- Volume 95, Issue 2, 2014

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Moreover, the virus causes outbreaks in poultry and other animals, such as pigs, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy. Here, we review entry of this important pathogen into target cells, an essential process by which viral genomes are delivered from extracellular virions to sites of transcription/replication in the cell nucleus. We summarize current knowledge on the interaction of influenza viruses with their receptor, sialic acid, and highlight the ongoing search for additional receptors. We describe receptor-mediated endocytosis and the recently discovered macropinocytosis as alternative virus uptake pathways, and illustrate the subsequent endosomal trafficking of the virus with advanced live microscopy techniques. Release of virus from the endosome and import of the viral ribonucleoproteins into the host cell nucleus are also outlined. Although a focus has been on viral protein function during entry, recent studies have revealed exciting information on cellular factors required for influenza virus entry. We highlight these, and discuss established entry inhibitors targeting viral and host factors, as well as the latest prospects for designing novel ‘anti-entry’ compounds. New entry inhibitors are of particular importance for current efforts to develop the next generation of anti-influenza drugs – entry is the first essential step of virus replication and is an ideal target to block infection efficiently.

Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now accepted widely as a major cause of acute gastroenteritis in both developed and developing countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our knowledge of norovirus biology has improved significantly over the past decade as a result of numerous technological advances. The use of a HuNV replicon, improved biochemical and cell-based assays, combined with the discovery of a murine norovirus capable of replication in cell culture, has improved greatly our understanding of the molecular mechanisms of norovirus genome translation and replication, as well as the interaction with host cell processes. In this review, the current state of knowledge of the intracellular life of noroviruses is discussed with particular emphasis on the mechanisms of viral gene expression and viral genome replication.

The majority of infections, including those caused by respiratory syncytial virus (RSV), occur at mucosal surfaces. As no RSV vaccine is available our goal is to produce an effective subunit vaccine with an adjuvant suitable for mucosal delivery and cross-presentation. A truncated secreted version of the RSV fusion (ΔF) protein formulated with polyI : C, an innate defence regulator peptide and polyphosphazene, induced local and systemic immunity, including affinity maturation of RSV F-specific IgG, IgA and virus-neutralizing antibodies, and F-specific CD8+ T-cells in the lung, when delivered intranasally. Furthermore, this ΔF protein formulation promoted the production of CD8+ central memory T-cells in the mediastinal lymph nodes and provided protection from RSV challenge. Formulation of ΔF protein with this adjuvant combination enhanced uptake by lung dendritic cells and trafficking to the draining lymph nodes. The ΔF protein formulation was confirmed to be highly efficacious and safe in cotton rats.

Viral hemorrhagic septicaemia virus (VHSV) is an important viral pathogen in European rainbow trout farming. Isolates from wild marine fish and freshwater trout farms show highly different virulence profiles: isolates from marine fish species cause little or no mortality in rainbow trout following experimental waterborne challenge, whilst challenge with rainbow trout isolates results in high levels of mortality. Phylogenetic analyses have revealed that the highly virulent trout-derived isolates from freshwater farms have evolved from VHSV isolates from marine fish host species over the past 60 years. Recent isolates from rainbow trout reared in marine zones show intermediate virulence. The present study aimed to identify molecular virulence markers that could be used to classify VHSV isolates according to their ability to cause disease in rainbow trout. By a reverse genetics approach using a VHSV-related novirhabdovirus [infectious hematopoietic necrosis virus (IHNV)], four chimaeric IHNV–VHSV recombinant viruses were generated. These chimaeric viruses included substitution of the IHNV glyco- (G) or non-structural (Nv) protein with their counterparts from either a trout-derived or a marine VHSV strain. Comparative challenge experiments in rainbow trout fingerlings revealed similar levels of survival induced by the recombinant (r)IHNV–VHSV chimaeric viruses regardless of whether the G or Nv genes originated from VHSV isolated from a marine fish species or from rainbow trout. Interestingly, recombinant IHNV gained higher virulence following substitution of the G gene with those of the VHSV strains, whilst the opposite was the case following substitution of the Nv genes.

Recently, a number of broad-spectrum human antibodies binding to the stalk region of influenza A haemagglutinin (HA) have been isolated. As this region tends to develop substitutions at a slower rate than other regions of HA, a vaccine eliciting such antibodies could have a longer effective life. But this begs a question: is the stalk resistant to change even in the face of evolutionary pressure? In this paper, we analysed the known epitopes in the H3 stalk and, utilizing a collection of 3440 sequences, present a novel approach for detecting putative B-cell epitopes in regions such as this, in which mutations occur infrequently. We concluded that there have been periods of activity in the stalk that are consistent with the evolution of antigenic escape. This work casts light on the presence of stalk-binding antibodies in the population as a whole and, through the analysis of antigenically active regions in the stalk, may contribute to the identification of epitopes that are refractive to change and hence useful for vaccine development.

Rodents and shrews are known to harbour various viruses. Paramyxoviruses have been isolated from Asian and Australian rodents, but little is known about them in African rodents. Recently, previously unknown paramyxovirus sequences were found in South African rodents. To date, there have been no reports related to the presence and prevalence of paramyxoviruses in shrews. We found a high prevalence of paramyxoviruses in wild rodents and shrews from Zambia. Semi-nested reverse transcription-PCR assays were used to detect paramyxovirus RNA in 21 % (96/462) of specimens analysed. Phylogenetic analysis revealed that these viruses were novel paramyxoviruses and could be classified as morbillivirus- and henipavirus-related viruses, and previously identified rodent paramyxovirus-related viruses. Our findings suggest the circulation of previously unknown paramyxoviruses in African rodents and shrews, and provide new information regarding the geographical distribution and genetic diversity of paramyxoviruses.

We modified the haemagglutinin–neuraminidase (HN) glycoprotein of the virulent Newcastle disease virus (NDV) strain Banjarmasin/010/10 (Ban/010) by adding C-terminal extensions similar to those found in certain avirulent NDV strains. Extension of the 571 aa wt Ban/010 HN protein to 577 and 616 aa by removal of one or two translational stop codons moderately reduced HN function and viral pathogenicity in 1-day-old and 3-week-old chickens. Substantially greater reductions were achieved by altering the 616 aa form by introducing a R596C mutation or by replacing the C-terminal extension with that of avirulent strain Ulster, which naturally contains the amino acid 596C. These results showed that extension of the C terminus of HN reduces NDV pathogenicity, and that this effect is substantially increased by the presence of 596C. These results indicate that this attenuating mechanism in avirulent strains such as Ulster can be applied directly to a highly virulent strain recently in circulation.

The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.

Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1′ protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a −1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1′ protein through the production of JEV NS1′-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1′ protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1′ protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1′ enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r 1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

A total of 139 stool samples from wild chimpanzees, gorillas and bonobos in Cameroon and Democratic Republic of Congo (DRC) were screened for enteroviruses (EVs) by reverse transcription PCR. Enterovirus RNA was detected in 10 % of samples, comprising eight from 58 sampled chimpanzees (13.8 %), one from 40 bonobos (2.5 %) and five from 40 gorillas (12.2 %). Three viruses isolated from chimpanzees grouped with human isolate EV-A89 and four (four chimpanzees, one gorilla) represented a newly identified type, EV-A119. These species A virus types overlapped with those circulating in human populations in the same area. The remaining six strains comprised a new species D type, EV-D120, infecting one chimpanzee and four gorillas, and a single EV variant infecting a bonobo that was remarkably divergent from other EVs and potentially constitutes a new enterovirus species. The study demonstrates both the circulation of genetically divergent EV variants in apes and monkeys as well as those shared with local human populations.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging highly pathogenic virus causing almost 50 % lethality in infected individuals. The development of a small-animal model is critical for the understanding of this virus and to aid in development of countermeasures against MERS-CoV. We found that BALB/c, 129/SvEv and 129/SvEv STAT1 knockout mice are not permissive to MERS-CoV infection. The lack of infection may be due to the low level of mRNA and protein for the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4), in the lungs of mice. The low level of DPP4 in the lungs likely contributes to the lack of viral replication in these mouse models and suggests that a transgenic mouse model expressing DPP4 to higher levels is necessary to create a mouse model for MERS-CoV.

Subclinical infection of murine norovirus (MNV) was detected in a mixed breeding group of WT and Stat1 −/− mice with no outward evidence of morbidity or mortality. Investigations revealed the presence of an attenuated MNV variant that did not cause cytopathic effects in RAW264.7 cells or death in Stat1 −/− mice. Histopathological analysis of tissues from WT, heterozygous and Stat1 −/− mice revealed a surprising spectrum of lesions. An infectious molecular clone was derived directly from faeces (MNV-O7) and the sequence analysis confirmed it was a member of norovirus genogroup V. Experimental infection with MNV-O7 induced a subclinical infection with no weight loss in Stat1 −/− or WT mice, and recapitulated the clinical and pathological picture of the naturally infected colony. Unexpectedly, by day 54 post-infection, 50 % of Stat1 −/− mice had cleared MNV-O7. In contrast, all WT mice remained infected persistently. Most significantly, this was associated with liver lesions in all the subclinically infected WT mice. These data confirmed that long-term persistence in WT mice is established with specific variants of MNV and that despite a subclinical presentation, active foci of acute inflammation persist within the liver. The data also showed that STAT1-dependent responses are not required to protect mice from lethal infection with all strains of MNV.

Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×106 immunofluorescent focus-forming units ml−1, which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.