Avian bornaviruses (ABVs) are a group of genetically diverse viruses within the Bornaviridae family that can infect numerous avian species and represent the causative agents of proventricular dilatation disease, an often fatal disease that is widely distributed in captive populations of parrots and related species. The current study was designed to assess the antigenic variability of the family Bornaviridae and to determine its impact on ABV diagnosis by employing fluorescent antibody assays. It was shown that polyclonal rabbit sera directed against recombinant bornavirus nucleoprotein, X protein, phosphoprotein, and matrix protein provided sufficient cross-reactivity for the detection of viral antigen from a broad range of bornavirus genotypes grown in cell culture. In contrast, a rabbit anti-glycoprotein serum and 2 monoclonal antibodies directed against nucleoprotein and phosphoprotein proteins reacted more specifically. Antibodies were readily detected in sera from avian patients infected with known ABV genotypes if cells persistently infected with a variety of different bornavirus genotypes were used for analysis. For all sera, calculated antibody titers were highest when the homologous or a closely related target virus was used for the assay. Cross-reactivity with more distantly related genotypes of other phylogenetic groups was usually reduced, resulting in titer reduction of up to 3 log units. The presented results contribute to a better understanding of the antigenic diversity of family Bornaviridae and further emphasize the importance of choosing appropriate diagnostic tools for sensitive detection of ABV infections.
Several studies indicated that TLR as well as retinoic acid-inducible gene I-like helicase (RLH) signaling contribute to vesicular stomatitis virus (VSV)-mediated triggering of type I IFN (IFN-I) responses. Nevertheless, TLR-deficient MyD88(-/-)Trif(-/-) mice and RLH-deficient caspase activation and recruitment domain adaptor inducing IFN-? (Cardif)(-/-) mice showed only marginally enhanced susceptibility to lethal VSV i.v. infection. Therefore, we addressed whether concomitant TLR and RLH signaling, or some other additional mechanism, played a role. To this end, we generated MyD88(-/-)Trif(-/-)Cardif(-/-) (MyTrCa(-/-)) mice that succumbed to low-dose i.v. VSV infection with similar kinetics as IFN-I receptor-deficient mice. Three independent approaches (i.e., analysis of IFN-?/? serum levels, experiments with IFN-? reporter mice, and investigation of local IFN-stimulated gene induction) revealed that MyTrCa(-/-) mice did not mount IFN-I responses following VSV infection. Of note, treatment with rIFN-? protected the animals, qualifying MyTrCa(-/-) mice as a model to study the contribution of different immune cell subsets to the production of antiviral IFN-I. Upon adoptive transfer of wild-type plasmacytoid dendritic cells and subsequent VSV infection, MyTrCa(-/-) mice displayed significantly reduced viral loads in peripheral organs and showed prolonged survival. On the contrary, adoptive transfer of wild-type myeloid dendritic cells did not have such effects. Analysis of bone marrow chimeric mice revealed that TLR and RLH signaling of radioresistant and radiosensitive cells was required for efficient protection. Thus, upon VSV infection, plasmacytoid dendritic cell-derived IFN-I primarily protects peripheral organs, whereas concomitant TLR and RLH signaling of radioresistant stroma cells as well as of radiosensitive immune cells is needed to effectively protect against lethal disease.
The transcription factor STAT1 is essential for interferon (IFN)-mediated immunity in humans and mice. STAT1 function is tightly regulated, and both loss- and gain-of-function mutations result in severe immune diseases. The two alternatively spliced isoforms, STAT1? and STAT1?, differ with regard to a C-terminal transactivation domain, which is absent in STAT1?. STAT1? is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1?. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1? or STAT1?. As expected, the functions of STAT1? and STAT1? in IFN-?/?- and IFN-?-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1? is transcriptionally active in response to IFN-?. In the absence of STAT1?, STAT1? shows more prolonged IFN-?-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-?-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiencies. Our data shed new light on the potential contributions of the individual STAT1 isoforms to STAT1-dependent immune responses. Knowledge of STAT1?'s function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity.
Avian bornaviruses (ABV) are the causative agents of proventricular dilatation disease (PDD), a widely distributed disease of parrots. Distinct ABV lineages were also found in various non-psittacine avian species, such as canaries, but the pathogenic role of ABV in these species is less clear. Despite the wide distribution of ABV in captive parrots and canaries, its mode of transmission is poorly understood: both horizontal transmission via the urofaecal-oral route and vertical transmission are discussed to play a role. In this study we investigated pathology and horizontal transmission of ABV in domestic canaries (Serinus canaria forma domestica) and cockatiels (Nymphicus hollandicus), two natural host species commonly used for experimental ABV infections. ABV inoculation resulted in persistent infection of all inoculated animals from both species. ABV-infected cockatiels exhibited PDD-like symptoms, such as neurologic signs or shedding of undigested seeds. In contrast, infected domestic canaries did not develop clinical disease. Interestingly, we did not detect viral RNA in cloacal swabs and organ samples or ABV-specific antibodies in serum samples of contact-exposed sentinel birds from either species at any time during a four months observation period. Our results strongly indicate that horizontal transmission of ABV by direct contact is inefficient in immunocompetent fully fledged domestic canaries and cockatiels.
Avian bornaviruses (ABV) are known to be the causative agent of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). A broad range of ABV genotypes has been detected not only in psittacine birds, but also in other avian species including canary birds (Serinus canaria forma domestica) and Bengalese finches (Lonchura striata f. dom.), which are both members of the order songbirds (Passeriformes). During this study 286 samples collected from captive and wild birds of various passerine species in different parts of Germany were screened for the presence of ABV. Interestingly, only three ABV-positive samples were identified by RT-PCR. They originated from one yellow-winged pytilia (Pytilia hypogrammica) and two black-rumped waxbills (Estrilda troglodytes) from a flock of captive estrildid finches in Saxony. The ABV isolates detected here were only distantly related to ABV isolates found in passerine species in Germany and Japan and form a new genotype tentatively called ABV-EF (for "estrildid finches").
The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-?, IFN-?, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-? agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-?-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.
The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-?) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-?. In contrast, the IFN-? response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-? response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-?. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-? response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-?. The lack of mouse hepatocyte response to IFN-? was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-?. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-? in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-? in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment.
Interferons (IFNs) are essential components of the antiviral defense system of vertebrates. In mammals, functional receptors for type III IFN (IFN-?) are mainly found on epithelial cells and IFN-? was demonstrated to play a crucial role in limiting viral infections of mucosal surfaces. To determine whether IFN-? plays a similar role in birds, we produced recombinant chicken IFN-? (chIFN-?) and we used the replication-competent retroviral RCAS vector system to generate mosaic-transgenic chicken embryos that constitutively express chIFN-?. We could demonstrate that chIFN-? markedly inhibited replication of various virus strains, including highly pathogenic influenza A viruses, in ovo and in vivo, as well as in epithelium-rich tissue and cell culture systems. In contrast, chicken fibroblasts responded poorly to chIFN-?. When applied in vivo to three-week-old chickens, recombinant chIFN-? strongly induced the IFN-responsive Mx gene in epithelium-rich organs such as lung, trachea and intestinal tract. Correspondingly, these organs were found to express high transcript levels of the putative chIFN-? receptor alpha chain (chIL28RA) gene. Transfection of chicken fibroblasts with a chIL28RA expression construct rendered these cells responsive to chIFN-? treatment, indicating that receptor expression determines cell type specificity of IFN-? action in chickens. Surprisingly, mosaic-transgenic chickens perished soon after hatching, demonstrating a detrimental effect of constitutive chIFN-? expression. Our data highlight fundamental similarities between the IFN-? systems of mammals and birds, and suggest that type III IFN might play a role in defending mucosal surfaces against viral intruders in most if not all vertebrates.
Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFN?, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called "priming loop", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28R?(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.
The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.
Influenza A viruses circulating in humans from ?1950 to ?1987 featured a nonstructural (NS1) protein with a C-terminal extension of seven amino acids. The biological significance of this NS1 elongation remained elusive. We observed that replication kinetics of the wild-type virus A/Hong Kong/01/68 (H3N2) and a mutant encoding a truncated NS1 were indistinguishable in most experimental systems. However, wild-type virus outcompeted the mutant during mixed infections, suggesting that the NS1 extension conferred minor growth advantages.
We established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly.
The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses or bacteria. Nanoparticles (NPs) are considered an efficient tool for inducing potent immune responses. In this study, we describe a novel vaccination approach with biodegradable calcium phosphate (CaP) NPs that serve as carrier of immunoactive TLR9 ligand (CpG) combined with a viral Ag from the influenza A virus hemagglutinin. Functionalized CaP NPs were efficiently taken up by dendritic cells in vivo and elicited a potent T cell-mediated immune response in immunized mice with high numbers of IFN-?-producing CD4(+) and CD8(+) effector T cells. Most importantly, both i.p. and intranasal immunization with these NPs offered protection in a mouse model of influenza virus infection. This study demonstrates the great potential of CaP NPs as a novel vaccination tool that offers substantial flexibility for several infection models.
The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-?-coding region. IFN-?-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-? responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c(+) cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-? at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.
Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.
Avian bornavirus (ABV) was identified in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. In addition, ABV variants were detected in wild waterfowl and in a canary bird. PDD-like diseases were also reported in various other avian species, but it remains unknown whether ABV is involved. In this study we detected ABV in 12 of 30 tested canary bird flocks (40%), indicating a wide distribution of ABV in captive canary birds in Germany. Sequence analysis identified several distinct ABV genotypes which differ markedly from the genotypes present in psittacine birds. Some canaries naturally infected with ABV exhibited gastrointestinal and neurological symptoms which resembled PDD in psittacines, while others did not show signs of disease. Canaries experimentally inoculated with ABV developed infections of the brain and various other organs. The experimentally infected canaries transmitted the virus to sentinel birds kept in the same aviary, but did not show any clinical signs during a five month observation period. Embryonated eggs originating from ABV-infected hens contained ABV-specific RNA, but virus could not be re-isolated from embryonic tissue. These results indicate that ABV is widely distributed in canary birds and due to its association to clinical signs should be considered as a potential pathogen of this species.
Types I and III interferons (IFNs) elicit protective antiviral immune responses during influenza virus infection. Although many cell types can synthesize IFN in response to virus infection, it remains unclear which IFN sources contribute to antiviral protection in vivo. We found that mice carrying functional alleles of the Mx1 influenza virus resistance gene partially lost resistance to infection with a highly pathogenic H7N7 influenza A virus strain if Toll-like receptor 7 (TLR7) signalling was compromised. This effect was achieved by deleting either the TLR7 gene or the gene encoding the TLR7 adaptor molecule MyD88. A similar decrease of influenza virus resistance was observed when animals were deprived of plasmacytoid dendritic cells (pDCs) at day 1 post-infection. Our results provide in vivo proof that pDCs contribute to the protection of the lung against influenza A virus infections, presumably via signals from TLR7.
The type I interferon (IFN) system plays an important role in antiviral defense against influenza A viruses (FLUAV), which are natural chicken pathogens. Studies of mice identified the Mx1 protein as a key effector molecule of the IFN-induced antiviral state against FLUAV. Chicken Mx genes are highly polymorphic, and recent studies suggested that an Asn/Ser polymorphism at amino acid position 631 determines the antiviral activity of the chicken Mx protein. By employing chicken embryo fibroblasts with defined Mx-631 polymorphisms and retroviral vectors for the expression of Mx isoforms in chicken cells and embryonated eggs, we show here that neither the 631Asn nor the 631Ser variant of chicken Mx was able to confer antiviral protection against several lowly and highly pathogenic FLUAV strains. Using a short interfering RNA (siRNA)-mediated knockdown approach, we noted that the antiviral effect of type I IFN in chicken cells was not dependent on Mx, suggesting that some other IFN-induced factors must contribute to the inhibition of FLUAV in chicken cells. Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.
From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-?) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.
Type I and type III IFNs bind to different cell-surface receptors but induce identical signal transduction pathways, leading to the expression of antiviral host effector molecules. Despite the fact that type III IFN (IFN-?) has been shown to predominantly act on mucosal organs, in vivo infection studies have failed to attribute a specific, nonredundant function. Instead, a predominant role of type I IFN was observed, which was explained by the ubiquitous expression of the type I IFN receptor. Here we comparatively analyzed the role of functional IFN-? and type I IFN receptor signaling in the innate immune response to intestinal rotavirus infection in vivo, and determined viral replication and antiviral gene expression on the cellular level. We observed that both suckling and adult mice lacking functional receptors for IFN-? were impaired in the control of oral rotavirus infection, whereas animals lacking functional receptors for type I IFN were similar to wild-type mice. Using Mx1 protein accumulation as marker for IFN responsiveness of individual cells, we demonstrate that intestinal epithelial cells, which are the prime target cells of rotavirus, strongly responded to IFN-? but only marginally to type I IFN in vivo. Systemic treatment of suckling mice with IFN-? repressed rotavirus replication in the gut, whereas treatment with type I IFN was not effective. These results are unique in identifying a critical role of IFN-? in the epithelial antiviral host defense.
The terminal structures of the Borna disease virus (BDV) genome (vRNA) and antigenome (cRNA) differ from those of other negative strand RNA viruses, as both molecules possess four nucleotides at the 3 terminus without an apparent template at the 5 end of the opposite strand. Consequently, the v- and cRNA molecules are not perfect mirror images, a situation that is not compatible with conventional strategies to maintain genetic information. We show here that recombinant viruses recovered from cDNA lacking the nontemplated nucleotides efficiently reconstitute the 3 overhangs. Analyses of recombinant viruses encoding genetic markers in potential alternative template sequences demonstrated that the BDV v- and cRNA molecules are extended by a realign-and-elongation process on internal template motifs located in close proximity to the 3 ends of v- and cRNA, respectively. The data further suggest that cRNA elongation is restricted to a single template motif of the nascent strand, whereas elongation of vRNA might use multiple template motifs. We propose that the elongation of the 3 termini supports the terminal integrity of the genomic RNA molecules during BDV persistence, and furthermore provides an elegant strategy to eliminate the triphosphate groups from the 5 termini of the BDV v- and cRNA without compromising the genetic information of the virus.
Infection of mice with pneumonia virus of mice (PVM) provides a convenient experimental pathogenesis model in a natural host for a human respiratory syncytial virus-related virus. Extending our previous work showing that the PVM nonstructural (NS) proteins were pathogenicity factors in mice, we identify both the NS1 and NS2 proteins as antagonists of alpha/beta interferon (IFN-?/?) and IFN-? by use of recombinant PVM (rPVM) with single and combined deletions of the NS proteins (?NS1, ?NS2, and ?NS1 ?NS2). Wild-type and NS deletion PVMs were evaluated for growth and pathogenesis by infecting knockout mice that lack functional receptors to IFN-?/?, IFN-?, or both. The absence of the receptor to IFN-?/? (IFNAR) or IFN-? (interleukin-28 receptor ? chain [IL-28R?]) individually did not reverse the attenuated virulence of the NS deletion viruses although loss of IFNAR partially restored replication efficiency. When both receptors were deleted, replication and virulence were largely rescued for rPVM ?NS1 and were significantly but not completely rescued for rPVM ?NS2. As for rPVM ?NS1 ?NS2, the effect was mostly limited to partial enhancement of replication. This indicates that both IFN-?/? and IFN-? contributed to restricting the NS deletion viruses, with the former playing the greater role. Interestingly, the replication and virulence of wild-type PVM were completely unaffected by the presence or absence of functional receptors to IFN-?/? and IFN-?, indicating that both systems are strongly suppressed during infection. However, pretreatment of mice with IFN-?/? was protective against lethal rPVM challenge, whereas pretreatment with IFN-? delayed but did not prevent disease and, in some cases, reduced mortality. The fact that virulence of rPVM lacking NS2 was not recovered completely when both interferon receptors were deleted suggests that NS2 may have further functions outside the IFN system.
We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.
Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.
The recently discovered type III interferons (IFNs), also known as IFN-lambda, are part of the early innate immune response against viral infections. The IFN-lambda system closely resembles the type I IFN (IFN-alpha/beta) system in terms of expression after virus infection as well as intracellular signaling and activation of antiviral host factors in susceptible cells. However, in contrast to type I IFN, which signals through a universally expressed cell surface receptor, IFN-lambda uses a distinct receptor complex (IL28R) for signaling, which is expressed on a limited range of cell types. Until recently both the contribution of type III IFN to antiviral resistance as well as the exact nature of IL28R-expressing cells in vivo remained elusive. In this review we discuss data obtained from the experiments with IL28Ralpha(0/0) mice that demonstrated the role of IFN-lambda in viral defense in vivo. We further discuss the experiments that identified the cell types in various organs that express functional IFN-lambda receptors.
Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.
Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-alpha, IFN-beta, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-lambda) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-lambda plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-lambda receptor defect. Careful analysis revealed that expression of functional IFN-lambda receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-lambda contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.
Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.
Thanks to new technologies which enable rapid and unbiased screening for viral nucleic acids in clinical specimens, an impressive number of previously unknown viruses have recently been discovered. Two research groups independently identified a novel negative-strand RNA virus, now designated avian bornavirus (ABV), in parrots with proventricular dilatation disease (PDD), a severe lymphoplasmacytic ganglioneuritis of the gastrointestinal tract of psittacine birds that is frequently accompanied by encephalomyelitis. Since its discovery, ABV has been detected worldwide in many captive parrots and in one canary with PDD. ABV induced a PDD-like disease in experimentally infected cockatiels, strongly suggesting that ABV is highly pathogenic in psittacine birds. Until the discovery of ABV, the Bornaviridae family consisted of a single species, classical Borna disease virus (BDV), which is the causative agent of a progressive neurological disorder that affects primarily horses, sheep, and some other farm animals in central Europe. Although ABV and BDV share many biological features, there exist several interesting differences, which are discussed in this review.
Like other pathogens that readily persist in animal hosts, members of the Bornaviridae family have evolved effective mechanisms to evade the innate immune response. The prototype of this virus family, Borna disease virus employs an unusual replication strategy that removes the triphosphates from the 5 termini of the viral RNA genome. This strategy allows the virus to avoid activation of RIG-I and other innate immune response receptors in infected cells. Here we determined whether the newly discovered avian bornaviruses (ABV) might use a similar strategy to evade the interferon response. We found that de novo infection of QM7 and CEC32 quail cells with two different ABV strains was efficiently inhibited by exogenous chicken IFN-?. IFN-? also reduced the viral load in QM7 and CEC32 cells persistently infected with both ABV strains, suggesting that ABV is highly sensitive to type I IFN. Although quail cells persistently infected with ABV contained high levels of viral RNA, the supernatants of infected cultures did not contain detectable levels of biologically active type I IFN. RNA from cells infected with ABV failed to induce IFN-? synthesis if transfected into human cells. Furthermore, genomic RNA of ABV was susceptible to 5-monophosphate-specific RNase, suggesting that it lacks 5-triphospates like BDV. These results indicate that bornaviruses of mammals and birds use similar strategies to evade the host immune response.
Influenza viruses lacking the interferon (IFN)-antagonistic non-structural NS1 protein are strongly attenuated. Here, we show that mutants of a highly virulent variant of A/PR/8/34 (H1N1) carrying either a complete deletion or C-terminal truncations of NS1 were far more potent inducers of IFN in infected mice than NS1 mutants derived from standard A/PR/8/34. Efficient induction of IFN correlated with successful initial virus replication in mouse lungs, indicating that the IFN response is boosted by enhanced viral activity. As the new NS1 mutants can be handled in standard biosafety laboratories, they represent convenient novel tools for studying virus-induced IFN expression in vivo.
Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-beta as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-beta gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-beta following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-beta is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-beta under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.
The X protein of Borna disease virus (BDV) is an essential factor that regulates viral polymerase activity and inhibits apoptosis of persistently infected cells. We observed that a BDV mutant which carries an additional X gene replicated well in cell culture only after acquiring second-site mutations that selectively reduced expression of the endogenous X gene. In rat brains, the virus acquired additional mutations which inactivated the ectopic X gene or altered the sequence of X. These results demonstrate that BDV readily acquires mutations if strong selection pressure is applied. They further indicate that fine-tuning of X expression determines viral fitness.
High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.
T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.
Avian bornaviruses (ABV), representing a new genus within the family Bornaviridae, were recently discovered in parrots from North America and Israel with proventricular dilatation disease (PDD). We show here that closely related viruses are also present in captive European parrots of various species with PDD. The six ABV strains that we identified in clinically diseased birds are new members of the previously defined ABV genotypes 2 and 4. Viruses of both genotypes readily established persistent, noncytolytic infections in quail and chicken cell lines but did not grow in cultured mammalian cells in which classical Borna disease virus strains replicate very efficiently. ABV antigens were present in both the cytoplasm and nucleus of infected cells, suggesting nuclear replication of ABV. The genome organization of avian and mammalian bornaviruses is highly conserved except that ABV lacks a distinct control element in the 5 noncoding region of the bicistronic mRNA encoding the viral proteins X and P. Reverse transcription-PCR analysis demonstrated the presence of virus in many, if not all, organs of birds with PDD. Viral nucleic acid was also found in feces of diseased birds, suggesting virus transmission by the fecal-oronasal route. Immunohistochemical analysis of organs from birds with PDD revealed that infection with ABV is not restricted to cells of the nervous system. Thus, ABV exhibits a broad tissue and cell tropism that is strikingly different from classical Borna disease virus.
Borna disease virus (BDV) is a neurotropic member of the order Mononegavirales with noncytolytic replication and obligatory persistence in cultured cells and animals. Here we show that the accessory protein X of BDV represents the first mitochondrion-localized protein of an RNA virus that inhibits rather than promotes apoptosis induction. Rat C6 astroglioma cells persistently infected with wild-type BDV were significantly more resistant to death receptor-dependent and -independent apoptotic stimuli than uninfected cells or cells infected with a BDV mutant expressing reduced amounts of X. Confocal microscopy demonstrated that X colocalizes with mitochondria and expression of X from plasmid DNA rendered human 293T and mouse L929 cells resistant to apoptosis induction. A recombinant virus encoding a mutant X protein unable to associate with mitochondria (BDV-X(A6A7)) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-X(A6A7) developed severe neurological symptoms and died around day 30 postinfection, whereas all animals infected with wild-type BDV remained healthy and became persistently infected. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed a significant increase in the number of apoptotic cells in the brain of BDV-X(A6A7)-infected animals, whereas the numbers of CD3(+) T lymphocytes were comparable to those detected in animals infected with wild-type BDV. Our data thus indicate that inhibition of apoptosis by X promotes noncytolytic viral persistence and is required for the survival of cells in the central nervous system of BDV-infected animals.
The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-alpha against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-alpha several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-alpha substantially increased the protective effect of the cytokine treatment. IFN-alpha did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-alpha can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-alpha as an emergency drug against pandemic influenza A is warranted.
Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the increasing genetic diversity among these viruses and continued outbreaks in avian species underscore the need for more effective measures for the control and prevention of human H5N1 virus infection. Additional small animal models with which therapeutic approaches against virulent influenza viruses can be evaluated are needed. In this study, we used the guinea pig model to evaluate the relative virulence of selected avian and human influenza A viruses. We demonstrate that guinea pigs can be infected with avian and human influenza viruses, resulting in high titers of virus shedding in nasal washes for up to 5 days postinoculation (p.i.) and in lung tissue of inoculated animals. However, other physiologic indicators typically associated with virulent influenza virus strains were absent in this species. We evaluated the ability of intranasal treatment with human alpha interferon (alpha-IFN) to reduce lung and nasal wash titers in guinea pigs challenged with the reconstructed 1918 pandemic H1N1 virus or a contemporary H5N1 virus. IFN treatment initiated 1 day prior to challenge significantly reduced or prevented infection of guinea pigs by both viruses, as measured by virus titer determination and seroconversion. The expression of the antiviral Mx protein in lung tissue correlated with the reduction of virus titers. We propose that the guinea pig may serve as a useful small animal model for testing the efficacy of antiviral compounds and that alpha-IFN treatment may be a useful antiviral strategy against highly virulent strains with pandemic potential.
Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.
STAT1-deficient mice are more susceptible to infection with severe acute respiratory syndrome coronavirus (SARS-CoV) than type I interferon (IFN) receptor-deficient mice. We used mice lacking functional receptors for both type I and type III IFN (double knockout, dKO) to evaluate the possibility that type III IFN plays a decisive role in SARS-CoV protection. We found that viral peak titres in lungs of dKO and STAT1-deficient mice were similar, but significantly higher than in wild-type mice. The kinetics of viral clearance from the lung were also comparable in dKO and STAT1-deficient mice. Surprisingly, however, infected dKO mice remained healthy, whereas infected STAT1-deficient mice developed liver pathology and eventually succumbed to neurological disease. Our data suggest that the failure of STAT1-deficient mice to control initial SARS-CoV replication efficiently in the lung is due to impaired type I and type III IFN signalling, whereas the failure to control subsequent systemic viral spread is due to unrelated defects in STAT1-deficient mice.
Beta interferon (IFN-?) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-? production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-? promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-?-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-? in LACV-infected mouse brains. They further indicate that IFN-? synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.
Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales, designated Nyavirus. The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae. We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae. NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5 monophosphorylated and has a protruding 3 terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.
Mononuclear phagocytes are an important component of an innate immune system perceived as a system ready to react upon encounter of pathogens. Here, we show that in response to microbial stimulation, mononuclear phagocytes residing in nonmucosal lymphoid organs of germ-free mice failed to induce expression of a set of inflammatory response genes, including those encoding the various type I interferons (IFN-I). Consequently, NK cell priming and antiviral immunity were severely compromised. Whereas pattern recognition receptor signaling and nuclear translocation of the transcription factors NF-?B and IRF3 were normal in mononuclear phagocytes of germ-free mice, binding to their respective cytokine promoters was impaired, which correlated with the absence of activating histone marks. Our data reveal a previously unrecognized role for postnatally colonizing microbiota in the introduction of chromatin level changes in the mononuclear phagocyte system, thereby poising expression of central inflammatory genes to initiate a powerful systemic immune response during viral infection.
Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-?) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-? genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-? genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-? genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-?. We also found that IFN-? genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-?2/?3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-?2. Together, these results suggest that IFN-? may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.
The viral haemagglutinin (HA) and the viral polymerase complex determine the replication fitness of a highly virulent variant of influenza A virus strain A/PR/8/34 (designated hvPR8) and its high pathogenicity in mice. We report here that the HA of the hvPR8 differs from the HA of a low virulent strain (lvPR8) by the efficiency of receptor binding and membrane fusion. hvPR8 bound to 2,6-linked as well as 2,3-linked sialic acid-containing receptors, whereas lvPR8 bound exclusively to 2,3-linked sialic acids with high avidity. Remarkably, hvPR8 infected its target cells faster than lvPR8 and tolerated an elevated pH for efficient membrane fusion. In spite of these differences, both viruses targeted type II but not type I pneumocytes in the lung of infected mice. The HA of hvPR8 differs from that of lvPR8 by 16 aa substitutions and one insertion. Mutational analyses revealed that amino acid at HA position 190 (H3 numbering) primarily determined the specificity of receptor binding, while the insertion at position 133 influenced the avidity of receptor binding. Both amino acid positions also strongly influenced viral virulence. Furthermore, leucine at position 78 and glutamine at position 354 were critical determinants of increased fusion activity and virulence of hvPR8. Our data suggest that the HA of hvPR8 enhances virulence by mediating optimal receptor binding and membrane fusion thereby promoting rapid and efficient viral entry into host cells.
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