Antibodies to influenza viruses (including the human A2-Asian-57 strain) in sera from Australian shearwaters (Puffinus pacificus)

Sera were collected from 320 shearwaters (Puffinus pacificus chlororhynchus) nesting on two islands off the east coast of northern Australia in December 1969. About 10% of these sera specifically inhibited the neuraminidase of the 1957 strain of human influenza (A2/Asian/57), some to high titre.     

Characterization and ecology of a type A influenzavirus isolated from a sheawater

An influenzavirus isolated from a shearwater bird nesting on Tryon Island on the Australian Great Barrier Reef in 1971 has been more extensively characterized. Haemagglutinin subunits were isolated from the shearwater virus and from the antigenically related avian influenzaviruses A/turkey/Mass./65 (Hav6N2) and A/duck/Penn./69 (Hav6N1). Maps of the tryptic peptides from the heavy polypeptides (HA1) of the haemagglutinin subunits of the three viruses showed a number of differences, but peptide maps of the light polypeptides (HA2) were almost identical, suggesting that these had almost the same amino acid sequence. Extensive tests confirmed that the neuraminidase of the shearwater virus was not related antigenically to any known neuraminidase. The sera collected from pelagic birds nesting on islands in the Capricorn—Bunker group in 1970 were devoid of any antibodies to the shearwater virus, while a high proportion of the sera collected from birds on the same islands in 1972 (one year after the isolation of the shearwater virus) had antibodies to the haemagglutinin and neuraminidase of the shearwater virus, some to a high titre. Thus, the shearwater virus appeared to have only recently been introduced into the area from where it was isolated.     

Comparison of influenza viruses isolated from man and from whales.

Four isolates of influenza virus strains from Moscow and Habarovsk that caused outbreaks of influenza in November and December 1977 in several cities of the USSR were studied and their haemagglutinins and neuraminidases were compared with those of other human and animal influenza viruses including A/whale/Pacific Ocean/76. In H1 tests these isolates, designated A/USSR/77, reacted with immune serum against A/FM/1/47 (H1N1) to the homologous titre, and with antiserum against A/whale/PO/19/76 virus to 1/8 of the homologous titre. In neuraminidase inhibition tests all A/USSR/77 isolates showed the presence of human N1 type neuraminidase, more closely related to A/sw/New Jersey/76 (Hsw1N1) than to A/FM/1/47 (H1N1) virus. The haemagglutinin of A/whale/Pacific Ocean/19/76 virus occupies an intermediate position between H0 and H1, but its neuraminidase is close to Nav2. The virus from whales multiplies better at low (28°C) and at high (40°C) temperatures than do the viruses of human origin that were tested.     

Antigenic variants of influenza A virus obtained in vitro.

The aim of this study was to investigate antigenic “drift” in the haemagglutinin and neuraminidase antigens of influenza A virus in vitro under immunological pressure. Variants of the “Asian” influenza strains A/England/12/64 (H2N2) and A/Tokyo/3/67 (H2N2) were isolated in the allantois-on-shell system in the presence of homologous postinfection ferret sera. For each of these two viruses three generations of variants were isolated and characterized. It was found that the successive antigenic variants of A/Eng/12/64 did not resemble A/Tokyo/3/67. Thus it is probable that the pathway of antigenic drift in vitro was not the same as that which occurred in nature during the evolution of A/Tokyo/3/67 from A/Eng/12/64. In addition, A/Tokyo/3/67, which was the last strain to be prevalent before the A/Hong Kong subtype appeared, underwent significant antigenic drift from “junior” to “senior” variants. This finding did not support the concept that, when antigenic drift occurs, resulting in the appearence of viruses with new haemagglutinin antigen subtypes, the previously prevalent strain has no capacity for further antigenic drift. The study did not result in the production of strains that were identifiable as “bridging” viruses between the H2 and H3 haemagglutinin subtypes. The present paper includes the first report of antigenic variation in the neuraminidase antigens of influenza A viruses occurring in vitro under immunological pressure.     

Intercontinental spread of Asian-origin H7 avian influenza viruses by captive bird trade in 1990's

Wild bird migration and illegal trade of infected poultry, eggs, and poultry products have been associated with the spread of avian influenza viruses (AIV). During 1992–1996, H7N1 and H7N8 low pathogenic AIV (LPAIV) were identified from captive wild birds; such as Pekin robin (Leiothrix lutea), magpie robin (Copsychus saularis), flycatcher sp. (genus Empidonax), a species of softbill and parakeet, sun conure (Aratinga solstitialis), painted conure (Pyrrhura picta), fairy bluebird (Irena puella), and common iora (Aegithina tiphia), kept in aviaries or quarantine stations in England, The Netherlands, Singapore and the United States (U.S.). In this study, we sequenced these H7 viruses isolated from quarantine facilities and aviaries using next-generation sequencing and conducted a comparative phylogenetic analysis of complete genome sequences to elucidate spread patterns. The complete genome sequencing and phylogenetic analysis suggested that H7 viruses originated from a common source, even though they were obtained from birds in distant geographical regions. All H7N1 and H7N8 viruses were LPAIV, except a H7N1 highly pathogenic AIV (HPAIV), A/Pekin robin/California/30412/1994(H7N1) virus. Our results support the continued need for regulation of the captive wild bird trade to reduce the risk of introduction and dissemination of both LPAIV and HPAIV throughout the world.     

Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates

The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q–G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L–S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q–G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.     

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.     

Generation and Characterization of Recombinant Influenza A(H1N1) Viruses Resistant to Neuraminidase Inhibitors

ObjectivesTo examine the effect of neuraminidase (NA) mutations on the NA inhibitor (NAI) resistance phenotype, the recombinant influenza A/Chungbuk/4448/2008(H1N1) virus isolated in South Korea during the 2008–2009 season was generated by reverse genetics.MethodsSite-directed mutagenesis was introduced on the NA gene of A/Chungbuk/4448/2008(H1N1) virus, and a total of 23 single, double, and triple mutants were generated. Resistance phenotype of these recombinant viruses was determined by NA-inhibition (NAI) assays based on a fluorometric method using two NAIs (oseltamivir and zanamivir).ResultsNA-inhibition assays showed that all the single and double mutants containing the Y275 except the single Y275-E119V mutant conferred important levels of resistance to oseltamivir, whereas all the single, double, and triple mutants containing the E119V mutation were associated with the resistance to zanamivir.ConclusionConsidering the effect of mutations in NA gene on the resistance to NAIs, it is important to monitor the possible emergence and dissemination of multidrug-resistant variants in the human population due to amino acid changes at NA gene as well as to develop novel NAIs.     

The importance of antineuraminidase antibodies in resistance to influenza A and immunologic memory for their synthesis.

Eight hundred and seventy-seven sera from 360 adults aged 18-50 who were under permanent observation from October 1980 to March 1981 have been studied by haemagglutination-inhibition (HI) and erythrocyte elution-inhibition (EI) tests--a simplified method of antineuraminidase antibody titration. It was demonstrated in some subjects infected with influenza A H1N1 and H3N2 viruses that the antibody rise was to one of the surface antigens only--haemagglutinin or neuraminidase. These subjects made up 5.2-25.8% of all examinees. The protective effect of antibodies to neuraminidase was similar to that of antihaemagglutinins. Interaction of both types of antibodies was observed in protection against the disease. Data have been obtained on the influence of antineuraminidase antibodies in decreasing the severity of natural infection with influenza A. A study of heterologous immunologic responses to haemagglutinin and neuraminidase among persons immunized with live influenza A H1N1 and H3N2 vaccines and among children naturally infected with influenza A H3N2 demonstrated the presence of immunologic memory for antineuraminidase antibody synthesis. Thus, the suggestion of a common antigenic structure for neuraminidase N1 and N2 is made.     

Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.     

Characteristics of a swine recombinant influenza virus isolated in 1980: recombination between swine and the earliest Hong Kong (H3N2) viruses

A recombinant (H1N2, formerly Hsw1N2), A/swine/Ehime/1/80 was found to possess antigenic, biological and genomic characteristics different from those of a previous A/swine/Kanagawa/2/78 (H1N2) strain¹. Five monoclonal antibodies to A/NJ/8/76 differentiated the haemagglutinin molecules of the former virus from the latter, showing that these viruses differed at two-antigenic determinants at least. Immuno-double diffusion tests with antisera to the isolated neuraminidase and neuraminidase-inhibition tests with monoclonal antibodies to different H2N2 and H3N2 viruses revealed that A/swine/Ehime/1/80 strain contained a neuraminidase subunit very similar to that of late human Asian (H2N2) and the earliest Hong Kong (H3N2) viruses. RNA analysis by oligonucleotide mapping suggested that A/swine/Ehime/1/80 may be a recombinanat between A/swine/Shizuoka/1/78-like and A/Aichi/2/68 (H3N2)-like viruses. To determine further the gene constellation of this recombinant virus, DNA-RNA hybridizations were performed using DNA segments complementary for swine (H1N1) virus RNA and the entire RNA of three viruses. The molecular hybridization could define the genomic composition of the recombinant, indicating that only the neutraminidase gene of this virus is derived from the earliest Hong Kong (H3N2)-like virus and remaining seven genes are derived from swine (H1N1) virus.     

Recycling of H1N1 influenza A virus in man--a haemagglutinin antibody study.

Sera from people born between 1883 and 1930 and collected in 1977 were tested for the presence of HI antibodies to A/FM/1/47 (H1N1) virus and three recently (1977 and 1978) isolated influenza A-H1N1 viruses. The highest frequency of high-titred antibody to the four H1N1 viruses was detected in sera from people born in 1903-4, i.e. 42, 54, 38, and 22% had antibody against A/FM/1/47, A/Hong Kong/117/77, A/Brazil/11/78, and A/Fukushima/103/78 respectively. The birthdate groups 1896-1907 showed a higher percentage of HI antibody titres greater than or equal to 18, greater than or equal to 50, greater than or equal to 100 or greater than or equal to 1600 against the four H1N1 viruses than the birthdate groups 1907-30. This indicates the existence of an era, 1908-18, in which, apart from the H3N2 virus (1900-18), the H1N1 virus was epidemic among the human population.     

Lead,mercury, and cadmium in feathers of tropical terns in Puerto Rico and Australia

Levels of lead, cadmium, and mercury were examined in breast feathers of terns nesting on offshore islets near Culebra, Puerto Rico and on Michaelmas Cay and Heron Island on the Great Barrier Reef, Australia. Levels of all metals in these tropical terns were predicted to be lower than those of terns nesting in temperate regions, because the tropical species feed offshore of non-industrial areas where contamination should be less than for temperate-nesting species that feed in inshore estuaries near industrialized areas. This prediction was not supported by the evidence. In Puerto Rico, lead and cadmium levels were highest in bridled tern (Sterna anaethetus), and mercury levels were highest in sooty (S. fuscata) and roseate tern (S. dougallii). In Australia, levels of lead and mercury were higher in black noddy (A. minutus) and lower for sooty tern; and cadmium levels were highest for brown noddy (A. stolidus) and sooty tern, and lowest for black noddy. Metal levels for the tropical terns nesting in Puerto Rico and Australia generally were not lower than levels reported for temperate-nesting or mainland nesting birds (except for mercury in Australia).     

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant region of hepatitis B core antigen: Broad protective efficacy of particles carrying four copies of M2e

A long-term objective when designing influenza vaccines is to create one with broad cross-reactivity that will provide effective control over influenza, no matter which strain has caused the disease. Here we summarize the results from an investigation into the immunogenic and protective capacities inherent in variations of a recombinant protein, HBc/4M2e. This protein contains four copies of the ectodomain from the influenza virus protein M2 (M2e) fused within the immunodominant loop of the hepatitis B virus core antigen (HBc). Variations of this basic design include preparations containing M2e from the consensus human influenza virus; the M2e from the highly pathogenic avian A/H5N1 virus and a combination of two copies from human and two copies from avian influenza viruses. Intramuscular delivery in mice with preparations containing four identical copies of M2e induced high IgG titers in blood sera and bronchoalveolar lavages. It also provoked the formation of memory T-cells and antibodies were retained in the blood sera for a significant period of time post immunization. Furthermore, these preparations prevented the death of 75–100% of animals, which were challenged with lethal doses of virus. This resulted in a 1.2–3.5 log 10 decrease in viral replication within the lungs. Moreover, HBc particles carrying only “human” or “avian” M2e displayed cross-reactivity in relation to human (A/H1N1, A/H2N2 and A/H3N2) or A/H5N1 and A(H1N1)pdm09 viruses, respectively; however, with the particles carrying both “human” and “avian” M2e this effect was much weaker, especially in relation to influenza virus A/H5N1. It is apparent from this work that to quickly produce vaccine for a pandemic it would be necessary to have several variations of a recombinant protein, containing four copies of M2e (each one against a group of likely influenza virus strains) with these relevant constructs housed within a comprehensive collection Escherichia coli-producers and maintained ready for use.     

Human H5N2 avian influenza infection in Japan and the factors associated with high H5N2-neutralizing antibody titer

Background

H5N2 avian influenza virus infection of humans has not been reported thus far. The first H5N2 avian influenza infection of poultry in Japan occurred in Ibaraki.

Methods

The subjects were workers at 35 chicken farms in Ibaraki Prefecture, where the H5N2 virus or antibody was isolated from chickens. None of the subjects exhibited influenza symptoms. The H5N2-neutralizing antibody titers of the first and second paired sera samples were compared. To investigate the possible factors for this increase, the H5N2-neutralizing antibody titer (1:40 or more) was calculated for the second samples. A logistic regression analysis was performed to examine the association of these factors with H5N2-neutralizing antibody positivity.

Results

We performed Wilcoxon matched-pairs signed-ranked test on data collected from 257 subjects, and determined that the H5N2 antibody titers of the second paired sera samples were significantly higher than those of the first samples (P < 0.001). The H5N2 antibody titers of paired sera of 13 subjects without a history of seasonal influenza vaccination within the previous 12 months increased 4-fold or more. The percentage of antibody positivity was 32% for subjects with a history of seasonal influenza vaccination (28% of all subjects) and 13% for those without a history of the same. The adjusted odds ratio of H5N2-neutralizing antibody positivity was 4.6 (95% confidence interval: 1.6-13.7) for those aged over 40 and 3.1 (95% confidence interval: 1.6-6.1) for those with a history of seasonal influenza vaccination within the previous 12 months.

Conclusion

The results suggest that this may have been the first avian influenza H5N2 infection of poultry to affect humans. A history of seasonal influenza vaccination might be associated with H5N2-neutralizing antibody positivity.Key words: Influenza, Human Influenza, H5N2, Neutralization Tests, Vaccination     

Antigenicity in hamsters of inactivated vaccines prepared from recombinant influenza viruses.

Inactivated vaccines prepared form influenza virus strains obtained by the recombination of A/PR/8/34 (H1N1) or A/FM/1/47 (H1N1) viruses with A/Victoria/3/75 (H3N2) virus, were tested for their antigenicity in hamsters. The parental origin of the genes of each cloned recombinant virus was determined by polyacrylamide gel electrophoresis, and vaccines prepared from each strain by concentration, purification on sucrose density gradients and inactivation with formalin. All the recombinant strains used in these studies possessed surface haemagglutinin and neuraminidase antigens derived from the A/Victoria/75 parent strain. On inoculation into hamsters, at equivalent concentrations, these vaccines varied in their ability to induce haemagglutination-inhibiting (HI) antibodies in the serum. This variation was not dependent on concentration and was observed using neutralization and single radial haemolysis, as well as HI. The possible reasons for the findings are discussed.     

Oseltamivir-Resistant Influenza A(H1N1)pdm09 Viruses,United States, 2013–14

We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013–14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance–conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.     

Evaluation of the zoonotic potential of a novel reassortant H1N2 swine influenza virus with gene constellation derived from multiple viral sources

In 2011–2012, contemporary North American-like H3N2 swine influenza viruses (SIVs) possessing the 2009 pandemic H1N1 matrix gene (H3N2pM-like virus) were detected in domestic pigs of South Korea where H1N2 SIV strains are endemic. More recently, we isolated novel reassortant H1N2 SIVs bearing the Eurasian avian-like swine H1-like hemagglutinin and Korean swine H1N2-like neuraminidase in the internal gene backbone of the H3N2pM-like virus. In the present study, we clearly provide evidence on the genetic origins of the novel H1N2 SIVs virus through genetic and phylogenetic analyses. In vitro studies demonstrated that, in comparison with a pre-existing 2012 Korean H1N2 SIV [A/swine/Korea/CY03-11/2012 (CY03-11/2012)], the 2013 novel reassortant H1N2 isolate [A/swine/Korea/CY0423/2013 (CY0423-12/2013)] replicated more efficiently in differentiated primary human bronchial epithelial cells. The CY0423-12/2013 virus induced higher viral titers than the CY03-11/2012 virus in the lungs and nasal turbinates of infected mice and nasal wash samples of ferrets. Moreover, the 2013 H1N2 reassortant, but not the intact 2012 H1N2 virus, was transmissible to naïve contact ferrets via respiratory-droplets. Noting that the viral precursors have the ability to infect humans, our findings highlight the potential threat of a novel reassortant H1N2 SIV to public health and underscore the need to further strengthen influenza surveillance strategies worldwide, including swine populations.     

Antibody status to influenza A/Singapore/1/57(H2N2) in Finland during a period of outbreaks caused by H3N2 and H1N1 subtype viruses.

The incidence of haemagglutination inhibition (HI) antibody (titre greater than or equal to 12) to influenza A/Singapore/1/57(H2N2) in sera collected from a Finnish population in the summer of 1981 was 58%. Subjects born after 1968 were essentially seronegative, and a comparable low HI antibody status was also recorded among the elderly, the lowest being in people born during the period 1901-10. A small increase in antibody titre to the H2N2 virus was observed in the different age groups after infections with the H3N2, but not the H1N1, subtype influenza A viruses. The heterotypic response, which could be due to HI or NA antibodies, was restricted almost exclusively to subjects already exhibiting this antibody in acute phase sera. Moreover, the anamnestic boosting was not as strong as that described in earlier studies from samples collected at the beginning of the present era of H3N2 viruses. At population level, the HI antibody status to H2N2 was about the same at the beginning and end of the follow-up period which covered eight epidemic seasons. The results are discussed with respect to the doctrine of ''original antigenic sin'' and to the threat of re-emergence of the H2N2 viruses.