivity was assessed, leading to the identification of sites essential for the functions of IBV S protein. Further characterization of these and other uncharacterized sites may reveal novel aspects of N-linked glycosylation in coronavirus replication and pathogenesis.
Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins Virology (IF 3.353) Pub Date : 2017-10-13 Yong Wah Tan, To Sing Fung, Hongyuan Shen, Mei Huang, Ding Xiang Liu
The cleavage products from coronavirus polyproteins, known as the non-structural proteins (nsps), are believed to make up the major components of the viral replication/transcription complex. In this study, several nsps encoded by avian gammacoronavirus infectious bronchitis virus (IBV) were screened for RNA-binding activity and interaction with its RNA-dependent RNA polymerase, nsp12. Nsp2, nsp5, nsp8, nsp9 and nsp10 were found to bind to untranslated regions (UTRs), while nsp8 was confirmed to interact with nsp12. Nsp8 has been reported to interact with nsp7 and functions as a primase synthesizing RNA primers for nsp12. Further characterization revealed that nsp8-nsp12 interaction is independent of the UTRs of viral RNA, and nsp8 interacts with both the N- and C-terminal regions of nsp12. These results have prompted a proposal of how the nsp7-nsp8 complex could possibly function in tandem with nsp12, forming a highly efficient complex that could synthesize both the RNA primer and viral RNA during coronavirus infection.
An easy way to detect dengue virus using nanoparticle-antibody conjugates Virology (IF 3.353) Pub Date : 2017-10-14 Caroline R. Basso, Claudia C. Tozato, Bruno P. Crulhas, Gustavo R. Castro, João Pessoa A. Junior, Valber A. Pedrosa
The aim of the present research is to propose a new method based on localized surface plasmon resonance (LSPR) for fast dengue virus detection. A pool with four dengue serotypes (DENV-1, -2, -3, -4) was detected through antigen-antibody binding using gold nanoparticles (AuNPs) as signaling antibody carriers. Such result was confirmed through surface plasmon resonance (SPR), transmission electron microcopy (TEM), and dynamic light scattering (DLS) techniques. The limit of detection was calculated for TCID50 107 demonstrating a linear correlation between viral concentration and number of cells with an r2 value of > 0.993. The assay presented good sensibility and reproducibility of results and the negative controls were not mistakenly detected. This design requires no pretreatment or high trained person. In the future, it can be used in commercial antibody detection kits.
Soybean-derived Bowman-Birk inhibitor (BBI) blocks HIV entry into macrophages Virology (IF 3.353) Pub Date : 2017-10-16 Tong-Cui Ma, Le Guo, Run-Hong Zhou, Xu Wang, Jin-Biao Liu, Jie-Liang Li, Yu Zhou, Wei Hou, Wen-Zhe Ho
Bowman-Birk inhibitor (BBI) is a soybean-derived protease inhibitor that has anti-inflammation and anti-HIV effect. Here, we further investigated the anti-HIV action of BBI in macrophages, focusing on its effect on viral entry. We found that BBI could significantly block HIV entry into macrophages. Investigation of the mechanism(s) of the BBI action on HIV inhibition showed that BBI down-regulated the expression of CD4 receptor (as much as 80%) and induced the production of the CC chemokines (up to 60 folds at protein level) in macrophages. This inhibitory effect of BBI on HIV entry could be blocked by the neutralization antibodies to CC chemokines. These findings indicate that BBI may have therapeutic potential as a viral entry inhibitor for the prevention and treatment of HIV infection.
Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry Virology (IF 3.353) Pub Date : 2017-10-12 Shirley Qiu, Anders Leung, Yuxia Bo, Robert A. Kozak, Sai Priya Anand, Corina Warkentin, Fabiola D.R. Salambanga, Jennifer Cui, Gary Kobinger, Darwyn Kobasa, Marceline Côté
For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P2-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P2 production in cells to promote efficient delivery to NPC1.
Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape Virology (IF 3.353) Pub Date : 2017-10-12 Adrien Lion, Mathilde Richard, Evelyne Esnault, Emmanuel Kut, Denis Soubieux, Vanaïque Guillory, Mélody Germond, Caroline Blondeau, Rodrigo Guabiraba, Kirsty R. Short, Daniel Marc, Pascale Quéré, Sascha Trapp
Endotheliotropism is a hallmark of gallinaceous poultry infections with highly pathogenic avian influenza (HPAI) viruses and a feature that distinguishes HPAI from low pathogenic avian influenza (LPAI) viruses. Here, we used chicken aortic endothelial cells (chAEC) as a novel in vitro infection model to assess the susceptibility, permissiveness, and host response of chicken endothelial cells (EC) to infections with avian influenza (AI) viruses. Our data show that productive replication of AI viruses in chAEC is critically determined by hemagglutinin cleavability, and is thus an exclusive trait of HPAI viruses. However, we provide evidence for a link between limited (i.e. trypsin-dependent) replication of certain LPAI viruses, and the viruses’ ability to dampen the antiviral innate immune response in infected chAEC. Strikingly, this cell response pattern was also detected in HPAI virus-infected chAEC, suggesting that viral innate immune escape might be a prerequisite for robust AI virus replication in chicken EC.
Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer Virology (IF 3.353) Pub Date : 2017-10-12 J. Emanuel Finet, Xiaoping Wan, J. Kevin Donahue
AAV-mediated gene therapy has become a promising therapeutic strategy for chronic diseases. Its clinical utilization, however, is limited by the potential risk of off-target effects. In this work we attempt to overcome this challenge, hypothesizing that cardiac ion channel-specific ligands could be fused onto the AAV capsid, and narrow its tropism to cardiac myocytes. We successfully fused the cardiac sodium channel (Nav1.5)-binding toxin Anthopleurin-B onto the AAV2 capsid without compromising virus integrity, and demonstrated increased specificity of cardiomyocyte attachment. Although virus attachment to Nav1.5 did not supersede the natural heparan-mediated virus binding, heparan-binding ablated vectors carrying Anthopleurin-B eliminated hepatic and other extracardiac gene transfer, while preserving cardiac myocyte gene transfer. Virus binding to the cardiac sodium channel transiently decreased sodium current density, but did not cause any arrhythmias. Our findings expand the knowledge of attachment, infectivity, and intracellular processing of AAV vectors, and present an alternative strategy for vector retargeting.
Modulation of the NF-κB signaling pathway by the HIV-2 envelope glycoprotein and its incomplete BST-2 antagonism Virology (IF 3.353) Pub Date : 2017-10-11 François E. Dufrasne, Mara Lucchetti, Anandi Martin, Emmanuel André, Géraldine Dessilly, Benoit Kabamba, Patrick Goubau, Jean Ruelle
Differential interaction between human and murine Crm1 and lentiviral Rev proteins Virology (IF 3.353) Pub Date : 2017-10-10 Yan Yue, Ayse K. Coskun, Navneet Jawanda, Jim Auer, Richard E. Sutton
Mice have multiple obstacles to HIV replication, including a block of unspliced and partially spliced viral mRNA nuclear export. In human, Rev binds to the Rev-response element and human (h) Crm1, facilitating nuclear export of RRE-containing viral RNAs. Murine (m) Crm1 is less functional than hCrm1 in this regard. Here we demonstrated that in biochemical experiments mCrm1 failed to interact with HIV Rev whereas hCrm1 did. In genetic experiments in human cells, we observed a modest but significant differential effect between mCrm1 and hCrm1, which was also true of other lentiviral Revs tested. Triple mutant hCrm1 P411T-M412V-F414S behaved similarly to mCrm1, whereas mCrm1 with T411P-V412M-S414F regained some activity, although contribution of additional residues to its function can not be excluded. Similar results were observed in murine cells. This suggests a differential interaction between hCrm1 and mCrm1 and many lentiviral Revs, which may partially explain the HIV replicative defect in mice.
HIV-1 subtype CRF01_AE and B differ in utilization of low levels of CCR5, Maraviroc susceptibility and potential N-glycosylation sites Virology (IF 3.353) Pub Date : 2017-10-09 Anjali Joshi, Emily K. Cox, Melina J. Sedano, Erin B. Punke, Raphael TC Lee, Sebastian Maurer-Stroh, Palvinder Kaur, Oon Tek Ng, Himanshu Garg
HIV subtypes not only predominate in different geographical regions but also differ in key phenotypic characteristics. To determine if genotypic and/or phenotypic differences in the Envelope (Env) glycoprotein can explain subtype related differences, we cloned 37 full length Envs from Subtype B and AE HIV infected individuals from Singapore. Our data demonstrates that CRF01_AE Envs have lower Potential N Glycosylation Sites and higher risk of ×4 development. Phenotypically, CRF01_AE were less infectious than subtype B Envs in cells expressing low levels of CCR5. Moreover, the Maraviroc IC50 was higher for subtype B Envs and correlated with infectivity in low CCR5 expressing cells as well as PNGS. Specifically, the glycosylation site N301 in the V3 loop was seen less frequently in AE subtype and CXCR4 topic viruses. CRF01_AE differs from B subtype in terms of CCR5 usage and Maraviroc susceptibility which may have implications for HIV pathogenesis and virus evolution.
Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines Virology (IF 3.353) Pub Date : 2017-10-09 Ajay B. Maghodia, Donald L. Jarvis
Sf-rhabdovirus was only recently identified as an adventitious agent of Spodoptera frugiperda (Sf) cell lines used as hosts for baculovirus vectors. As such, we still know little about its genetic variation, infectivity, and the potential impact of variation on the Sf-rhabdovirus-host interaction. Here, we characterized Sf-rhabdoviruses from two widely used Sf cell lines to confirm and extend information on Sf-rhabdovirus variation. We then used our novel Sf-rhabdovirus-negative (Sf-RVN) Sf cell line to assess the infectivity of variants with and without a 320 bp X/L deletion and found both established productive persistent infections in Sf-RVN cells. We also assessed their infectivity using heterologous insect and mammalian cell lines and found neither established productive persistent infections in these cells. These results are the first to directly demonstrate Sf-rhabdoviruses are infectious for Sf cells, irrespective of the X/L deletion. They also confirm and extend previous results indicating Sf-rhabdoviruses have a narrow host range.
Full genomic characterization of California serogroup viruses, genus Orthobunyavirus, family Peribunyaviridae including phylogenetic relationships Virology (IF 3.353) Pub Date : 2017-10-03 Holly R. Hughes, Robert S. Lanciotti, Carol D. Blair, Amy J. Lambert
Thorough molecular characterization of reference viruses supports the detection of emerging human pathogens as well as studies of evolutionary relationships. However, full characterization of the tripartite RNA genomes of many viruses of the clinically important family Peribunyaviridae remains incomplete, making it difficult to identify emerging strains. Here, we report the full genome sequences of nine viruses belonging to the California serogroup and describe multi-segment analyses of these and previously published California serogroup strain data to determine the role of segment reassortment in the evolution of this serogroup. Phylogenetic trees from the small, medium, and large segments suggest long term, independent evolution of the majority of strains. However, trees from each segment were not entirely congruent and evidence of reassortment among some strains is presented. Of unique interest, the L segment phylogeny reveals divergent branching patterns for encephalitic versus non-encephalitic viruses in both major clades of the California serogroup.
Adverse fetal outcomes in pregnant rabbits experimentally infected with rabbit hepatitis E virus Virology (IF 3.353) Pub Date : 2017-10-02 Hee-Seop Ahn, Sang-Hoon Han, Yong-Hyun Kim, Byung-Joo Park, Dong-Hwi Kim, Joong-Bok Lee, Seung-Yong Park, Chang-Seon Song, Sang-Won Lee, Changsun Choi, Jinjong Myoung, In-Soo Choi
Hepatitis E virus (HEV) causes severe hepatitis in pregnant women, with associated poor fetal outcomes. To study HEV viral pathogenesis, pregnant rabbits were infected with low- and high-dose rabbit HEV at 2 weeks gestation. HEV was identified in the serum, feces, and liver tissue of infected rabbits, and dose-dependent fetal mortality rates ranging from 67% to 80% were observed. The aspartate transaminase (AST)/alanine transaminase ratio was significantly higher (P < 0.01) in high-dose infected rabbits than low-dose infected and negative control rabbits 14 days post infection (dpi). Tumor necrosis factor-α (TNF-α) was significantly higher in low-dose (P < 0.01) and high-dose infected rabbits (P < 0.001) than in negative controls 7 dpi. High-dose HEV-infected rabbits produced significantly more interferon-γ (IFN-γ; P < 0.05) than negative control rabbits at 7 and 14 dpi. High levels of AST, TNF-α, and IFN-γ may substantially influence adverse fetal outcomes in pregnant rabbits infected with high-dose HEV.
Antibody-based immunotherapy of aciclovir resistant ocular herpes simplex virus infections Virology (IF 3.353) Pub Date : 2017-10-03 Dirk Bauer, Jessica Keller, Mira Alt, Axel Schubert, Ulrich Wilhelm Aufderhorst, Vivien Palapys, Maren Kasper, Christiane Silke Heilingloh, Ulf Dittmer, Björn Laffer, Anna Maria Eis-Hübinger, Georges M. Verjans, Arnd Heiligenhaus, Michael Roggendorf, Adalbert Krawczyk
The increasing incidence of aciclovir- (ACV) resistant strains in patients with ocular herpes simplex virus (HSV) infections is a major health problem in industrialized countries. In the present study, the humanized monoclonal antibody (mAb) hu2c targeting the HSV-1/2 glycoprotein B was examined for its efficacy towards ACV-resistant infections of the eye in the mouse model of acute retinal necrosis (ARN). BALB/c mice were infected by microinjection of an ACV-resistant clinical isolate into the anterior eye chamber to induce ARN and systemically treated with mAb hu2c at 24 h prior (pre-exposure prophylaxis) or at 24, 40, and 56 h after infection (post-exposure immunotherapy). Mock treated controls and ACV-treated mice showed pronounced retinal damage. Mice treated with mAb hu2c were almost completely protected from developing ARN. In conclusion, mAb hu2c may become a reliable therapeutic option for drug/ACV-resistant ocular HSV infections in humans in order to prevent blindness.
Selective recruitment of nucleoporins on vaccinia virus factories and the role of Nup358 in viral infection Virology (IF 3.353) Pub Date : 2017-09-28 Deepak Khuperkar, Ashish Kamble, Aditi Singh, Arya Ghate, Renuka Nawadkar, Arvind Sahu, Jomon Joseph
Vaccinia virus (VACV), a member of the Poxviridae family, uses cytoplasmic factories for its replication. Recent studies indicated that VACV infection requires a set of nucleoporins. However, how the nucleoporins contribute to viral life cycle remains unclear. Here, we report that the nucleoporins Nup62 and Nup358 localize to the cytoplasmic viral factories (VFs). Nup358 was targeted to the VFs at 6 h post-infection (hpi), whereas Nup62, along with the previously reported translation factors such as eIF4E, eIF3η and G3BP1, was recruited to the VFs at 8 hpi. Nup358 depletion led to a decrease in the size and number of viral factories and reduction in viral yield. Further studies showed that Nup358 is involved in recruiting Nup62 and eIF4E to the VFs. Collectively, our results reveal spatio-temporal regulation in the recruitment of nucleoporins and translation factors to VFs, and particularly the importance of Nup358 in VACV infection.
Oxidation-sensitive polymersomes as vaccine nanocarriers enhance humoral responses against Lassa virus envelope glycoprotein Virology (IF 3.353) Pub Date : 2017-09-28 Clara Galan-Navarro, Marcela Rincon-Restrepo, Gert Zimmer, Erica Ollmann Saphire, Jeffrey A. Hubbell, Sachiko Hirosue, Melody A. Swartz, Stefan Kunz
Lassa virus (LASV) causes severe hemorrhagic fever with high mortality, yet no vaccine currently exists. Antibodies targeting viral attachment proteins are crucial for protection against many viral infections. However, the envelope glycoprotein (GP)−1 of LASV elicits weak antibody responses due to extensive glycan shielding. Here, we explored a novel vaccine strategy to enhance humoral immunity against LASV GP1. Using structural information, we designed a recombinant GP1 immunogen, and then encapsulated it into oxidation-sensitive polymersomes (PS) as nanocarriers that promote intracellular MHCII loading. Mice immunized with adjuvanted PS (LASV GP1) showed superior humoral responses than free LASV GP1, including antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. Together, these data demonstrate the potential of our nanocarrier vaccine platform for generating virus-specific antibodies against weakly immunogenic viral antigens.
Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells Virology (IF 3.353) Pub Date : 2017-09-28 Devi Rajan, Raghavan Chinnadurai, Evan O. Keefe, Seyhan Boyoglu-Barnum, Sean O. Todd, Tina V. Hartert, Jacques Galipeau, Larry J. Anderson
RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.
Adenovirus E1A TRRAP-targeting domain-mediated enhancement of MYC association with the NuA4 complex activates a panel of MYC target genes enriched for gene expression and ribosome biogenesis Virology (IF 3.353) Pub Date : 2017-09-28 Ling-Jun Zhao, Paul M. Loewenstein, Maurice Green
Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere Virology (IF 3.353) Pub Date : 2017-06-23 V.Gregory Chinchar, Thomas B. Waltzek, Kuttichantran Subramaniam
Members of the family Iridoviridae, collectively referred to as iridovirids, are large, double-stranded DNA-containing viruses that infect invertebrates and cold-blooded (ectothermic) vertebrates. Infections in the former often lead to massive levels of virus replication resulting in iridescence of the infected animal and ultimately death. Among the latter, infections target a variety of organs and are capable of causing high levels of morbidity and mortality among commercially and ecologically important fish and amphibian species. The viral replication strategy has been elucidated primarily through the study of frog virus 3 (FV3) with additional input from other iridovirids of ecological or commercial importance. Replication occurs within both nuclear and cytoplasmic compartments and involves synthesis of genome length and concatemeric DNA, extensive methylation of the viral genome (among vertebrate viruses only), coordinate expression of three classes of viral gene products, and formation of icosahedral virions within cytoplasmic viral assembly sites. Phylogenetic analyses delineate five genera within the family and suggest that members of the families Iridoviridae, Ascoviridae, and Marseilleviridae compromise a monophyletic lineage in which ascoviruses are most closely related to invertebrate iridoviruses.
From fish to frogs and beyond: Impact and host range of emergent ranaviruses Virology (IF 3.353) Pub Date : 2017-08-30 Stephen J. Price, Ellen Ariel, Alicia Maclaine, Gonçalo M. Rosa, Matthew J. Gray, Jesse L. Brunner, Trenton W.J. Garner
Ranaviruses are pathogens of ectothermic vertebrates, including amphibians. We reviewed patterns of host range and virulence of ranaviruses in the context of virus genotype and postulate that patterns reflect significant variation in the historical and current host range of three groups of Ranavirus: FV3-like, CMTV-like and ATV-like ranaviruses. Our synthesis supports previous hypotheses about host range and jumps: FV3s are amphibian specialists, while ATVs are predominantly fish specialists that switched once to caudate amphibians. The most recent common ancestor of CMTV-like ranaviruses and FV3-like forms appears to have infected amphibians but CMTV-like ranaviruses may circulate in both amphibian and fish communities independently. While these hypotheses are speculative, we hope that ongoing efforts to describe ranavirus genetics, increased surveillance of host species and targeted experimental assays of susceptibility to infection and/or disease will facilitate better tests of the importance of hypothetical evolutionary drivers of ranavirus virulence and host range.
Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor via modulating host inflammation response Virology (IF 3.353) Pub Date : 2017-07-06 Yepin Yu, Youhua Huang, Songwei Ni, Lingli Zhou, Jiaxin Liu, Jingcheng Zhang, Xin Zhang, Yin Hu, Xiaohong Huang, Qiwei Qin
Virus encoded tumor necrosis factor receptor (TNFR) homologues are usually involved in immune evasion by regulating host immune response or cell death. Singapore grouper iridovirus (SGIV) is a novel ranavirus which causes great economic losses in aquaculture industry. Previous studies demonstrated that SGIV VP51, a TNFR-like protein regulated apoptotic process in VP51 overexpression cells. Here, we developed a VP51-deleted recombinant virus Δ51-SGIV by replacing VP51 with puroR-GFP. Deletion of VP51 resulted in the decrease of SGIV virulence, evidenced by the reduced replication in vitro and the decreased cumulative mortalities in Δ51-SGIV challenged grouper compared to WT-SGIV. Moreover, VP51 deletion significantly increased virus induced apoptosis, and reduced the expression of pro-inflammatory cytokines in vitro. In addition, the expression of several pro-inflammatory genes were decreased in Δ51-SGIV infected grouper compared to WT-SGIV. Thus, we speculate that SGIV VP51 functions as a critical virulence factor via regulating host cell apoptosis and inflammation response.
Characterization of a PKR inhibitor from the pathogenic ranavirus, Ambystoma tigrinum virus, using a heterologous vaccinia virus system Virology (IF 3.353) Pub Date : 2017-09-14 Trung P. Huynh, James K. Jancovich, Latha Tripuraneni, Michael C. Heck, Jeffrey O. Langland, Bertram L. Jacobs
Ambystoma tigrinum virus (ATV) (family Iridoviridae, genus Ranavirus) was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi) from the San Rafael Valley in southern Arizona, USA in 1996. Genomic sequencing of ATV, as well as other members of the genus, identified an open reading frame that has homology to the eukaryotic translation initiation factor, eIF2α (ATV eIF2α homologue, vIF2αH). Therefore, we asked if the ATV vIF2αH could also inhibit PKR. To test this hypothesis, the ATV vIF2αH was cloned into vaccinia virus (VACV) in place of the well-characterized VACV PKR inhibitor, E3L. Recombinant VACV expressing ATV vIF2αH partially rescued deletion of the VACV E3L gene. Rescue coincided with rapid degradation of PKR in infected cells. These data suggest that the salamander virus, ATV, contains a novel gene that may counteract host defenses, and this gene product may be involved in the presentation of disease caused by this environmentally important pathogen.
The Ambystoma tigrinum virus (ATV) RNase III gene can modulate host PKR activation and interferon production Virology (IF 3.353) Pub Date : 2017-08-23 Alexander G. Allen, Scott Morgans, Eric Smith, Mariah M. Aron, James K. Jancovich
The iridovirus RNase III gene is one of 26 conserved core genes among the family Iridoviridae. Initial studies suggest this viral protein functions to suppress RNA interference pathways that may attack viral RNA during infection. Therefore, to determine if the Ambystoma tigrinum virus (ATV) RNase III-like gene (ORF 25R) can modulate the host innate immune response fish and human cells ectopically expressing 25R were treated with polyI:C and monitored for interferon synthesis and phosphorylation of eIF2α and PKR. We found a decrease in cellular IFN production and modulation of the PKR pathway. In addition, ATV deleted of the RNase III gene (ATVΔ25R) shows reduced pathogenicity in tiger salamanders. Collectively our data suggest that the ATV 25R protein is a pathogenesis factor that may function to help evade the host's immune response by masking activators of the IFN pathway.
Xenopus-FV3 host-pathogen interactions and immune evasion Virology (IF 3.353) Pub Date : 2017-06-16 Robert Jacques, Eva-Stina Edholm, Sanchez Jazz, Torres-Luquis Odalys, De Jesús Andino Francisco
We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene.
Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV) Virology (IF 3.353) Pub Date : 2017-08-14 Paul M. Hick, Kuttichantran Subramaniam, Patrick M. Thompson, Thomas B. Waltzek, Joy A. Becker, Richard J. Whittington
Low genetic diversity of Epizootic haematopoietic necrosis virus (EHNV) was determined for the complete genome of 16 isolates spanning the natural range of hosts, geography and time since the first outbreaks of disease. Genomes ranged from 125,591–127,487 nucleotides with 97.47% pairwise identity and 106–109 genes. All isolates shared 101 core genes with 121 potential genes predicted within the pan-genome of this collection. There was high conservation within 90,181 nucleotides of the core genes with isolates separated by average genetic distance of 3.43 × 10−4 substitutions per site. Evolutionary analysis of the core genome strongly supported historical epidemiological evidence of iatrogenic spread of EHNV to naïve hosts and establishment of endemic status in discrete ecological niches. There was no evidence of structural genome reorganization, however, the complement of non-core genes and variation in repeat elements enabled fine scale molecular epidemiological investigation of this unpredictable pathogen of fish.
Ranavirus phylogenomics: Signatures of recombination and inversions among bullfrog ranaculture isolates Virology (IF 3.353) Pub Date : 2017-08-10 Sieara C. Claytor, Kuttichantran Subramaniam, Nelmarie Landrau-Giovannetti, V. Gregory Chinchar, Matthew J. Gray, Debra L. Miller, Carla Mavian, Marco Salemi, Samantha Wisely, Thomas B. Waltzek
Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China. The later outbreak was due to a chimeric ranavirus that displayed a novel genome arrangement and a DNA backbone typical for Frog virus 3 (FV3) strains except for interspersed fragments acquired through recombination with the CMTV isolated earlier. Both bullfrog ranaviruses are more pathogenic than wild-type FV3 suggesting recombination may have resulted in the increased pathogenicity observed in the ranavirus isolated in the later outbreak. Our study underscores the role international trade in farmed bullfrogs may have played in the global dissemination of highly pathogenic ranaviruses.
Hairpin structures with conserved sequence motifs determine the 3′ ends of non-polyadenylated invertebrate iridovirus transcripts Virology (IF 3.353) Pub Date : 2017-07-11 İkbal Agah İnce, Gorben P. Pijlman, Just M. Vlak, Monique M. van Oers
Live baculovirus acts as a strong B and T cell adjuvant for monomeric and oligomeric protein antigens Virology (IF 3.353) Pub Date : 2017-08-24 Suvi Heinimäki, Kirsi Tamminen, Maria Malm, Timo Vesikari, Vesna Blazevic
Recombinant proteins produced by baculovirus (BV) expression systems contain residual BV after crude purification. We studied adjuvant effect of BV on antibody and T cell responses against two model antigens, monomeric ovalbumin (OVA) protein and oligomeric norovirus (NoV) virus-like particles (VLPs). BALB/c mice were immunized intradermally with OVA alone or OVA formulated with live or inactivated BV, and VLP formulations comprised of chromatographically purified NoV GII.4 VLPs alone or mixed with BV, or of crude purified VLPs containing BV impurities from expression system. Live BV improved immunogenicity of NoV VLPs, sparing VLP dose up to 10-fold. Moreover, soluble OVA protein induced IgG2a antibodies and T cell response only when co-administered with live BV. BV adjuvant effect was completely abrogated by removal or inactivation of BV. These findings support the usage of crude purified proteins containing residual BV as vaccine antigens.
Effects of simultaneously elevated temperature and CO2 levels on Nicotiana benthamiana and its infection by different positive-sense RNA viruses are cumulative and virus type-specific Virology (IF 3.353) Pub Date : 2017-09-29 Francisco J. del Toro, Farshad Rakhshandehroo, Beatriz Larruy, Emmanuel Aguilar, Francisco Tenllado, Tomás Canto
We have studied how simultaneously elevated temperature and CO2 levels [climate change-related conditions (CCC) of 30 °C, 970 parts-per-million (ppm) of CO2 vs. standard conditions (SC) of 25 °C, ~ 405 ppm CO2] affect physiochemical properties of Nicotiana benthamiana leaves, and also its infection by several positive-sense RNA viruses. In previous works we had studied effects of elevated temperature, CO2 levels separately. Under CCC, leaves of healthy plants almost doubled their area relative to SC but contained less protein/unit-of-area, similarly to what we had found under conditions of elevated CO2 alone. CCC also affected the sizes/numbers of different foliar cell types differently. Under CCC, infection outcomes in titers and symptoms were virus type-specific, broadly similar to those observed under elevated temperature alone. Under either condition, infections did not significantly alter the protein content of leaf discs. Therefore, effects of elevated temperature and CO2 combined on properties of the pathosystems studied were overall cumulative.
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