- Volume 96, Issue 11, 2015

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that establishes persistent infection in the nucleus. Although BDV forms viral inclusion bodies, termed viral speckles of transcripts (vSPOTs), which are associated with chromatin in the nucleus, the host factors involved in the maintenance of vSPOTs remain largely unknown. In this study, we identified X-linked RNA-binding motif protein (RBMX) as a nuclear factor interacting with BDV nucleoprotein. Interestingly, knockdown of RBMX led to disruption of the formation of vSPOTs and reduced both transcription and replication of BDV. Our results indicate that RBMX is involved in the maintenance of the structure of the virus factory in the nucleus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFκB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-β, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-β-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-β-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFN-stimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 (‘Qinghai’ lineage) in 2005.

NK-cells have traditionally been viewed as innate effector lymphocytes that serve as a first line of defence against a range of viruses and tumours. More recently, the importance of NK-cell immunoregulatory functions has been highlighted. NK-cells can inhibit antiviral T-cell responses, and also play an important role in controlling harmful T-cell activity in autoimmunity and transplantation settings. Moreover, immunopathological effects of NK-cells during infection have been reported. Nevertheless, the phenotype and function of NK-cells in the thymus during influenza virus infection is not understood. In the present study, we demonstrated that influenza A virus (IAV) infection in mice led to severe thymic atrophy caused by increased thymic T-cell apoptosis and suppressed proliferation.  We found that NK-cells played a critical role in this phenotype. IFN-γ production by NK-cells was a contributing factor for thymic atrophy during IAV infection. Taken together, our data indicate that NK-cells are involved in the thymic atrophy associated with IAV infection.

Chronic infection of the liver by hepatitis C virus (HCV) induces a range of host factors including IFN-stimulated genes such as ISG15. ISG15 functions as an antiviral factor that limits virus replication. Previous studies have suggested that ISG15 could influence HCV replication in both a positive and a negative manner. In this report, we determined the effect of ISG15 on HCV RNA replication in two independent cell lines that support viral genome synthesis by inhibiting ISG15 expression through small interfering RNA, short-hairpin RNA and CRISPR/Cas9 gene knockout approaches. Our results demonstrated that ISG15 impairs HCV RNA replication in both the presence and absence of IFN stimulation, consistent with an antiviral role for ISG15 during HCV infection. ISG15 conjugation to protein substrates typically requires the E3 ligase, HERC5. Our results showed that the inhibitory effect of ISG15 on HCV RNA replication does not require its conjugation to substrates by HERC5.

Chikungunya virus (CHIKV), an alphavirus of the family Togaviridae, causes fever, polyarthritis and rash. There are three genotypes: West African, Asian and East/Central/South African (ECSA). The latter two genotypes have caused global outbreaks in recent years. Recent ECSA CHIKV outbreaks have been associated with severe neurological disease, but it is not known if different CHIKV genotypes are associated with different neurovirulence. In this study, the neurovirulence of Asian (MY/06/37348) and ECSA (MY/08/065) strains of CHIKV isolated in Malaysia were compared. Intracerebral inoculation of either virus into suckling mice was followed by virus titration, histopathology and gene expression analysis of the harvested brains. Both strains of CHIKV replicated similarly, yet mice infected with MY/06/37348 showed higher mortality. Histopathology findings showed that both CHIKV strains spread within the brain (where CHIKV antigen was localized to astrocytes and neurons) and beyond to skeletal muscle. In MY/06/37348-infected mice, apoptosis, which is associated with neurovirulence in alphaviruses, was observed earlier in brains. Comparison of gene expression showed that a pro-apoptotic gene (eIF2αK2) was upregulated at higher levels in MY/06/37348-infected mice, while genes involved in anti-apoptosis (BIRC3), antiviral responses and central nervous system protection (including CD40, IL-10RA, MyD88 and PYCARD) were upregulated more highly in MY/08/065-infected mice. In conclusion, the higher mortality observed following MY/06/37348 infection in mice is due not to higher viral replication in the brain, but to differentially expressed genes involved in host immune responses. These findings may help to identify therapeutic strategies and biomarkers for neurological CHIKV infections.

Infection with hepatitis E virus (HEV) can be clinically inapparent or produce symptoms and signs of hepatitis of varying severity and occasional fatality. This variability in clinical outcomes may reflect differences in host susceptibility or the presence of virally encoded determinants of pathogenicity. Analysis of complete genome sequences supports the division of HEV genotype 3 (HEV-3) variants into three major clades: 3ra comprising HEV isolates from rabbits, and 3efg and 3abchij comprising the corresponding named subtypes derived from humans and pigs. Using this framework, we investigated associations between viral genetic variability of HEV-3 in symptomatic and asymptomatic infections by comparing HEV-3 subgenomic sequences previously obtained from blood donors with those from patients presenting with hepatitis in the UK (54 blood donors, 148 hepatitis patients), the Netherlands (38 blood donors, 119 hepatitis patients), France (24 blood donors, 55 hepatitis patients) and Germany (14 blood donors, 36 hepatitis patients). In none of these countries was evidence found for a significant association between virus variants and patient group (P>0.05 Fisher's exact test). Furthermore, within a group of 123 patients in Scotland with clinically apparent HEV infections, we found no evidence for an association between variants of HEV-3 and disease severity or alanine aminotransferase level. The lack of detectable virally encoded determinants of disease outcomes in HEV-3 infection implies a more important role for host factors in its clinical phenotype.

Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50–59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages.

Ultra-deep pyrosequencing (UDPS) was used to analyse the dynamics of quasispecies and resistant mutations during telbivudine (LDT) treatment of hepatitis B patients. Twenty-six HBeAg-positive chronic hepatitis B patients were treated with LDT for a period of 104 weeks and were characterized as 16 responders, six partial responders and four viral breakthrough patients based on hepatitis B virus (HBV) DNA levels. The plasma samples were subjected to UDPS of the reverse transcriptase (RT) region of HBV. Mutations rtM204I, rtL80I and rtL80V were detected in at least three of the four viral breakthrough patients, indicating the significant roles of the mutations in resistance to LDT. The degree of complexity of viral quasispecies remained in a steady state in the absence of selection pressure, but increased after the LDT treatment. The complexity in the responder group at week 12 was significantly higher than that in the group comprising partial responders and viral breakthrough patients. In vitro replication efficiency analyses showed that the RT mutations had different impacts on HBV replication, with a tendency of rtM204I>rtL80V>rtL80I. Furthermore, double mutations rtL80I/M204I and rtL80V/M204V had replication efficiency similar to that of rtL80I and rtL80V, respectively. Consistent with previous studies, mutation rtM204I was found to be highly resistant to LDT. However, in contrast with their sensitivity to lamivudine, rtL80I and rtL80V were moderately resistant to LDT. Our results indicated that rtL80I and rtL80V may not only serve as replication complementary mutations to rtM204I, but also directly contribute to the LDT resistance.

Herpes simplex virus type 1 (HSV-1) glycoprotein M (gM/UL10) is a 473 aa type III transmembrane protein that resides in various membrane compartments. HSV-1 gM contains several putative trafficking motifs, but their functional relevance remains to be elucidated. We show here that transiently expressed gM 19–343 was sufficient for transport to the trans-Golgi network (TGN), whilst gM 133–473, where the first two transmembrane domains were deleted, and gM 1–342, which lacked the final residue of the last transmembrane domain, were retained in the endoplasmic reticulum (ER), indicating that all transmembrane domains are required for proper folding and ER exit. A series of bacterial artificial chromosome mutants revealed that in addition to the authentic start codon, translation of gM can be initiated at methionine 19 and 133/135. Whilst a protein lacking the first 18 residues supported WT-like growth, gM 133/135–473 resulted in reduced plaque diameters resembling a UL10 deletion mutant. An HSV-1 mutant encoding gM 1–342 showed similar growth characteristics and accumulated non-enveloped cytoplasmic particles, whilst gM 1–343 resulted in a gain of function, indicating that all transmembrane domains of the protein are important for viral growth. A C-terminal extension further supported viral propagation; however, the C-terminal trafficking motifs (residues 423–473) were completely dispensable. We propose a functional core within gM 19–343 comprised of all transmembrane domains that is sufficient to target the protein to the TGN, a favoured site for envelopment, and to support viral functions.

Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

Singapore grouper iridovirus (SGIV) is a major viral pathogen of grouper aquaculture, and has caused heavy economic losses in China and South-east Asia. In this study, we generated four ssDNA aptamers against SGIV-infected grouper spleen (GS) cells using SELEX (systematic evolution of ligands by exponential enrichment) technology. Four aptamers exhibited high affinity to SGIV-infected GS cells, in particular the Q2 aptamer. Q2 had a binding affinity of 12.09 nM, the highest of the four aptamers. These aptamers also recognized SGIV-infected tissues with high levels of specificity. Protease treatment and flow cytometry analysis of SGIV-infected cells revealed that the target molecules of the Q3, Q4 and Q5 aptamers were trypsin-sensitive proteins, whilst the target molecules of Q2 might be membrane lipids or surface proteins that were not trypsin-sensitive. The generated aptamers appeared to inhibit SGIV infection in vitro. Aptamer Q2 conferred the highest levels of protection against SGIV and was able to inhibit SGIV infection in a dose-dependent manner. In addition, Q2 was efficiently internalized by SGIV-infected GS cells and localized at the viral assembly sites. Our results demonstrated that the four novel aptamers we generated were specific for SGIV-infected cells and could potentially be applied as rapid molecular diagnostic test reagents or therapeutic drugs targeting SGIV.

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces a fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle, and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+T cells are unknown. Many γ-HVs express microRNAs (miRNAs). These small non-coding RNAs can regulate expression of host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. The AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, using cloning and sequencing of small RNAs we identified 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and quantitative reverse transcription PCR in lymphoid organs of MCF-developing calves or rabbits. To determine the concerted contribution in MCF of 28 viral miRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro or MCF induction in rabbits, indicating that the AlHV-1 miRNAs clustered in this non-protein-coding genomic region are dispensable for MCF induction.

Metabolomics analysis of biofluids is increasingly being recognized as a useful tool for the diagnosis and management of a number of infectious diseases. Here we showed that plasma metabolomics profiling by untargeted 1H nuclear magnetic resonance may allow the anticipation of the occurrence of cytomegalovirus (CMV) DNAemia in allogeneic stem cell transplant. For this purpose, key discriminatory metabolites were total glutathione, taurine, methylamine, trimethylamine N-oxide and lactate, all of which were upregulated in patients eventually developing CMV DNAemia. The overall classification accuracy (predictability) of the projection to latent structure discriminant analysis (PLS-DA) model in cross-validation technical replicates was 73 %. Increased levels of alanine, lactate and total fatty acids, and a shift in the fatty acid profile towards unsaturated species, were observed in patients with detectable CMV DNA in plasma. The classification accuracy of this PLS-DA model in cross-validation technical replicates was 81 %. Plasma metabolomics profiling may prove useful for identifying patients at highest risk for CMV DNAemia thus allowing early inception of antiviral therapy.

Reverse transcription (RT) is one of the hallmark features of retroviruses. During RT, virus-encoded reverse transcriptase (RTase) must transfer from one end to the other end of the viral genome on two separate occasions to complete RT and move on to the production of proviral DNA. In addition, multiple strand-transfer events between homologous regions of the dimerized viral genome by RTase are also observed, and such recombination events serve as one of the driving forces behind human immunodeficiency virus (HIV) genome sequence diversity. Although retroviral recombination is widely considered to be important, several features of its mechanism are still unclear. We constructed an HIV-1 vector system to examine the target sequences required for virus recombination, and elucidated other necessary prerequisites to harbour recombination, such as the length, homology and the stability of neighbouring structures around the target sequences.