Molecular Basis of Live-Attenuated Influenza Virus

Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.     

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates.     

Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.     

Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

Background

Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes.

Methodology/Principal Findings

LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7.

Conclusions/Significance

Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for distribution by WHO to vaccine manufacturers.     

Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.     

Immune response of noninbred mice to subvirion influenza vaccines with various antigen and sorbent loads

The variants of splitted and subunit influenza monovaccines from virus strains A/Leningrad/385/80R (H3N2) and A/Kiev/59/79R (H1N1), adsorbed on aluminium hydroxide and having the varying content of hemogglutinin and the carrier, have been studied. The immune response of noninbred mice to a single and double injections of these vaccines have been evaluated, the concentrations of the antigen and the carrier inducing a high response in the animals, have been determined. Differences in the immunological potency of hemagglutinins H1 and H3 have been noted.     

Antigenic anachronism of influenza viruses A(H2N2) in Leningrad in 1980. II. The laboratory characteristics of influenza A/Leningrad/80 viruses

As indicated by the results of the hemagglutination inhibition (HAI) test, influenza viruses A/Leningrad/80 contain hemagglutinin (HA), similar to that of virus A/Singapore/1/57 (H2N2). Neuraminidase contained in viruses A/Leningrad/80 belongs to serological subtype N2 and is similar to that of virus A/Singapore/1/57 (H2N2). No differences in the polypeptide composition of the virus-induced proteins of viruses A/Leningrad/527/80, A/Leningrad/549/80, A/Leningrad/553/80 and virus A/Singapore/1/57 used as reference have been detected in the study of their electrophoretic mobility in polyacrylamide gel, as well as the mobility of duplexes obtained by the hybridization of the virion and complement RNA of viruses A/Leningrad/553/80 and A/Singapore/1/57. The results of the HAI test with antisera to purified HA indicate that virus A/Leningrad/549/80 contains HA similar to that of viruses A(H2N2) isolated in 1957, but not in 1964. The HAI test with the sera of polecats having the infection permits the differentiation of viruses A/Leningrad/80 from epidemic viruses A(H2N2) isolated in 1957-1965, including reference virus A/Singapore/1/57. In relation to the latter, the isolates of 1980 are older antigenic mutants. The isolates of 1980 are distinguished from virus A(H2N2), isolated in 1975 from the system of persisting influenza infection in a tissue culture, by mutation in NS-gene and the properties of RNA-polymerase. The authenticity of the isolation of viruses A(H2N2) in Leningrad in 1980 has been proved.     

Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.Human infections with the swine origin influenza virus A (H1N1) were first detected in April 2009 and spread across the globe, resulting in WHO declaring a pandemic on 12 June 2009 for the first time in the past 41 years. More than 296,471 people have had confirmed infections with this novel H1N1 virus, and there have been at least 3,486 deaths as of September 18, 2009. In the last century, an influenza H1N1 virus caused the devastating 1918-1919 pandemic; this pandemic was characterized by a mild outbreak in the spring of 1918, followed by a lethal wave globally in the fall of that year which killed as many as 50 million people worldwide (20, 29). The 2009 H1N1 viruses circulating globally since April 2009 have not caused a significant rise in mortality related to influenza. Nucleotide sequence analysis suggested that E627 in PB2, a deletion of the PDZ ligand domain in NS1, and the lack of the PB1-F2 open reading frame in the 2009 H1N1 viruses may contribute to the relatively mild virulence (20, 26, 27). Recent animal studies have shown that the 2009 H1N1 influenza viruses did not replicate in tissues beyond the respiratory tract and did not cause significant mortality in the ferret model; however, the 2009 H1N1 viruses are capable of infecting deep in the lung tissues and caused more significant lesions in the lung tissues of animals, including nonhuman primates, than typical seasonal strains (13, 17, 19). Children and young adults are particularly susceptible to the 2009 H1N1 virus infection because they have no or low immunity to the novel 2009 H1N1 strains (11, 13). The widespread and rapid distribution of the 2009 H1N1 viruses in humans raises a concern about the evolution of more virulent strains during passage in the population. One fear is that mutant forms of the 2009 H1N1 viruses may exhibit significantly increased virulence (2, 19). Therefore, there is an urgent need to develop an effective vaccine to control the influenza pandemic caused by the swine origin H1N1 viruses.Live attenuated influenza vaccine (LAIV) has been licensed in the United States annually since 2003. The seasonal vaccine protects against influenza illness and elicits both systemic and mucosal immune responses, including serum hemagglutination inhibition (HAI) antibodies that react to antigenically drifted strains (3, 4). A critical attribute of an effective pandemic vaccine is its capability to elicit an immune response in immunonaive individuals; LAIV has been shown to offer protection following a single dose in young children. However, two doses of vaccines are recommended for children younger than 9 years of age who have never been immunized with influenza vaccines. In order to produce LAIV to protect against the newly emerged swine origin H1N1 influenza virus, we have produced several 6:2 reassortant candidate vaccine strains that express the hemagglutinin (HA) and neuraminidase (NA) gene segments from influenza virus A/California/4/09 (A/CA/4/09) (H1N1) or A/CA/7/09 (H1N1), as well as the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) from cold-adapted A/Ann Arbor/6/60 (H2N2) (AA60) virus, which is the master donor virus for all influenza virus A strains in trivalent seasonal LAIV. Initial evaluation of these candidate vaccine strains indicated that they did not replicate as efficiently as seasonal H1N1 influenza vaccine strains in embryonated chicken eggs. In this report, we describe directed modifications of the HA gene segment that improved vaccine yields in eggs, resulting in a number of vaccine candidates that are available for human use.     

Development and Pre-Clinical Evaluation of Two LAIV Strains against Potentially Pandemic H2N2 Influenza Virus

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.     

Immunization with a Live Attenuated H7N9 Influenza Vaccine Protects Mice against Lethal Challenge

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.     

Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.     

Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.     

Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.     

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.     

2006～2007年流行流感病毒减毒疫苗株的构建

选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为骨架病毒,对其6个内部基因片段进行了全基因合成,同时人工引入5个氨基酸突变(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA来源于2006-2007当年流行株A/New Caledonia/20/99(H1N1).8个基因片段通过与改造后的转录载体pAD3000连接,构建8个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS、pMDV-A-HA、pMDV-A-NA.6质粒与当年流行株的表面基因HA和NA进行"6 2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝性的冷适应减毒的重组A型人流感病毒.鸡胚尿囊液中重组病毒的血凝效价为l:279-1:210.构建的A/AA/6/60 6个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发奠定了基础.     

Characteristics of incomplete reproduction cycle of ts-mutant of influenza virus A at non-permissive temperature

Incomplete reproduction cycle of influenza virus A/134/17/57, the attenuation donor being used for preparation of recombinant vaccine strains, hs been analyzed with the use of molecular biology methods. Virus A/134/17/57 with two mutations in P3, NP and M genes remains capable of synthesis of viral polypeptides that are devoid of ability to be inserted into cellular plasma membrane, when the virus is propagated in MDCK culture at non-permissive temperature. The process is preceded by defects in the process of formation of RNP structures connected, evidently, with deficient synthesis of viral RNA due to mutations in the gene coding for P3 protein.     

Haemagglutination-inhibiting (HI) antibodies against strains of influenza A virus in horse and pig sera in Nigeria

Sera from horses and pigs obtained from Lagos and Ibadan respectively were examined for haemagglutination-inhibiting (HI) antibodies to two strains each of H3N2 and H1N1 subtypes of influenza A virus. More horse sera had HI antibodies to the H3N2 than the H1N1 strains while pig sera reacted almost equally with strains of both subtypes. All the horse sera had HI antibodies to the two strains of H3N2 subtype (A/Mississippi/1/85 and A/Leningrad/360/86), while 87% and 14% of the horses examined were positive to A/Taiwan/1/86 and A/Chile/1/83. On the other hand HI antibody prevalence to the two subtypes in pigs are as follows, for H3N2 A/Mississippi/1/85 (86%), A/Victoria/3/75 (94%); for H1N1 A/Chile/1/83 (87%) and A/Taiwan 1/86 (79%). Analysis of the data by the Chi-square test showed significant difference between the prevalence of HI antibodies to the influenza A virus strains in horse sera examined while there was no significant difference between HI antibody prevalence to the four strains in pigs. The study shows that horses and pigs circulate influenza A virus in Nigeria and may serve as origin of human epidemics.     

海南省2016-2018年A(H1N1)pdm09亚型流感病毒基因特性分析

2009年A(H1N1)pdm09亚型流感病毒在墨西哥暴发,之后在全世界流行。为了解海南省2016-2018年A(H1N1)pdm09亚型流感病毒流行态势,分析血凝素(HA)与神经氨酸酶(NA)基因遗传进化特征与变异情况,本研究从中国流感监测信息系统获取海南省2016-2018年流感病毒病原学监测数据,选取5家流感监测网络实验室分离鉴定的37株A(H1N1)pdm09亚型流感毒株进行HA与NA基因测序,利用MEGA 10.1.8构建HA与NA基因种系进化树,并分析其氨基酸变异情况。结果显示,2016-2018年共出现3次A(H1N1)pdm09亚型流感病毒活动高峰。2017年10月份以后的分离株(4/8)与2018年大部分分离株(21/22)独立于疫苗株A/Michigan/45/2015聚为一个小支,发生20余处HA与NA氨基酸位点变异。与疫苗株A/California/7/2009(2010-2016)相比,2016-2018年流感病毒分离株在HA基因抗原决定簇上发生7处氨基酸变异并有一个潜在糖基化位点,未发现HA基因受体结合位点变异与NA基因耐药性变异。本研究提示,2016-2018年,A(H1N1)pdm09亚型流感病毒逐步发生规律性进化,氨基酸变异频率有增加趋势,今后应持续加强流感病毒病原学监测,密切追踪A(H1N1)pdm09亚型流感病毒基因变异情况,为科学防控提供理论依据。     

Antigenic anachronism of the influenza A(H2N2) viruses in Leningrad in 1980. I. The epidemiological and serological characteristics of the influenzal infection caused by A/Leningrad/80 (H2N2) viruses

In April-May 1980 a number of unrelated outbreaks of influenza-like diseases were registered in Leningrad in an infant home (50 out of 68 children under observation, aged 3 months to 2 years, were affected) and among the pupils of a boarding school (13 out of 50 adolescents under observation, aged 15-17 years, were affected). 5 strains of influenza A virus were isolated from 3 sick children and 1 clinically healthy child. A similar virus was isolated from a sick adolescent in a boarding school, as well as from a female patient aged 24 years at a domiciliary focus of infection (a sporadic case). In the subsequent laboratory investigation all these 7 strains were identified as viruses A/H2N2. Isolated cases of seroconversion to hemagglutinin H2 were definitely registered in 6 patients during February--May 1980. In 3 cases, including the 24-year old female patient with an acute respiratory disease, seroconversion to hemagglutinin H2 was observed in combination with the release of influenza viruses A/H2N2 from these patients. 2 influenza virus strains with this antigenic characteristic were isolated from a young female patient at an interval of 3 days. Among the patients admitted to the clinics of the Research Institute of Influenza in Leningrad on account of acute respiratory diseases isolated cases of diagnostically significant seroconversion to hemagglutinin H2 constituted 3.5% among children and 4.5% among adults. The study of the level of antihemagglutinins in the population revealed that in 1980 persons aged 18-50 years showed a high level of antihemagglutinins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.