Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundamental techniques essential for procuring such knowledge are IAV isolation and culture from experimental and surveillance samples. Here we present a detailed protocol for IAV sample collection and processing, amplification in chicken eggs or mammalian cells, and identification from samples containing unknown pathogens. This protocol is robust, and it allows for the generation of virus cultures that can be used for downstream analyses. Once experimental or surveillance samples are obtained, virus cultures can be generated and the presence of IAVs can be verified in 3-5 d via reverse-transcription (RT)-PCR or hemagglutination assay. Increased time frames may be required for less experienced laboratory personnel, or when large numbers of samples will be processed.
The influenza viral polymerase complex affects host tropism and pathogenicity. In particular, several amino acids in the PB2 polymerase subunit are essential for the efficient replication of avian influenza viruses in mammals. The PA polymerase subunit also contributes to host range and pathogenicity. Here, we report that the PA proteins of several highly pathogenic avian H5N1 viruses have attenuating properties in mammalian cells and that the attenuating phenotype is conferred by strain-specific amino acid changes. Specifically, lysine at position 185 of A/duck/Vietnam/TY165/2010 (TY165; H5N1) PA induced strongly attenuating effects in vitro and in vivo. More importantly, the introduction of the arginine residue commonly found at this position in PA significantly increased the viral polymerase activity of TY165 in mammalian cells and its virulence and pathogenicity in mice. These findings demonstrate that the PA protein plays an important role in influenza virulence and pathogenicity.
Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4(+) and CD8(+) T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective.
Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases.
Highly pathogenic avian H5N1 influenza viruses have sporadically transmitted to humans causing high mortality. The mechanistic basis for adaptation is still poorly understood, although several residues in viral protein PB2 are known to be important for this event. Here, we demonstrate that three residues, 147T, 339T and 588T, in PB2 play critical roles in the virulence of avian H5N1 influenza viruses in a mammalian host in vitro and in vivo and, together, result in a phenotype comparable to that conferred by the previously known PB2-627K mutation with respect to virus polymerase activity. A virus with the three residues and 627K in PB2, as has been isolated from a lethal human case, is more pathogenic than viruses with only the three residues or 627K in PB2. Importantly, H5N1 viruses bearing the former three PB2 residues have circulated widely in recent years in avian species in nature.
Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines.
TCR signal strength during priming is a key determinant of CD4 T cell activation, but its impact on effector CD4 T functions in vivo remains unclear. In this study, we compare the functionality of CD4 T cell responses induced by peptides displaying varying binding half-lives with MHC class II before and after influenza virus infection. Although significant quantitative and qualitative differences in CD4 T cell responses were observed before infection between mice vaccinated with low- or high-stability peptides, both mice mounted robust early Th1 effector cytokine responses upon influenza challenge. However, only effector CD4 T cells induced by low-stability peptides proliferated and produced IL-17A after influenza challenge. In contrast, effector T cells elicited by higher-stability peptides displayed a terminally differentiated phenotype and divided poorly. This defective proliferation was T cell intrinsic but could not be attributed to a reduced expression of lymph node homing receptors. Instead, we found that CD4 T cells stimulated with higher-stability peptides exhibited decreased responsiveness to low levels of Ag presentation. Our study reveals the critical role of TCR signal strength during priming for the function and Ag sensitivity of effector CD4 T cells during viral challenge.
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
The outcome of respiratory virus infection is determined by a complex interplay of viral and host factors. Some potentially important host factors for the antiviral response, whose functions remain largely unexplored, are long non-coding RNAs (lncRNAs). Here we systematically inferred the regulatory functions of host lncRNAs in response to influenza A virus and severe acute respiratory syndrome coronavirus (SARS-CoV) based on their similarity in expression with genes of known function. We performed total RNA-Seq on viral-infected lungs from eight mouse strains, yielding a large data set of transcriptional responses. Overall 5,329 lncRNAs were differentially expressed after infection. Most of the lncRNAs were co-expressed with coding genes in modules enriched in genes associated with lung homeostasis pathways or immune response processes. Each lncRNA was further individually annotated using a rank-based method, enabling us to associate 5,295 lncRNAs to at least one gene set and to predict their potential cis effects. We validated the lncRNAs predicted to be interferon-stimulated by profiling mouse responses after interferon-? treatment. Altogether, these results provide a broad categorization of potential lncRNA functions and identify subsets of lncRNAs with likely key roles in respiratory virus pathogenesis. These data are fully accessible through the MOuse NOn-Code Lung interactive database (MONOCLdb).
Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor ?, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.
Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome.
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.
Avian influenza viruses rarely infect humans, but the recently emerged avian H7N9 influenza viruses have caused sporadic infections in humans in China, resulting in 440 confirmed cases with 122 fatalities as of 16 May 2014. In addition, epidemiologic surveys suggest that there have been asymptomatic or mild human infections with H7N9 viruses. These viruses replicate efficiently in mammals, show limited transmissibility in ferrets and guinea pigs, and possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. In this review, we summarize the characteristic features of the novel H7N9 viruses and assess their pandemic potential.
The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications.
The domestic ferret (Mustela putorius furo) is an important animal model for multiple human respiratory diseases. It is considered the 'gold standard' for modeling human influenza virus infection and transmission. Here we describe the 2.41 Gb draft genome assembly of the domestic ferret, constituting 2.28 Gb of sequence plus gaps. We annotated 19,910 protein-coding genes on this assembly using RNA-seq data from 21 ferret tissues. We characterized the ferret host response to two influenza virus infections by RNA-seq analysis of 42 ferret samples from influenza time-course data and showed distinct signatures in ferret trachea and lung tissues specific to 1918 or 2009 human pandemic influenza virus infections. Using microarray data from 16 ferret samples reflecting cystic fibrosis disease progression, we showed that transcriptional changes in the CFTR-knockout ferret lung reflect pathways of early disease that cannot be readily studied in human infants with cystic fibrosis disease.
The sensitivity of influenza rapid diagnostic tests (IRDTs) currently available in Japan for various influenza virus strains, including human H7N9 and H5N1 isolates, were compared and it was found that all of the IRDTs examined detected these viruses; however, their detection sensitivities differed.
Shedding of the pandemic virus during an influenza pandemic is thought to persist longer than shedding of influenza viruses during annual influenza seasons, because people have much less immunity against a pandemic influenza. A correlation is thought to exist between the length of virus shedding and the clinical severity of influenza illness.
Several arenaviruses are known to cause viral hemorrhagic fever (VHF) in sub-Saharan Africa and South America, where VHF is a major public health and medical concern. The biosafety level 4 categorization of these arenaviruses restricts their use and has impeded biological studies, including therapeutic drug and/or vaccine development. Due to difficulties associated with handling live viruses, pseudotype viruses, which transiently bear arenavirus envelope proteins based on vesicular stomatitis virus (VSV) or retrovirus, have been developed as surrogate virus systems. Here, we report the development of a pseudotype VSV bearing each envelope protein of various species of arenaviruses (AREpv), including the newly identified Lujo virus (LUJV) and Chapare virus. Pseudotype arenaviruses generated in 293T cells exhibited high infectivity in various mammalian cell lines. The infections by New World and Old World AREpv were dependent on their receptors (human transferrin receptor 1 [hTfR1] and ?-dystroglycan [?DG], respectively). However, infection by pseudotype VSV bearing the LUJV envelope protein (LUJpv) occurred independently of hTfR1 and ?DG, indicating that LUJpv utilizes an unidentified receptor. The pH-dependent endocytosis of AREpv was confirmed by the use of lysosomotropic agents. The fusion of cells expressing these envelope proteins, except for those expressing the LUJV envelope protein, was induced by transient treatment at low pH values. LUJpv infectivity was inhibited by U18666A, a cholesterol transport inhibitor. Furthermore, the infectivity of LUJpv was significantly decreased in the Niemann-Pick C1 (NPC1)-deficient cell line, suggesting the necessity for NPC1 activity for efficient LUJpv infection.
Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ?1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo.
Novel influenza A viruses of the H7N9 subtype [A(H7N9)] emerged in the spring of 2013 in China and had infected 163 people as of 10 January 2014; 50 of them died of the severe respiratory infection caused by these viruses. Phylogenetic studies have indicated that the novel A(H7N9) viruses emerged from reassortment of H7, N9, and H9N2 viruses. Inspections of protein sequences from A(H7N9) viruses and their immediate predecessors revealed several amino acid changes in A(H7N9) viruses that may have facilitated transmission and replication in the novel host. Since mutations that occurred more ancestrally may also have contributed to the genesis of A(H7N9) viruses, we inferred historical evolutionary events leading to the novel viruses. We identified a number of amino acid changes on the evolutionary path to A(H7N9) viruses, including substitutions that may be associated with host range, replicative ability, and/or host responses to infection. The biological significance of these amino acid changes can be tested in future studies.
Herpesviruses have evolved a unique mechanism for nuclear egress of nascent progeny nucleocapsids: the nucleocapsids bud through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes (primary envelopment), and enveloped nucleocapsids then fuse with the outer nuclear membrane to release nucleocapsids into the cytoplasm (de-envelopment). We have shown that the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (or VP13/VP14) is a novel regulator for HSV-1 nuclear egress. In particular, we demonstrated the following: (i) UL47 formed a complex(es) with HSV-1 proteins UL34, UL31, and/or Us3, which have all been reported to be critical for viral nuclear egress, and these viral proteins colocalized at the nuclear membrane in HSV-1-infected cells; (ii) the UL47-null mutation considerably reduced primary enveloped virions in the perinuclear space although capsids accumulated in the nucleus; and (iii) UL47 was detected in primary enveloped virions in the perinuclear space by immunoelectron microscopy. These results suggested that UL47 promoted HSV-1 primary envelopment, probably by interacting with the critical HSV-1 regulators for viral nuclear egress and by modulating their functions.
HIV-1 vaccines based on recombinant vectors have been developed to elicit immune responses; however, the failure of the STEP HIV-1 vaccine trial has caused concern regarding the impact on vaccine efficacy of pre-existing vector seropositivity in humans. By using a mouse model of infection, we evaluated the immune responses elicited by intranasal and vaginal immunization with the recombinant influenza virus WSN/CKG carrying the PCLUS3-P18 peptide and a Gag epitope in its hemagglutinin, and the impact of pre-existing vector immunity on protection against recombinant vaccinia virus challenge. We found that despite the protective immunity induced in naïve mice by the WSN/CKG virus via either route, the vaginal immunization of mice with pre-existing influenza immunity restricted vPE16 replication more significantly in the ovaries than intranasal immunization. Thus, successful vaccination strategies under limiting conditions, such as pre-existing vector immunity, require the local induction of mucosal immunity at the site of virus infection.
Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature.
Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.
Influenza virus is rich in variation and mutations. It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations. Here, we established a histochemical imaging assay for influenza virus sialidase activity in living cells by using a new fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-?-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). The BTP3-Neu5Ac assay histochemically visualized influenza virus-infected cells regardless of viral hosts and subtypes. Influenza virus neuraminidase-expressed cells, viral focus formation, and virus-infected locations in mice lung tissues were easily, rapidly, and sensitively detected by the BTP3-Neu5Ac assay. Histochemical visualization with the BTP3-Neu5Ac assay is extremely useful for detection of influenza viruses without the need for fixation or a specific antibody. This novel assay should greatly improve the efficiency of detection, titration, and isolation of influenza viruses and might contribute to research on viral sialidase.
Influenza infection of humans remains an important public health problem. Vaccine strategies result in a significant but only partial control (65-85%) of infection. Thus, chemotherapeutic approaches are needed to provide a solution both for vaccine failures and to limit infection in the unvaccinated population. Previously (Walsh et al., 2011; Teijaro et al., 2011) documented that sphingosine-1-phosphate 1 receptor (S1P1R) agonists significantly protected mice against pathogenic H1N1 influenza virus by limiting immunopathologic damage while allowing host control of the infection. Here we extend that observation by documenting S1P1R agonist can control pathogenic H1N1 influenza infection in ferrets. S1P1R agonist was more effective in reducing pulmonary injury than the antiviral drug oseltamivir but, importantly, combined therapy was significantly more effective than either therapy alone.
Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian-adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells, and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells, but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals.
To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs.
Two ferret-adapted H5N1 viruses capable of respiratory droplet transmission have been reported with mutations in the hemagglutinin receptor-binding site and stalk domains. Glycan microarray analysis reveals that both viruses exhibit a strong shift toward binding to "human-type" ?2-6 sialosides but with notable differences in fine specificity. Crystal structure analysis further shows that the stalk mutation causes no obvious perturbation of the receptor-binding pocket, consistent with its impact on hemagglutinin stability without affecting receptor specificity.
Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.
The influenza A virus possesses an eight-segmented, negative-sense, single-stranded RNA genome (vRNA). Each vRNA segment binds to multiple copies of viral nucleoproteins and a small number of heterotrimeric polymerase complexes to form a rod-like ribonucleoprotein complex (RNP), which is essential for the transcription and replication of the vRNAs. However, how the RNPs are organized within the progeny virion is not fully understood. Here, by focusing on polymerase complexes, we analyzed the fine structure of purified RNPs and their configuration within virions by using various electron microscopies (EM). We confirmed that the individual RNPs possess a single polymerase complex at one end of the rod-like structure and that, as determined using immune EM, some RNPs are incorporated into budding virions with their polymerase-binding ends at the budding tip, whereas others align with their polymerase-binding ends at the bottom of the virion. These data further our understanding of influenza virus virion morphogenesis.
Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.
Since the end of March 2013, avian a influenza viruses of the H7N9 subtype have caused more than 130 human cases of infection in China, many of which were severe, resulting in 43 fatalities. Although this A(H7N9) virus outbreak is now under control, the virus (or one with similar properties) could reemerge as winter approaches. To better assess the pandemic threat posed by A(H7N9) viruses, NIAID/NIH Centers of Excellence in Influenza Research and Surveillance (CEIRS) investigators and other expert laboratories in China and elsewhere have characterized the wild-type avian A(H7N9) viruses in terms of host range, virulence, and transmission, and are evaluating the effectiveness of antiviral drugs and vaccine candidates. However, to fully assess the potential risk associated with these novel viruses, there is a need for additional research including experiments that may be classified as "gain-of-function" (GOF). Here, we outline the aspects of the current situation that most urgently require additional research, our proposed studies, and risk-mitigation strategies.
The influenza A virus genome comprises eight single-stranded negative-sense RNA segments (vRNAs). All eight vRNAs are selectively packaged into each progeny virion via so-called segment-specific genome-packaging signal sequences that are located in the noncoding and terminal coding regions of both the 3 and the 5 ends of the vRNAs. However, it remains unclear how these signals ensure that eight different vRNAs are packaged. Here, by using a reverse genetics system, we demonstrated that, in the absence of the other seven vRNAs, a recombinant NP vRNA bearing only a reporter gene flanked by the noncoding NP regions was incorporated into virus-like particles (VLPs) as efficiently as a recombinant NP vRNA bearing the reporter gene flanked by the complete NP packaging signals (i.e., the noncoding sequences and the terminal coding regions). Viruses that comprised a recombinant NP vRNA whose packaging signal was disrupted, and the remaining seven authentic vRNAs, did not undergo multiple cycles of replication; however, a recombinant NP vRNA with only the noncoding regions was readily incorporated into VLPs, suggesting that the packaging signal as currently defined is not necessarily essential for the packaging of the vRNA in which it resides; rather, it is required for the packaging of the full set of vRNAs. We propose that the 3 and 5 noncoding regions of each vRNA bear a virion incorporation signal for that vRNA and that the terminal coding regions serve as a bundling signal that ensures the incorporation of the complete set of eight vRNAs into the virion.
Influenza is a common infectious disease caused by influenza viruses. Annual epidemics cause severe illnesses, deaths, and economic loss around the world. To better defend against influenza viral infection, it is essential to understand its mechanisms and associated host responses. Many studies have been conducted to elucidate these mechanisms, however, the overall picture remains incompletely understood. A systematic understanding of influenza viral infection in host cells is needed to facilitate the identification of influential host response mechanisms and potential drug targets.
To gain insight into the ecology of avian influenza viruses (AIV), we conducted active influenza virus surveillance in domestic ducks on farms located on the flyway of migratory birds in the Dongting Lake region of Hunan Province, China, from winter 2011 until spring 2012. Specimens comprising 3,030 duck swab samples and 1,010 environmental samples were collected from 101 duck farms. We isolated AIV of various HA subtypes, including H3, H4, H5, H6, H9, H10, H11, and H12. We sequenced the entire coding sequences of the genomes of 28 representative isolates constituting 13 specific subtypes. When the phylogenetic relationships among these isolates were examined, we observed that extensive reassortment events had occurred. Among the 28 Dongting Lake viruses, 21 genotypes involving the six internal genes were identified. Furthermore, we identified viruses or viral genes introduced from other countries, viral gene segments of unknown origin, and a novel HA/NA combination. Our findings emphasize the importance of farmed domestic ducks in the Dongting Lake region to the genesis and evolution of AIV and highlight the need for continued surveillance of domestic ducks in this region.
Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.
Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.
Ebola virus (EBOV) protein L (EBOL) acts as a viral RNA-dependent RNA polymerase. To better understand the mechanisms underlying the transcription and replication of the EBOV genome, we sought to identify cellular factors involved in these processes via their coimmunoprecipitation with EBOL and by mass spectrometry. Of 65 candidate proteins identified, we focused on DNA topoisomerase 1 (TOP1), which localizes to the nucleus and unwinds helical DNA. We found that in the presence of EBOL, TOP1 colocalizes and interacts with EBOL in the cytoplasm, where transcription and replication of the EBOV genome occur. Knockdown of TOP1 markedly reduced virus replication and viral polymerase activity. We also found that the phosphodiester bridge-cleaving and recombination activities of TOP1 are required for the polymerase activity of EBOL. These results demonstrate that TOP1 is an important cellular factor for the transcription and replication of the EBOV genome and, as such, plays a key role in the EBOV life cycle.
Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.
Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live attenuated influenza vaccines are approved in several countries, yet no vaccine is licensed for HPIV. We previously showed that a replication-incompetent PB2-knockout (PB2-KO) virus that possesses a reporter gene in the coding region of the PB2 segment can serve as a platform for a bivalent vaccine. To develop a bivalent vaccine against influenza and parainfluenza virus, here, we generated a PB2-KO virus possessing the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV type 3 (HPIV3), a major surface antigen of HPIV, in its PB2 segment. We confirmed that this virus replicated only in PB2-expressing cells and expressed HN. We then examined the efficacy of this virus as a bivalent vaccine in a hamster model. High levels of virus-specific IgG antibodies in sera and IgA, IgG, and IgM antibodies in bronchoalveolar lavage fluids against both influenza virus and HPIV3 were detected from hamsters immunized with this virus. The neutralizing capability of these serum antibodies was also confirmed. Moreover, the immunized hamsters were completely protected from virus challenge with influenza virus or HPIV3. These results indicate that PB2-KO virus expressing the HN of HPIV3 has the potential to be a novel bivalent vaccine against influenza and human parainfluenza viruses.
Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.
Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators - and the relative contributions of each toward viral pathogenicity - is not well-understood.
The emergence of human-transmissible H5N1 avian influenza viruses poses a major pandemic threat. H5N1 viruses are thought to be highly genetically diverse both among and within hosts; however, the effects of this diversity on viral replication and transmission are poorly understood. Here we use deep sequencing to investigate the impact of within-host viral variation on adaptation and transmission of H5N1 viruses in ferrets. We show that, although within-host genetic diversity in haemagglutinin (HA) increases during replication in inoculated ferrets, HA diversity is dramatically reduced upon respiratory droplet transmission, in which infection is established by only 1-2 distinct HA segments from a diverse source virus population in transmitting animals. Moreover, minor HA variants present in as little as 5.9% of viruses within the source animal become dominant in ferrets infected via respiratory droplets. These findings demonstrate that selective pressures acting during influenza virus transmission among mammals impose a significant bottleneck.
Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology.
H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3.
Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel "crowd-based" approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.
An influenza A virus that originated in pigs caused a pandemic in 2009. The sialidase activity of the neuraminidase (NA) of previous pandemic influenza A viruses are stable at low pH (?5). Here, we identified the amino acids responsible for this property. We found differences in low-pH stability at pH 5.0 among pandemic (H1N1) 2009 viruses, which enhanced the replication of these viruses. Low-pH-stable NA enhancement of virus replication may have contributed to the rapid worldwide spread and adaptation to humans of pandemic (H1N1) 2009 viruses during the early stages of the 2009 pandemic.
Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem ?-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.
Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.
Influenza A virus matrix protein (M1) plays an important role in virus assembly and budding. Besides a well-characterized basic amino acid-rich nuclear localization signal region at positions 101 to 105, M1 contains another basic amino acid stretch at positions 76-78 that is highly conserved among influenza A and B viruses, suggesting the importance of this stretch. To understand the role of these residues in virus replication, we mutated them to either lysine (K), alanine (A), or aspartic acid (D). We could generate viruses possessing either single or combination substitutions with K or single substitution with A at any of these positions, but not those with double substitutions with A or a single substitution with D. Viruses with the single substitution with A exhibited slower growth and had lower nucleoprotein/M1 quantitative ratio in virions compared to the wild-type virus. In cells infected with a virus possessing the single substitution with A at position 77 or 78 (R77A or R78A, respectively), the mutated M1 localized in patches at the cell periphery where nucleoprotein and hemagglutinin colocalized more often than the wild-type did. Transmission electron microscopy showed that virus possessing M1 R77A or R78A, but not the wild-type virus, was present in vesicular structures, indicating a defect in virus assembly and/or budding. The M1 mutations that did not support virus generation exhibited an aberrant M1 intracellular localization and affected protein incorporation into virus-like particles. These results indicate that the basic amino acid stretch of M1 plays a critical role in influenza virus replication.
Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.
Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-?2-3-galactose [Sia?2-3Gal] linkages on sialyloligosaccharides)--and human (Sia?2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.
Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.
Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008.
Type I interferon (IFN) signaling is mediated through several signaling pathways, including the Janus kinase and signal transducer and activator (JAK-STAT) and p38 mitogen-activated protein (MAP) kinase pathways. The VP24 protein of Ebolavirus is an IFN antagonist, blocking type I IFN signaling through the JAK-STAT pathway. Here, we show that, in 293 cells, VP24 also interferes with the p38 MAP kinase pathway by blocking IFN-?-stimulated phosphorylation of p38-?. Similar inhibition was not observed in HeLa cells, suggesting cell type-specific differences in signal transduction.
Similar to other viruses, the viral proteins of Ebola virus (EBOV) interact with a variety of host proteins for its replication. Of the 7 structural proteins encoded in the EBOV genome, VP24 is the smallest and is multifunctional.
sGP, which is perceived as nonstructural, secretory glycoprotein, shares 295 amino acids at its N-terminal with GP(1,2), which include the specific residue necessary to interact with GP(2). In the present study, we tested whether the sGP protein of Zaire ebolavirus (ZEBOV) could substitute for GP(1) and form a complex with GP(2), thus serving as a structural protein.
Ebola virus VP35 is a cofactor of the viral RNA polymerase complex and, together with NP and VP24, is an essential component for nucleocapsid formation. In the present study, we examined the interactions between VP35 and NP and found that VP35 interacts with helical NP-RNA complexes through the C-terminus of NP. We also found that coexpression of excess VP35 with NP reduced the yields of NP-RNA complexes purified by CsCl gradient ultracentrifugation and inhibited the formation of the NP-induced inclusion bodies that typically form in Ebola virus-infected cells. These findings suggest that the NP to VP35 ratio is important in the Ebola virus replication cycle and advance our knowledge of nucleocapsid morphogenesis.
The matrix protein VP40 of Marburg virus promotes the formation and release of virus-like particles (VLPs). Marburg virus VP40 interacts with cellular Tsg101 via its L domain motif; however, mutation of this motif does not affect VLP budding or the accumulation of VP40 in multivesicular bodies (MVBs), which are platforms for virus particle formation. To identify regions of Marburg virus VP40 that are important for VLP budding, we examined deletion mutants and alanine-scanning mutants at the N- and C-terminus of VP40 for their involvement in VLP budding. VLPs were not detected in the presence of alanine-replacement mutants at Ile39 and Thr40, and the level of VLP budding for the alanine mutant at Asn297 was decreased. Moreover, these mutants did not accumulate in MVBs. Our results suggest the involvement of a novel host factor(s) in VLP budding and VP40 transport to MVBs.
Plasmid-based reverse genetics systems allow the artificial generation of viruses with cloned cDNA-derived genomes. Since the establishment of such systems for influenza virus, numerous attempts have been made to tame this pathogenic agent. In particular, several types of viruses expressing foreign genes have been generated and used to further our knowledge of influenza virus replication and pathogenicity and to develop novel influenza vaccines. Here, we review these achievements and discuss future perspectives.
The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.
A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.
During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.
Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.
Protein-protein interaction (PPI) databases are widely used tools to study cellular pathways and networks; however, there are several databases available that still do not account for cell type-specific differences. Here, we evaluated the characteristics of six interaction databases, incorporated tissue-specific gene expression information and finally, investigated if the most popular proteins of scientific literature are involved in good quality interactions.
Negatively stained influenza virions sometimes show irregular morphology and are often referred to as pleomorphic. However, this irregular morphology has not been visualized when ultrathin-section transmission and scanning electron microscopies are used. This study focused on the effects of ultracentrifugation on influenza A virion morphology, as negative staining often involves ultracentrifugation to concentrate or purify virions. The morphologies of unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed virions were quantitatively compared before and after ultracentrifugation, and it was found that, without chemical fixation, approximately 30% of virions were altered from oval to irregular shapes following ultracentrifugation. By contrast, most glutaraldehyde-fixed virions remained uniformly elliptical, even after ultracentrifugation. When a virus with an 11 aa deletion at the C terminus of its M2 cytoplasmic tail was ultracentrifuged, its morphology was appreciably deformed compared with that of the wild-type virus. These results demonstrate that the native morphology of influenza A virions is regular but is disrupted by ultracentrifugation, and that the cytoplasmic tail of M2 is important for virion integrity.
Influenza A virus uses cellular protein transport systems (e.g., CRM1-mediated nuclear export and Rab11-dependent recycling endosomes) for genome trafficking from the nucleus to the plasma membrane, where new virions are assembled. However, the detailed mechanisms of these events have not been completely resolved, and additional cellular factors are probably required. Here, we investigated the role of the cellular human immunodeficiency virus (HIV) Rev-binding protein (HRB), which interacts with influenza virus nuclear export protein (NEP), during the influenza virus life cycle. By using small interfering RNAs (siRNAs) and overexpression of a dominant negative HRB protein fragment, we show that cells lacking functional HRB have significantly reduced production of influenza virus progeny and that this defect results from impaired viral ribonucleoprotein (vRNP) delivery to the plasma membrane in late-stage infection. Since HRB colocalizes with influenza vRNPs early after their delivery to the cytoplasm, it may mediate a connection between the nucleocytoplasmic transport machinery and the endosomal system, thus facilitating the transfer of vRNPs from nuclear export to cytoplasmic trafficking complexes. We also found an association between NEP and HRB in the perinuclear region, suggesting that NEP may contribute to this process. Our results identify HRB as a second endosomal factor with a crucial role in influenza virus genome trafficking, suggest cooperation between unique endosomal compartments in the late steps of the influenza virus life cycle, and provide a common link between the cytoplasmic trafficking mechanisms of influenza virus and HIV.
Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NP(ma), ZEBOV-VP24(ma), and ZEBOV-NP/VP24(ma)) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24(ma)) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24(ma) was nearly the same as that to ZEBOV-NP/VP24(ma), the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality.
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