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Volume 96,
Issue 9,
2015
Volume 96, Issue 9, 2015
- Reviews
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Antiviral immunity in marine molluscs
More LessMarine molluscs, like all living organisms, are constantly exposed to viruses and have evolved efficient antiviral defences. We review here recent developments in molluscan antiviral immunity against viruses belonging to the order Herpesvirales. Emerging results suggest an interferon-like response and autophagy are involved in the antiviral defence of bivalves to viral infection. Multi-functional plasma proteins from gastropods and bivalves have been identified to have broad-spectrum antiviral activity against mammalian viruses. The antiviral defences present in molluscs can be enhanced by genetic selection, as shown by the presence of oyster strains specifically resistant to ostreid herpesvirus type 1. Whether varying amounts or different isoforms of these antiviral plasma proteins contributes to genetic resistance is worthy of further research. Other evolutionarily conserved antiviral mechanisms, such as RNA interference and apoptosis, still need further characterization.
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Alphavirus RNA synthesis and non-structural protein functions
More LessThe members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.
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- SGM Prize Lecture
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What's the host and what's the microbe? The Marjory Stephenson Prize Lecture 2015
More LessThe interchange between retroviruses and their hosts is an intimate one because retroviruses integrate proviral DNA into host chromosomal DNA as an obligate step in the replication cycle. This has resulted in the occasional transduction of host genes into retroviral genomes as oncogenes, and also led to the integration of viral genomes into the host germ line that gives rise to endogenous retroviruses. I shall reflect on the evolutionary consequences of these events for virus and host. Then, I shall discuss the emergence of non-viral infections of host origin, namely, how malignant cells can give rise to eukaryotic single cell ‘parasites’ that colonize new hosts and how these in turn have been colonized by host mitochondria.
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- Animal
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- Negative-strand RNA Viruses
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Replication and transmission of mammalian-adapted H9 subtype influenza virus in pigs and quail
Influenza A virus is a major pathogen of birds, swine and humans. Strains can jump between species in a process often requiring mutations and reassortment, resulting in outbreaks and, potentially, pandemics. H9N2 avian influenza is predominant in poultry across Asia and occasionally infects humans and swine. Pandemic H1N1 (H1N1pdm) is endemic in humans and swine and has a history of reassortment in pigs. Previous studies have shown the compatibility of H9N2 and H1N1pdm for reassortment in ferrets, a model for human infection and transmission. Here, the effects of ferret adaptation of H9 surface gene segments on the infectivity and transmission in at-risk natural hosts, specifically swine and quail, were analysed. Reassortant H9N1 and H9N2 viruses, carrying seven or six gene segments from H1N1pdm, showed infectivity and transmissibility in swine, unlike the wholly avian H9N2 virus with ferret-adapted surface genes. In quail, only the reassortant H9N2 with the six internal gene segments from the H1N1pdm strain was able to infect and transmit, although less efficiently than the wholly avian H9N2 virus with ferret-adapted surface genes. These results highlight that ferret-adapted mutations on the haemagglutinin of H9 subtype virus do not restrict the ability of the virus to infect swine and quail, and that the ability to transmit in these species depends on the context of the whole virus. As such, this study emphasizes the threat that H9N2 reassortant viruses pose to humans and agricultural species and the importance of the genetic constellation of the virus to its ability to replicate and transmit in natural hosts of influenza.
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H5N1 influenza A virus with K193E and G225E double mutations in haemagglutinin is attenuated and immunogenic in mice
Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin–Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.
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Preferential host switching and its relation with Hantavirus diversification in South America
More LessIn recent years, the notion of co-speciation between Hantavirus species and their hosts was discarded in favour of a more likely explanation: preferential host switching. However, the relative importance of this last process in shaping the evolutionary history of hantaviruses remains uncertain, given the present limited knowledge not only of virus–host relationships but also of the pathogen and reservoir phylogenies. In South America, more than 25 hantavirus genotypes were detected; several of them act as aetiological agents of hantavirus pulmonary syndrome (HPS). An understanding of the diversity of hantaviruses and of the processes underlying host switching is critical since human cases of HPS are almost exclusively the result of human–host interactions. In this study, we tested if preferential host switching is the main process driving hantavirus diversification in South America, by performing a co-phylogenetic analysis of the viruses and their primary hosts. We also suggest a new level of amino acid divergence to define virus species in the group. Our results indicate that preferential host switching would not be the main process driving virus diversification. The historical geographical proximity among rodent hosts emerges as an alternative hypothesis to be tested.
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CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells
Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.
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Sialic acid-dependent interactions between influenza viruses and Streptococcus suis affect the infection of porcine tracheal cells
More LessBacterial co-infections are a major complication in influenza-virus-induced disease in both humans and animals. Either of the pathogens may induce a host response that affects the infection by the other pathogen. A unique feature in the co-infection by swine influenza viruses (SIV) and Streptococcus suis serotype 2 is the direct interaction between the two pathogens. It is mediated by the haemagglutinin of SIV that recognizes the α2,6-linked sialic acid present in the capsular polysaccharide of Streptococcus suis. In the present study, this interaction was demonstrated for SIV of both H1N1 and H3N2 subtypes as well as for human influenza viruses that recognize α2,6-linked sialic acid. Binding of SIV to Streptococcus suis resulted in co-sedimentation of virus with bacteria during low-speed centrifugation. Viruses bound to bacteria retained infectivity but induced only tiny plaques compared with control virus. Infection of porcine tracheal cells by SIV facilitated adherence of Streptococcus suis, which was evident by co-staining of bacterial and viral antigen. Sialic-acid-dependent binding of Streptococcus suis was already detectable after incubation for 30 min. By contrast, bacterial co-infection had a negative effect on the replication of SIV as indicated by lower virus titres in the supernatant and a delay in the kinetics of virus release.
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Dynamics of virus shedding and antibody responses in influenza A virus-infected feral swine
Given their free-ranging habits, feral swine could serve as reservoirs or spatially dynamic ‘mixing vessels’ for influenza A virus (IAV). To better understand virus shedding patterns and antibody response dynamics in the context of IAV surveillance amongst feral swine, we used IAV of feral swine origin to perform infection experiments. The virus was highly infectious and transmissible in feral swine, and virus shedding patterns and antibody response dynamics were similar to those in domestic swine. In the virus-inoculated and sentinel groups, virus shedding lasted ≤ 6 and ≤ 9 days, respectively. Antibody titres in inoculated swine peaked at 1 : 840 on day 11 post-inoculation (p.i.), remained there until 21 days p.i. and dropped to < 1 : 220 at 42 days p.i. Genomic sequencing identified changes in wildtype (WT) viruses and isolates from sentinel swine, most notably an amino acid divergence in nucleoprotein position 473. Using data from cell culture as a benchmark, sensitivity and specificity of a matrix gene-based quantitative reverse transcription-PCR method using nasal swab samples for detection of IAV in feral swine were 78.9 and 78.1 %, respectively. Using data from haemagglutination inhibition assays as a benchmark, sensitivity and specificity of an ELISA for detection of IAV-specific antibody were 95.4 and 95.0 %, respectively. Serological surveillance from 2009 to 2014 showed that ∼7.58 % of feral swine in the USA were positive for IAV. Our findings confirm the susceptibility of IAV infection and the high transmission ability of IAV amongst feral swine, and also suggest the need for continued surveillance of IAVs in feral swine populations.
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Expression of Raf kinase inhibitor protein is downregulated in response to Newcastle disease virus infection to promote viral replication
Newcastle disease virus (NDV) causes a severe and economically significant disease affecting almost the entire poultry industry worldwide. However, factors that affect NDV replication in host cells are poorly understood. Raf kinase inhibitory protein (RKIP) is a physiological inhibitor of c-RAF kinase and NF-κB signalling, known for their functions in the control of immune response as well as tumour invasion and metastasis. In the present study, we investigated the consequences of overexpression of host RKIP during viral infection. We demonstrate that NDV infection represses RKIP expression thereby promoting virus replication. Experimental upregulation of RKIP in turn acts as a potential antiviral defence mechanism in host cells that restricts NDV replication by repressing the activation of Raf/MEK/ERK and IκBα/NF-κB signalling pathways. Our results not only extend the concept of linking NDV–host interactions, but also reveal RKIP as a new class of protein-kinase-inhibitor protein that affects NDV replication with therapeutic potential.
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PA-X is a virulence factor in avian H9N2 influenza virus
H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro- or antivirulence role of PA-X in influenza viruses is virus strain-dependent.
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- Positive-strand RNA Viruses
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Different functional sensitivity to mutation at intersubunit interfaces involved in consecutive stages of foot-and-mouth disease virus assembly
More LessSmall spherical viruses are paradigms of supramolecular self-assembly. Identifying the specific structural determinants for virus assembly provides guidelines to develop new antiviral drugs or engineer modified viral particles for medical or technological applications. However, very few systematic studies have been carried out so far to identify those chemical groups at interfaces between virus capsid subunits that are important for viral assembly and function. Foot-and-mouth disease virus (FMDV) and other picornaviruses are assembled in a stepwise process in which different protein–protein interfaces are formed: 5 protomeric subunits oligomerize to form a pentameric intermediate, and 12 of these stable pentameric building blocks associate to form a labile capsid. In this study, a systematic mutational analysis revealed that very few amino acid side chains involved in substantial interactions between protomers within each pentamer are individually required for virus infectivity. This result contrasts sharply with the previous finding that most amino acid side chains involved in interactions between pentamers during the next assembly step are individually required for infectivity. The dramatic difference in sensitivity to single mutations between the two types of protein–protein interfaces in FMDV is discussed in terms of possible structural strategies for achieving self-assembly and genome uncoating in the face of diverse selective constraints.
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A highly divergent picornavirus in an amphibian, the smooth newt (Lissotriton vulgaris)
More LessGenetically highly divergent picornavirus (Newt/2013/HUN, KP770140) was detected using viral metagenomics in faecal samples of free-living smooth newts (Lissotriton vulgaris). Newt picornavirus was identified by reverse transcription-polymerase chain reaction (RT-PCR) in six (25 %) of the 24 samples originating from individuals caught in two out of the six investigated natural ponds in Hungary. The first picornavirus in amphibians expands the host range of members of the Picornaviridae, and opens a new research field in picornavirus evolution in lower vertebrates. Newt picornavirus represents a novel species in a novel genus within the family Picornaviridae, provisionally named genus Ampivirus (amphibian picornavirus).
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Brunenders: a partially attenuated historic poliovirus type I vaccine strain
Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.
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Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence
Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE− vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE− -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE− replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.
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Assessment of cross-species transmission of hepatitis C virus-related non-primate hepacivirus in a population of humans at high risk of exposure
The recent discovery of hepatitis C virus (HCV)-related viruses in different animal species has raised new speculations regarding the origin of HCV and the possibility of a zoonotic source responsible for the endemic HCV transmission. As a consequence, these new findings prompt questions regarding the potential for cross-species transmissions of hepaciviruses. The closest relatives to HCV discovered to date are the non-primate hepaciviruses (NPHVs), which have been described to infect horses. To evaluate the risk of a potential zoonotic transmission, we analysed NPHV RNA and antibodies in humans with occupational exposure to horses in comparison with a low-risk group. Both groups were negative for NPHV RNA, even though low seroreactivities against various NPHV antigens could be detected irrespective of the group. In conclusion, we did not observe evidence of NPHV transmission between horses and humans.
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Arterivirus nsp12 versus the coronavirus nsp16 2′-O-methyltransferase: comparison of the C-terminal cleavage products of two nidovirus pp1ab polyproteins
The 3′-terminal domain of the most conserved ORF1b in three of the four families of the order Nidovirales (except for the family Arteriviridae) encodes a (putative) 2′-O-methyltransferase (2′-O-MTase), known as non structural protein (nsp) 16 in the family Coronaviridae and implicated in methylation of the 5′ cap structure of nidoviral mRNAs. As with coronavirus transcripts, arterivirus mRNAs are assumed to possess a 5′ cap although no candidate MTases have been identified thus far. To address this knowledge gap, we analysed the uncharacterized nsp12 of arteriviruses, which occupies the ORF1b position equivalent to that of the nidovirus 2′-O-MTase (coronavirus nsp16). In our in-depth bioinformatics analysis of nsp12, the protein was confirmed to be family specific whilst having diverged much further than other nidovirus ORF1b-encoded proteins, including those of the family Coronaviridae. Only one invariant and several partially conserved, predominantly aromatic residues were identified in nsp12, which may adopt a structure with alternating α-helices and β-strands, an organization also found in known MTases. However, no statistically significant similarity was found between nsp12 and the twofold larger coronavirus nsp16, nor could we detect MTase activity in biochemical assays using recombinant equine arteritis virus (EAV) nsp12. Our further analysis established that this subunit is essential for replication of this prototypic arterivirus. Using reverse genetics, we assessed the impact of 25 substitutions at 14 positions, yielding virus phenotypes ranging from WT-like to non-viable. Notably, replacement of the invariant phenylalanine 109 with tyrosine was lethal. We concluded that nsp12 plays an essential role during EAV replication, possibly by acting as a co-factor for another enzyme.
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Identification of the functional domain of the porcine epidemic diarrhoea virus receptor
More LessPorcine aminopeptidase N (pAPN) is a functional receptor for porcine epidemic diarrhoea virus (PEDV). Although PEDV is known to use the pAPN as the major receptor for cell entry, the crucial domain of the pAPN that interacts with the PEDV is still unknown. In the present study, in order to determine the crucial domain of the pAPN, the extracellular domain of pAPN was divided into three subdomains named SPA, SPB and SPC, based on its secondary structure. Recombinant plasmid pcDNA3.1 expressing SPA, SPB and SPC was constructed and introduced into Madin–Darby canine kidney (MDCK) cells by transfection. Following the detection of PEDV infection in transfected MDCK cells after PEDV challenge, we clearly demonstrated that the SPC subdomain plays a key role in cell entry of PEDV and its expression permits PEDV growth in transfected MDCK cells, while virus propagation can be inhibited by anti-SPC serum, indicating that the SPC subdomain appears to be a crucial functional domain in contributing to efficient PEDV infection.
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In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain
The characteristics of genotype V Japanese encephalitis virus (GV JEV) remain poorly understood as only two strains have been isolated to date. In this study, we examined the effects of the GV JEV Muar strain on in vitro growth and pathogenicity in mice; we also evaluated the efficacy of inactivated JEV vaccines against the Muar strain. Although growth of the Muar strain in mouse neuroblastoma N18 cells was clearly worse than that of the GIII Beijing-1 and GI Mie/41/2002 strains, neuroinvasiveness of the Muar strain was similar to that of the Beijing-1 strain and significantly higher than that of the Mie/41/2002 strain. The results of a plaque reduction neutralization test suggested that the neutralization ability of the JEV vaccines against the Muar strain was reduced compared with the GI and GIII strains. However, the protection potency of the JEV vaccine against the Muar strain was similar to that for the Beijing-1 strain in mice. Our data indicate that GV JEV has unique growth, virulence and antigenicity features.
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HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis
More LessWe previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.
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Volumes and issues
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Volume 106 (2025)
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