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We isolated 77 highly pathogenic avian influenza viruses during routine surveillance in live poultry markets in northern provinces of Vietnam from 2018 to 2021. These viruses are of the H5N6 subtype and belong to HA clades 2.3.4.4g and 2.3.4.4h. Interestingly, we did not detect viruses of clade 2.3.4.4b, which in recent years have dominated in different parts of the world. The viruses isolated in this current study do not encode major determinants of mammalian adaptation (e.g., PB2-E627K or PB1-D701N) but possess amino acid substitutions that may affect viral receptor-binding, replication, or the responses to human antiviral factors. Several of the highly pathogenic H5N6 virus samples contained other influenza viruses, providing an opportunity for reassortment. Collectively, our study demonstrates that the highly pathogenic H5 viruses circulating in Vietnam in 2018–2021 were different from those in other parts of the world, and that the Vietnamese H5 viruses continue to evolve through mutations and reassortment. [ABSTRACT FROM AUTHOR]

Current SARS-CoV-2 vaccines provide protection for COVID-19-associated hospitalization and death, but remain inefficient at inhibiting initial infection and transmission. Despite updated booster formulations, breakthrough infections and reinfections from emerging SARS-CoV-2 variants are common. Intranasal vaccination to elicit mucosal immunity at the site of infection can improve the performance of respiratory virus vaccines. We developed SARS-CoV-2 M2SR, a dual SARS-CoV-2 and influenza vaccine candidate, employing our live intranasal M2-deficient single replication (M2SR) influenza vector expressing the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein of the prototype strain, first reported in January 2020. The intranasal vaccination of mice with this dual vaccine elicits both high serum IgG and mucosal IgA titers to RBD. Sera from inoculated mice show that vaccinated mice develop neutralizing SARS-CoV-2 antibody titers against the prototype and Delta virus strains, which are considered to be sufficient to protect against viral infection. Moreover, SARS-CoV-2 M2SR elicited cross-reactive serum and mucosal antibodies to the Omicron BA.4/BA.5 variant. The SARS-CoV-2 M2SR vaccine also maintained strong immune responses to influenza A with high titers of anti H3 serum IgG and hemagglutination inhibition (HAI) antibody titers corresponding to those seen from the control M2SR vector alone. With a proven safety record and robust immunological profile in humans that includes mucosal immunity, the M2SR influenza viral vector expressing key SARS-CoV-2 antigens could provide more efficient protection against influenza and SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Current influenza vaccines demonstrate low vaccine efficacy, especially when the predominantly circulating strain and vaccine are mismatched. The novel influenza vaccine platform M2- or BM2-deficient single replication (M2SR and BM2SR) has been shown to safely induce strong systemic and mucosal antibody responses and provide protection against significantly drifted influenza strains. In this study, we demonstrate that both monovalent and quadrivalent (Quad) formulations of M2SR are non-pathogenic in mouse and ferret models, eliciting robust neutralizing and non-neutralizing serum antibody responses to all strains within the formulation. Following challenge with wildtype influenza strains, vaccinated mice and ferrets demonstrated reduced weight loss, decreased viral replication in the upper and lower airways, and enhanced survival as compared to mock control groups. Mice vaccinated with H1N1 M2SR were completely protected from heterosubtypic H3N2 challenge, and BM2SR vaccines provided sterilizing immunity to mice challenged with a cross-lineage influenza B virus. Heterosubtypic cross-protection was also seen in the ferret model, with M2SR vaccinated animals exhibiting decreased viral titers in nasal washes and lungs following the challenge. BM2SR-vaccinated ferrets elicited robust neutralizing antibodies toward significantly drifted past and future influenza B strains. Mice and ferrets that received quadrivalent M2SR were able to mount immune responses equivalent to those seen with each of the four monovalent vaccines, demonstrating the absence of strain interference in the commercially relevant quadrivalent formulation. [ABSTRACT FROM AUTHOR]

Background: We previously demonstrated that an intranasal dose of 108 TCID50 M2SR (M2-deficient single replication) influenza vaccine protected against highly drifted H3N2 influenza challenge in a subset of subjects who demonstrated ≥2-fold increase in microneutralization (MN) antibodies to Belgium2015 (the challenge strain) after vaccination. Here, we describe a phase 1b, observer-blinded, dose-escalation study demonstrating an increased proportion of responders with this signal of immune protection.Methods: Sero-susceptible subjects ages 18-49 years were randomized to receive two doses (108-109TCID50) of M2SR or placebo administered 28 days apart. Clinical specimens were collected before and after each dose. The primary objective was to demonstrate safety of M2SR vaccines (ClinicalTrials.gov number NCT03999554).Results: The vaccine was well-tolerated at all dose levels. Against Belgium2015,  ≥ 2-fold increases in MN antibodies were noted among 40% (95% CI 24.9-56.7) of subjects following a single 108 TCID50 M2SR dose and among 80.6% (95% CI 61.4-92.3) after 109 dose (P < 0.001). A single 109 TCID50 dose of M2SR generated ≥4-fold HAI seroconversion against the vaccine strain in 71% (95% CI 52.0-85.8) of recipients. Mucosal and cellular immune responses were also induced.Conclusions: These results indicate that M2SR may provide substantial protection against infection with highly drifted strains of H3N2 influenza. [ABSTRACT FROM AUTHOR]

Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic), whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines. [ABSTRACT FROM AUTHOR]

Background: Current influenza vaccines are strain specific and demonstrate low vaccine efficacy against H3N2 influenza disease, especially when vaccine is mismatched to circulating virus. The novel influenza vaccine candidate, M2-deficient single replication (M2SR), induces a broad, multi-effector immune response.Methods: A phase 2 challenge study was conducted to assess the efficacy of an M2SR vaccine expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (Bris2007 M2SR H3N2; clade 1). Four weeks after vaccination, recipients were challenged with antigenically distinct H3N2 virus (A/Belgium/4217/2015, clade 3C.3b) and assessed for infection and clinical symptoms.Results: Adverse events after vaccination were mild and similar in frequency for placebo and M2SR recipients. A single dose of Bris2007 M2SR induced neutralizing antibody to the vaccine (48% of recipients) and challenge strain (27% of recipients). Overall, 54% of M2SR recipients were infected after challenge, compared with 71% of placebo recipients. The subset of M2SR recipients with a vaccine-induced microneutralization response against the challenge virus had reduced rates of infection after challenge (38% vs 71% of placebo recipients; P = .050) and reduced illness.Conclusions: Study participants with vaccine-induced neutralizing antibodies were protected against infection and illness after challenge with an antigenically distinct virus. This is the first demonstration of vaccine-induced protection against a highly drifted H3N2 challenge virus. [ABSTRACT FROM AUTHOR]

In hamsters, SARS-CoV-2 infection at the same time as or before H3N2 influenza virus infection resulted in significantly reduced influenza virus titers in the lungs and nasal turbinates. This interference may be correlated with SARS-CoV-2-induced expression of MX1. [ABSTRACT FROM AUTHOR]

Worldwide outbreaks of influenza (pandemics) are caused by influenza A viruses to which persons lack protective immune responses. Currently, we are unable to predict which influenza virus strains may cause a pandemic. In this article, we summarize some of the information that will be needed to better assess the pandemic potential of influenza viruses, and we discuss our current gaps in knowledge. [ABSTRACT FROM AUTHOR]

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine. [ABSTRACT FROM AUTHOR]

Background: Treatment of immunocompromised, influenza virus-infected patients with the viral neuraminidase inhibitor oseltamivir often leads to the emergence of drug-resistant variants. Combination therapy with compounds that target different steps in the viral life cycle may improve treatment outcomes and reduce the emergence of drug-resistant variants.Methods: Here, we infected immunocompromised nude mice with an influenza A virus and treated them with neuraminidase (oseltamivir, laninamivir) or viral polymerase (favipiravir) inhibitors, or combinations thereof.Results: Combination therapy for 28 days increased survival times compared with monotherapy, but the animals died after treatment was terminated. Mono- and combination therapies did not consistently reduce lung virus titers. Prolonged viral replication led to the emergence of neuraminidase inhibitor-resistant variants, although viruses remained sensitive to favipiravir. Overall, favipiravir provided greater benefit than neuraminidase inhibitors.Conclusions: Collectively, our data demonstrate that combination therapy in immunocompromised hosts increases survival times, but does not suppress the emergence of neuraminidase inhibitor-resistant variants. [ABSTRACT FROM AUTHOR]

Antiviral compounds (eg, the neuraminidase inhibitor oseltamivir) are invaluable for the treatment of individuals infected with influenza A viruses of the H7N9 subtype (A[H7N9]), which have infected and killed hundreds of persons. However, oseltamivir treatment often leads to the emergence of resistant viruses in immunocompromised individuals. To better understand the emergence and properties of oseltamivir-resistant A(H7N9) viruses in immunosuppressed individuals, we infected immunosuppressed cynomolgus macaques with an A(H7N9) virus and treated them with oseltamivir. Disease severity and mortality were higher in immunosuppressed than in immunocompetent animals. Oseltamivir treatment at 2 different doses reduced A(H7N9) viral titers in infected animals, but even high-dose oseltamivir did not block viral replication sufficiently to suppress the emergence of resistant variants. Some resistant variants were not appreciably attenuated in cultured cells, but an oseltamivir-resistant A(H7N9) virus did not transmit among ferrets. These findings are useful for the control of A(H7N9) virus infections in clinical settings. [ABSTRACT FROM AUTHOR]

Highly pathogenic influenza viruses of the H5N1 subtype have infected more than 600 people since 1997, resulting in the deaths of approximately 60% of those infected. Multiple studies have established the viral hemagglutinin (HA) surface glycoprotein as the major determinant of H5N1 virulence. HA mediates host-specific virus binding to cells, and mutations that allow efficient binding to viral receptors on mammalian cells are critical (although not sufficient) for H5N1 transmissibility among mammals. The viral polymerase PB2 protein is also a critical virulence determinant, and adaptive mutations in this protein are crucial for efficient H5N1 virus replication in mammals. Additionally, viral proteins (such as NS1 and PB1-F2) with roles in innate immune responses also affect the virulence of highly pathogenic H5N1 viruses. [ABSTRACT FROM AUTHOR]

Influenza A viral ribonucleoprotein (vRNP) complexes comprise the eight genomic negative-sense RNAs, each of which is bound to multiple copies of the vRNP and a trimeric viral polymerase complex. The influenza virus life cycle centres on the vRNPs, which in turn rely on host cellular processes to carry out functions that are necessary for the successful completion of the virus life cycle. In this Review, we discuss our current knowledge about vRNP trafficking within host cells and the function of these complexes in the context of the virus life cycle, highlighting how structure contributes to function and the crucial interactions with host cell pathways, as well as on the information gaps that remain. An improved understanding of how vRNPs use host cell pathways is essential to identify mechanisms of virus pathogenicity, host adaptation and, ultimately, new targets for antiviral intervention. [ABSTRACT FROM AUTHOR]

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication. [ABSTRACT FROM AUTHOR]

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties. [ABSTRACT FROM AUTHOR]

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. A prime example is the recent emergence of swine-origin H1N1 viruses that have transmitted to and spread among humans, resulting in outbreaks internationally. Efforts to control these outbreaks and real-time monitoring of the evolution of this virus should provide us with invaluable information to direct infectious disease control programmes and to improve understanding of the factors that determine viral pathogenicity and/or transmissibility. [ABSTRACT FROM AUTHOR]

M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naïve and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects. [ABSTRACT FROM AUTHOR]

Influenza A viruses cause pandemics at random intervals. Pandemics are caused by viruses that contain a hemagglutinin (HA) surface glycoprotein to which human populations are immunologically naive. Such an HA can be introduced into the human population through reassortment between human and avian virus strains or through the direct transfer of an avian influenza virus to humans. The factors that determine the interspecies transmission and pathogenicity of influenza viruses are still poorly understood; however, the HA protein plays an important role in overcoming the interspecies barrier and in virulence in avian influenza viruses. Recently, the RNA polymerase (PB2) protein has also been recognized as a critical factor in host range restriction, while the nonstructural (NS1) protein affects the initial host immune responses. We summarize current knowledge of viral factors that determine host range restriction and pathogenicity of influenza A viruses. [ABSTRACT FROM AUTHOR]

The generation of vaccines for highly pathogenic avian influenza viruses, including those of the H5N1 subtype, relies on reverse genetics, which allows the production of influenza viruses from cloned cDNA. In the future, reverse genetics will likely be the method of choice for the generation of conventional influenza vaccine strains because gene reassortment by more traditional methods is cumbersome. Established systems for the artificial generation of influenza A viruses require transfection of cells with the eight to 12 plasmids that provide the eight influenza viral RNAs as well as the polymerase and nucleoproteins of the virus. However, cell lines appropriate for human vaccine production (e.g., Vero cells) cannot be transfected with high efficiencies. To overcome these problems, we established a reverse genetics system in which the eight RNA polymerase I transcription cassettes for viral RNA synthesis are combined on one plasmid. Similarly, two cassettes encoding the hemagglutinin and neuraminidase segments and six cassettes encoding the remaining proteins were combined. We also combined three RNA polymerase II transcription cassettes for the expression of the polymerase subunits. By combining these cassettes, we reduced the number of plasmids required for virus generation significantly and produced influenza A virus in Vero cells with higher efficiency than with the traditional 12 plasmid system. This new system is thus suitable for influenza virus vaccine production and may be applicable to other reverse genetics systems that rely on the introduction of several plasmids into eukaryotic cells. [ABSTRACT FROM AUTHOR]

Background Demonstration of protection by a M2SR (M2 deficient Single Replication) monovalent H3N2 vaccine was assessed in a phase 2a clinical trial in which the challenge virus was substantially drifted from the vaccine. M2SR is an investigational, live virus vaccine containing hemagglutinin (HA) and neuraminidase (NA) selected from targeted Type A influenza strains. M2SR undergoes only a single round of infection in the respiratory epithelium but evokes an immune response profile similar to wild-type influenza virus and protects ferrets against both homologous and heterologous influenza variants. Methods A blinded, randomized, placebo-controlled human challenge study (EudraCT #: 2017-004971-30) was conducted with M2SR containing HA and NA from A/Brisbane/10/2007 (H3N2). 18–55-year-old subjects received 1 IN dose of saline or 108 TCID50 of vaccine. 4 weeks later, 99 subjects were challenged IN with 106 TCID50 H3N2 A/Belgium/4217/2015 (Figures 1 and 2). Results Adverse events (AE) were similar between placebo (N = 51) and M2SR recipients (N = 48) during the 28 days after immunization. After challenge with A/Belgium/4217/2015, 35% of M2SR recipients experienced influenza infection and illness, compared with 49% of placebo subjects (Figure 3). An 18% reduction in viral load was noted after challenge for M2SR subjects. Serum microneutralization response to vaccine was detected in 54% of M2SR subjects (vs. 0/51 placebo subjects), and among these subjects a 34% reduction in viral load and 51% reduction in symptom scores was noted after challenge vs placebo. Among the 29% of subjects with post-vaccine response to both vaccine and challenge strains, a 62% reduction in viral load and 56% reduction in symptom scores was noted after challenge with highly drifted H3N2 (Figure 4). Conclusion One dose of M2SR protected healthy adults against influenza infection and illness with a highly drifted challenge strain. This is believed to be the first study to demonstrate protection against challenge with an influenza strain substantially different from the vaccine and indicates potential for improved breadth of protection by M2SR compared with current vaccines. The mild vaccine AE profile supports clinical trials of additional dose levels and regimens to enhance drifted strain protection by M2SR. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

• Waning immunity and new variants necessitate next generation SARS-CoV-2 vaccines. • The M2SR platform has a clear safety and immunogenic profile in clinical testing. • Intranasal dual influenza/COVID-19 vaccine induces systemic and mucosal antibodies. • SARS-CoV-2 M2SR protects hamsters from SARS-CoV-2 infection and lung pathology. • Dual vaccine maintains immunogenicity to matched and drifted influenza strains. The COVID-19 pandemic has highlighted the need for mucosal vaccines as breakthrough infections, short-lived immune responses and emergence of new variants have challenged the efficacy provided by the first generation of vaccines against SARS-CoV-2 viruses. M2SR SARS-CoV-2, an M2-deleted single-replication influenza virus vector modified to encode the SARS-CoV-2 receptor binding domain, was evaluated following intranasal delivery in a hamster challenge model for protection against Wuhan SARS-CoV-2. An adjuvanted inactivated SARS-CoV-2 whole virus vaccine administered intramuscularly was also evaluated. The intranasal M2SR SARS-CoV-2 was more effective than the intramuscular adjuvanted inactivated whole virus vaccine in providing protection against SARS-CoV-2 challenge. M2SR SARS-CoV-2 elicited neutralizing serum antibodies against Wuhan and Omicron SARS-CoV-2 viruses in addition to cross-reactive mucosal antibodies. Furthermore, M2SR SARS-CoV-2 generated serum HAI and mucosal antibody responses against influenza similar to an H3N2 M2SR influenza vaccine. The intranasal dual influenza/COVID M2SR SARS-CoV-2 vaccine has the potential to provide protection against both influenza and COVID. [ABSTRACT FROM AUTHOR]

Recombinant influenza virus neuraminidase (NA) is a promising broadly protective influenza vaccine candidate. However, the recombinant protein alone is not sufficient to induce durable and protective immune responses and requires the coadministration of immunostimulatory molecules. Here, we evaluated the immunogenicity and cross-protective potential of a recombinant influenza virus N2 neuraminidase vaccine construct, adjuvanted with a toll-like receptor 9 (TLR9) agonist (CpG 1018® adjuvant), and alum. The combination of CpG 1018 adjuvant and alum induced a balanced and robust humoral and T-cellular immune response against the NA, which provided protection and reduced morbidity against homologous and heterologous viral challenges in mouse and hamster models. This study supports Syrian hamsters as a useful complementary animal model to mice for pre-clinical evaluation of influenza virus vaccines. • N2-MPP provides cross-protection to heterologous seasonal H3N2 strains in two animal models. • N2-MPP vaccine provides protection to challenge with H1N2 swine influenza virus in mice. • Alum enhances the adjuvant effect of ODN 1018. [ABSTRACT FROM AUTHOR]

To study the influenza virus determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012 [QT1480]) and 2013 (A/duck/Vietnam/QT1728/2013 [QT1728]) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D (PA with the S-to-A change at position 343 and the E-to-D change at position 347) mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low-pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans. [ABSTRACT FROM AUTHOR]

Favipiravir, a viral RNA-dependent RNA polymerase inhibitor, has recently been approved in Japan for influenza pandemic preparedness. Here, we conducted a cell-based screening system to evaluate the susceptibility of influenza viruses to favipiravir. In this assay, the antiviral activity of favipiravir is determined by inhibition of virus-induced cytopathic effect, which can be measured by using a colorimetric cell proliferation assay. To demonstrate the robustness of the assay, we compared the favipiravir susceptibilities of neuraminidase (NA) inhibitor-resistant influenza A(H1N1)pdm09, A(H3N2), A(H7N9) and B viruses and their sensitive counterparts. No significant differences in the favipiravir susceptibilities were found between NA inhibitor-resistant and sensitive viruses. We, then, examined the antiviral susceptibility of 57 pairs of influenza viruses isolated from patients pre- and post-administration of favipiravir in phase 3 clinical trials. We found that there were no viruses with statistically significant reduced susceptibility to favipiravir or NA inhibitors, although two of 20 paired A(H1N1)pdm09, one of 17 paired A(H3N2) and one of 20 paired B viruses possessed amino acid substitutions in the RNA-dependent RNA polymerase subunits, PB1, PB2 and PA, after favipiravir administration. This is the first report on the antiviral susceptibility of influenza viruses isolated from patients after favipiravir treatment. [ABSTRACT FROM AUTHOR]

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans. [ABSTRACT FROM AUTHOR]