The pathogenicity and transmission of live bird market H2N2 avian influenza viruses in chickens, Pekin ducks, and guinea fowl.

H2N2 subtype low pathogenic avian influenza viruses (LPAIVs) have persisted in live bird markets (LBMs) in the Northeastern United States since 2014. Although unrelated to the 1957 pandemic H2N2 lineage, there is concern that the virus could have animal and public health consequences because of high contact with humans and numerous species in the LBM system. The pathogenicity, infectivity, and transmissibility of six LBM H2N2 viruses isolated from three avian species in LBMs were examined in chickens. Two of these isolates were also tested in Pekin ducks and guinea fowl. Full genome sequence was obtained from all 6 isolates and evaluated for genetic markers for host adaptation and pathogenicity in poultry. Clinical signs were not observed in any host with any of the isolates, however one recent isolate was shed at higher titers than the other isolates and had the lowest bird infectious dose of all the isolates tested in all three species. This isolate, A/chicken/NY/19-012787-1/2019, was also the only isolate with a deletion in the stalk region of the neuraminidase protein (NA). This supports the theory that the NA stalk deletion is evidence of adaptation to gallinaceous poultry.

In Vitro Neutralization Is Not Predictive of Prophylactic Efficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and CR9114 against Lethal H2 Influenza Virus Challenge in Mice.

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs.IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

[MECHANISMS OF ATTENUATION OF COLD-ADAPTED STRAIN A/KRASNO- DAR/101/35/59 (H2N2)].

AIM: Study of mechanisms of attenuation of cold-adapted (ca) influenza virus strain A/ Krasnodar/101/35/59 (H2N2), associated with disruption of NS1 protein functions. MATERIALS AND METHODS: Study of interferonogenic activity of ca strain A/Krasnodar/101/35/59 (H2N2), its parent variant A/Krasnodar/101/59 (H2N2), virulent strain A/WSN/33 (H1N1) and a number of single gene and multiple gene reassortants between these strains, obtained using reverse genetics, was carried out. Study of dynamics of IFNß gene expression was carried out by using a methodical approach of RT-PCR in real time mode. RESULTS: Inclusion of PB-1 gene of ca strain A/ Krasnodar/101/35/59 (H2N2) with reversion to wild type into genome composition of virulent strain A/WSN/33 (H1N1) does not result in a sharp change of interferonogenic activity of the reassortant. At the same time, similar inclusion of PB-1 gene of ca strain resulted in an incredible growth of interferonogenic activity of the reassortant. On the other hand, inclusion of NP-gene of wild type strain A/Krasnodar/101/59 (H2N2) into genome composition of the wild type strain A/WSN/33 did not differ by effect on interferonogenicity of the reassortant from inclusion of NP-gene of ca strain. CONCLUSION: Both constellations of genes of parent variants and mutations localized in these genes could affect formation of attenuation phenotype of reassortants. The data obtained allow to assume possible mechanisms of attenuation of ca strains, associated with disruption.of NS gene function.

[COMPARATIVE STUDY OF IMMUNOGENICITY AND PROTECTIVE EFFECT OF LIVE COLD-ADAPTED AND INACTIVATED VACCINES AGAINST TYPE A INFLUENZA].

AIM: Direct comparative studies of immunogenicity and protective effect of live cold-adapted (ca) and inactivated vaccines against type A influenza. MATERIALS AND METHODS: Groups of mice were immunized intramuscularly (i/m) or intranasally (i/n) twice with inactivated or live ca vaccines based on wild-type parent strain A/Krasnodar/101/59 (H2N2) and the corresponding ca donor strain A/Krasnodar/101/59/30CE/5MDCK/l/7/4 (H2N2), respectively. Immunogenicity was determined by HAI antibodies in sera and lungs (extracts) against both vaccine strains. Protective effect--by the level of wild-type strain in lungs of immunized mice after the infection. RESULTS: Live ca and inactivated vaccines based on similar strains increase immunogenicity and protective effect when administered via different routes in varying patterns. A significant increase of immunogenicity was only observed for i/m (sera antibodies) and i/n (lung antibodies) administration of the live ca vaccine, and could be determined by antigenic features of the vaccine strains. At the same time, all the vaccine variants and administration routes induced at least partial protection from infection compared with unimmunized control. However, complete protection from infection was only noted for the i/m administered live ca vaccine. CONCLUSION: A combination of immunization variant and vaccine type determines immunogenicity and protective effect, and their interconnection requires further studies using all the possible combinations of preparations and administration routes as well as determination of induction of various components of the immune system.

[COLD-ADAPTED A/KRASNODAR/101/35/59 (H2N2) STRAIN--A PROMISING STRAIN-DONOR OF ATTENUATION FOR PROCURATION OF LIVE INFLUENZA VACCINES].

AIM: Study of ts, ca, att phenotype, immunogenicity and protective effectiveness of reassortants obtained by a way of recombination of a new influenza cold-adapted (ca) strain donor of attenuation A/Krasnodar/101/35/59 (H2N2) and virulent strain of influenza virus. MATERIALS AND METHODS: Viruses were used: ca strain A/Krasnodar/101/35.59 (H2N2), virulent strains: A/Kumamoto/102/02 (H3N2) and A/Bern/07/95. For determination of ts and ca phenotype, titration of viruses in chicken embryos was carried out simultaneously at optimal, decreased and increased temperature. Protective effect of immunization was evaluated during intranasal infection of mice with a virulent strain of influenza virus. RESULTS: All the obtained reassortants possessed 6 internal genes from strain-donor of attenuation and 2 genes, coding HA and NA-proteins from virulent strains. Ca reassortants were characterized by ts and ca phenotype, had antigenic specificity and good immunogenicity, had high protective effectiveness. CONCLUSION: The data obtained indicate on the perspectiveness of ca strain A/Krasnodar/101/35/59 (H2N2)as a donor of attenuation for live influenza vaccines.

Risk assessment of H2N2 influenza viruses from the avian reservoir.

H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Reemergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data show that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cage mates. Markers of mammalian adaptation in hemagglutinin (HA) and PB2 proteins were absent from all isolates, and they retained a preference for avian-like α2,3-linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of prepandemic planning.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys.

The purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription-polymerase chain reactions (rRT-PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (C(t)) value obtained from the rRT-PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT-PCR for pooled and individually tested swabs (cloacal and buccal) vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT-PCR sensitivity by pooling a weak positive swab with negative swabs on the Ct values which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT-PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.

Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques.

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.

Pandemic influenza in Africa, lessons learned from 1968: a systematic review of the literature.

BACKGROUND: To help understand the potential impact of the 2009 H1N1 pandemic in Africa, we reviewed published data from Africa of the two previous influenza pandemics. METHODS: We conducted a systematic search of three biomedical databases for articles in any language on 1957 H2N2 or 1968 H3N2 pandemic influenza virus infection in Africa published from January 1950 through August 2008. RESULTS: We identified 1327 potentially relevant articles, and 298 warranted further review. Fourteen studies on 1968 H3N2 influenza met inclusion criteria, while two studies identified describing 1957 H2N2 were excluded for data limitations. Among these 14 studies, community attack rates for symptomatic infection during all 1968 pandemic waves were around 20%. However, the proportion infected in communities ranged from 6% in isolated communities to 100% in enclosed populations. A total of 22-64% of sampled clinic patients and 8-72% of hospitalized patients had evidence of 1968 H3N2 virus infection. After the second pandemic wave, up to 41-75% of persons tested had serological evidence of 1968 H3N2 virus infection. CONCLUSION: The 1968 H3N2 influenza pandemic, generally regarded as mild worldwide, appears to have had a substantial impact upon public health in Africa. Without more epidemiologic data the impact of the 2009 H1N1 pandemic in Africa cannot be assumed to have been mild. Assessment of the burden of 2009 H1N1 virus and future influenza pandemics in Africa should attempt to assess disease impact by a variety of methods, including substudies among specific populations.

Vaccination strategies to protect children against seasonal and pandemic influenza.

To protect children against infection with seasonal influenza viruses, this age group is vaccinated annually in some countries. However, currently used inactivated seasonal influenza vaccines do not protect well against antigenically distinct pandemic influenza virus strains. Furthermore, annual vaccination may prevent infection with seasonal influenza viruses and subsequently the induction of heterosubtypic immunity. Therefore, the development of influenza vaccines that induce broad protective immunity should be considered a priority. In the absence of such vaccines children that are vaccinated annually against seasonal influenza should in a pandemic scenario also receive pandemic vaccines as soon as these become available. In order to protect young infant under six months of age for which no vaccines are registered at present, vaccination of pregnant women should be considered. This would afford protection through maternally derived antibodies. In addition, vaccination of close family members of young infants is recommended, to prevent transmission within the household.

Functional macroautophagy induction by influenza A virus without a contribution to major histocompatibility complex class II-restricted presentation.

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigen-processing pathways have been reported, little is known about their relative contributions to global CD4(+) T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4(+) T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4(+) response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen.

Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic.

The emergence of the 2009 H1N1 virus pandemic was unexpected, since it had been predicted that the next pandemic would be caused by subtype H5N1. We also had to learn that a pandemic does not necessarily require the introduction of a new virus subtype into the human population, but that it may result from antigenic shift within the same subtype. The new variant was derived from human and animal viruses by genetic reassortment in the pig, supporting the concept that this animal is the mixing vessel for the generation of new human influenza viruses. Although it is generally believed that the 2009 outbreak was mild, there have been severe cases particularly among the young and the middle-aged. Pathogenicity and host range are determined to a large extent by the polymerase, the haemagglutinin and the NS1 protein of influenza A viruses. There is evidence that mutations of these proteins may change the pathogenicity of the new virus.

The PB2 subunit of the influenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon.

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-beta) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-beta and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence.

Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model.

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.

Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic.

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 A resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses.

Human influenza viruses derive their genes from avian viruses. The neuraminidase (NA) of the avian viruses has, in addition to the catalytic site, a separate sialic acid binding site (hemadsorption site) that is not present in human viruses. The biological significance of the NA hemadsorption activity in avian influenza viruses remained elusive. A sequence database analysis revealed that the NAs of the majority of human H2N2 viruses isolated during the influenza pandemic of 1957 differ from their putative avian precursor by amino acid substitutions in the hemadsorption site. We found that the NA of a representative pandemic virus A/Singapore/1/57 (H2N2) lacks hemadsorption activity and that a single reversion to the avian-virus-like sequence (N367S) restores hemadsorption. Using this hemadsorption-positive NA, we generated three NA variants with substitutions S370L, N400S and W403R that have been found in the hemadsorption site of human H2N2 viruses. Each substitution abolished hemadsorption activity. Although, there was no correlation between hemadsorption activity of the NA variants and their enzymatic activity with respect to monovalent substrates, all four hemadsorption-negative NAs desialylated macromolecular substrates significantly slower than did the hemadsorption-positive counterpart. The NA of the 1918 pandemic virus A/Brevig Mission/1/18 (H1N1) also differed from avian N1 NAs by reduced hemadsorption activity and less efficient hydrolysis of macromolecular substrates. Our data indicate that the hemadsorption site serves to enhance the catalytic efficiency of NA and they suggest that, in addition to changes in the receptor-binding specificity of the hemagglutinin, alterations of the NA are needed for the emergence of pandemic influenza viruses.

An introduction to influenza: lessons from the past in epidemiology, prevention, and treatment.

Better understanding of viral biology and the origins of influenza epidemics and pandemics may improve diagnosis and disease control. Advances to stop the spread of disease, including live-attenuated and inactivated vaccines and new antiviral agents, promise to reduce disease burden, mortality, and morbidity.

A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets.

The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials.

Increased survival after gemfibrozil treatment of severe mouse influenza.

Gemfibrozil, an agent that inhibits production of proinflammatory cytokines in addition to its clinically useful lipid-lowering activity, increased survival in BALB/c mice that were already ill from infection by influenza virus A/Japan/305/57 (H2N2). Gemfibrozil was administered intraperitoneally once daily from days 4 to 10 after intranasal exposure to the virus. Survival increased from 26% in vehicle-treated mice (n = 50) to 52% in mice given gemfibrozil at 60 mg/kg/day (n = 46) (P = 0.0026). If this principle translates to patients, a drug already approved for human use, albeit by a different route for another purpose, might be adapted relatively fast for use against influenza, conceivably including human infection with a derivative of the avian H5N1 strain.