Crystallization and peptide maps of neuraminidase “heads” from H2N2 and H3N2 influenza virus strains

Neuraminidase “heads” of two H2N2 influenza virus strains (A/Nederlands/68 and A/Korea/68) and one H3N2 strain (A/Hong Kong/68) were isolated by pronase digestion of recombinant viruses possessing neuraminidase molecules from these strains and H (NWS) hemagglutinin. The neuraminidase heads were purified and crystallized by dialysis against distilled water. Large flat crystals, which differed in appearance between the three strains, were obtained. Tryptic peptide maps of the crystallized neuraminidase heads of the Nederlands/68 and Hong Kong/68 strains were almost identical, suggesting that a virus closely related to the Nederlands/68 (H2N2) strain may have been the source of the neuraminidase for the formation of the H3N2 Hong Kong subtype.     

Persistence of Q strain of H2N2 influenza virus in avian species: Antigenic,biological and genetic analysis of avian and human H2N2 viruses

Summary The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5^–/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific -inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42° C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42° C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957.With 2 Figures     

Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003

Our aim was to establish the phylogenetic relation of H9N2 avian viruses in the Middle East to other Asian H9N2 lineages by characterization of 7 viruses isolated from United Arab Emirates (2000-2003). All these viruses had an additional basic amino acid at the hemagglutinin-connecting peptide; 6 contained a mutation associated with increased affinity toward human-like sialic acid substrates. The viruses' surface glycoproteins and most internal genes were >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) lineage. The hemadsorbing site of neuraminidase had up to 4 amino acid substitutions, as do human pandemic viruses. M2 sequence analysis revealed amino acid changes at 2 positions, with increasing resistance to amantadine in cell culture. They replicated efficiently in inoculated chickens and were successfully transmitted to contacts. They continue to maintain H5N1-like genes and may augment the spread of H5N1 viruses through regional co-circulation and inapparent infection. These viruses may present as potential pandemic candidates themselves.     

A novel means of identifying the neuraminidase type of currently circulating human A(H1) influenza viruses

With the recent emergence and spread of influenza A(H1N2) viruses which appear to have arisen by reassortment of circulating A(H1N1) and A(H3N2) strains, there is a need in epidemiological studies to determine the neuraminidase type in order to differentiate between influenza A(H1N2) and A(H1N1) strains. A fluorescence-based neuraminidase enzyme inhibition assay that has been developed to screen influenza viruses for potential resistance to the neuraminidase inhibitor drugs appears to be suitable for this purpose. When used with the neuraminidase inhibitor zanamivir the assay was able to provide a positive predictive value of 93.5% for the identification of neuraminidase type N1 or N2. This assay enables a large number of influenza A viruses to be screened at low cost to determine relative levels of A(H1N2) or A(H1N1) viruses circulating in the population.     

Mitogenicity of influenza hemagglutinin glycoproteins and influenza viruses bearing H2-hemagglutinin.

The hemagglutinin glycoprotein is responsible for the mitogenic effect of influenza A viruses of the H2N2 subtype. This was indicated by the ability of viruses bearing the H2-hemagglutinin glycoprotein, regardless of its associated neuraminidase, to induce lymphocyte proliferation in normal spleen cell suspensions and by the ability of antisera with specificity for the H2-hemagglutinin to block this response. Moreover, purified hemagglutinin from representative viruses from the H0N1, H1N1, H2N2, H3N2, and influenza B subtypes were also shown to be mitogenic.     

Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza A viruses.

Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza. A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6. The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 degrees C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals.     

Circulation of the influenza A virus of H13 serosubtype among seagulls in the Northern Caspian (1979-1985)

The results of seven-year ecologo-virological studies (1979-1985) of Laridae colonies on the island Zhemchuzhnyi, northern Kaspian Sea, showed annual isolation of influenza A viruses. Altogether, 95 hemagglutinating agent have been isolated. Strains with 4 different combinations of surface antigens were identified: H5N2, H13N2, H13N3, H13N6. The possibility of transovarial transmission is confirmed by the fact of isolation of an influenza virus strain A/black-headed herring gull/Astrakhan/458/85 (H13N6) from a nestling having no contacts with the environment. Simultaneous circulation of influenza A viruses (in 1983--H13N2 and H13N6, in 1985.--H13N3 and H13N6) and the presence in the virion of neuraminidase of human influenza virus (N2) allow to consider the isolates to be natural recombinants.     

Phylogenetic analysis of an H1N2 influenza A virus isolated from a pig in Korea

Summary. An influenza H1N2 virus was isolated from a pig during an severe outbreak of respiratory disease in a Korean herd. The neuraminidase (NA) and PB1 genes of the H1N2 isolate were of human origin, while the hemagglutinin (HA), matrix (M), nucleoprotein (NP), and non-structural (NS) genes were of swine origin and PA and PB2 gene were of avain origin. Phylogenetic results indicate that the Korean H1N2 isolate was closely related to H1N2 viruses isolated recently from pigs in the United States.     

Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray

A novel strain of influenza A (H1N1) virus was isolated in Mexico and the US in March and April 2009. This novel virus spread to many countries and regions in a few months, and WHO raised the level of pandemic alert from phase 5 to phase 6 on June 11, 2009. The accurate identification of H1N1 virus and other human seasonal influenza A viruses is very important for further treatment and control of their infections. In this study, we developed an oligonucleotide microarray to subtype human H1N1, H3N2 and H5N1 influenza viruses, which could distinguish the novel H1N1 from human seasonal H1N1 influenza viruses and swine H1N1 influenza viruses. The microarray utilizes a panel of primers for multiplex PCR amplification of the hemagglutinin (HA), neuraminidase (NA) and matrix (MP) genes of human influenza A viruses. The 59-mer oligonucleotides were designed to distinguish different subtypes of human influenza A viruses. With this microarray, we accurately identified and correctly subtyped the reference virus strains. Moreover, we confirmed 4 out of 39 clinical throat swab specimens from suspected cases of novel H1N1.     

Antiviral susceptibility profile of influenza A viruses; keep an eye on immunocompromised patients under prolonged treatment

There was an increase in severe and fatal influenza cases in Greece during the 2011–2015 post-pandemic period. To investigate causality, we determined neuraminidase (NA) inhibitor susceptibility and resistance-conferring NA and hemagglutinin (HA) mutations in circulating influenza type A viruses during the pandemic (2009–2010) and post-pandemic periods in Greece. One hundred thirty-four influenza A(H1N1)pdm09 and 95 influenza A(H3N2) viruses submitted to the National Influenza Reference Laboratory of Southern Greece were tested for susceptibility to oseltamivir and zanamivir. Antiviral resistance was assessed by neuraminidase sequence analysis, as well as the fluorescence-based 50 % inhibitory concentration (IC₅₀) method. Five influenza A(H1N1)pdm09 viruses (2.2 %) showed significantly reduced inhibition by oseltamivir (average IC₅₀ 300.60nM vs. 1.19nM) by Gaussian kernel density plot analysis. These viruses were isolated from immunocompromised patients and harbored the H275Y oseltamivir resistance-conferring NA substitution. All A(H1N1)pdm09 viruses were zanamivir-susceptible, and all A(H3N2) viruses were susceptible to both drugs. Oseltamivir-resistant viruses did not form a distinct cluster by phylogenetic analysis. Permissive mutations were detected in immunogenic and non immunogenic NA regions of both oseltamivir- resistant and susceptible viruses in the post-pandemic seasons. Several amino acid substitutions in the HA1 domain of the HA gene of post-pandemic viruses were identified. This study indicated low resistance to NAIs among tested influenza viruses. Antiviral resistance emerged only in immunocompromised patients under long-term oseltamivir treatment. Sequential sample testing in this vulnerable group of patients is recommended to characterise resistance or reinfection and viral evolution.     

A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1

The neuraminidase of influenza viruses is the target of the inhibitors oseltamivir and zanamivir. Recent reports on influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAI) are a cause for concern. Several amino acid substitutions, each as a consequence of one single nucleotide mutation, are known to confer resistance to NAI. An increase of NAI-resistant viruses appears to be likely as a result of a wider application of NAI for treatment and prophylaxis of seasonal influenza infections. Monitoring the occurrence and spread of resistant viruses is an important task. Therefore, RT-PCR assays were developed with subsequent pyrosequencing analysis (PSQ-PCR). These assays allow a rapid, high-throughput and cost-effective screening of subtype A/H1N1, A/H3N2, and A/H5N1 viruses. Various specimens such as respiratory swabs, allantoic fluid, or cell-propagated viruses can be used and results are available within hours. Several A/H1N1, A/H3N2, and A/H5N1 viruses isolated from human and avian specimens were tested to evaluate the method. Positive controls encoding resistance-associated mutations were created using site-directed mutagenesis. The results obtained with these controls showed that the assay can discriminate clearly the wild-type virus from a mutant virus. The detection limit of minor virus variants within the viral quasispecies amounts to 10%.     

深圳市2008-2009年A型H1N1季节性流感病毒神经氨酸酶抑制剂耐药性监测

目的 对深圳市2008-2009年分离到的H1N1季节性流感病毒神经氨酸酶(NA)抑制剂的耐药性进行监测.方法 根据原始临床样本的采集时间,按周抽取了55株2008-2009年分离到的H1N1季节性流感病毒,对其NA片段进行全长测序,选取WHO推荐的疫苗株和部分国内外分离到的H1N1季节性流感病毒作为参考株,运用Mega3.1软件进行种系发生树的构建、耐药相关位点及糖基化位点的分析.结果 对NA片段的序列分析发现2008年有2株(7.1%)出现了H275Y突变,但是2009年则有25株(92.6%)出现了该突变.提示H275Y达菲耐药突变株成为了2009年深圳市社区传播的优势株.同时还发现了一株Q136K变异株,显示对乐感清出现耐药.分子进化分析结果显示,H275Y变异成为了毒株在系统进化树上分布的主要依据.所有的深圳株NA片段上潜在的糖基化位点序列保守.结论 大量H275Y达菲耐药株的出现提示在今后的工作中应当密切关注流感病毒的耐药进展,进一步加强其耐药机制的研究.

Abstract：

Objective To analyze neuraminidase(NA) inhibitor resistance of seasonal H1N1 influenza A viruses isolated in Shenzhen during 2008 to 2009. Methods The NA gene of these viruses were sequenced. Phylogenetic analysis of the sequences was performed with Mega3. 1 software. Results In 2008, most isolates of the seasonal H1 N1 virus were susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance had been detected in 92.6% of the strains isolated in 2009. Furthermore, a strain with Q136K was found, which showed the resistance to Zanamivir. Conclusion In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in influenza virus should be a priority.     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.     

Genetic characterization of avian influenza viruses isolated in Israel during 2000–2006

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000–2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005–2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.     

Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March–April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.     

Antigenic specificity of neuraminidase and structure of neuraminidase gene of influenza A(H1N1) serotype Hsw1N1 viruses isolated from man and animals

The enzyme immunoassay and neuraminidase activity inhibition test using polyclonal and monospecific antineuraminidase sera were employed to establish the similarities and differences in the antigenic structure of neuraminidase of influenza A viruses (H1N1), serovariant Hsw1N1, isolated from man in Alma-Ata (USSR), 1984, New Jersey (USA), 1976, and Pazardjik (BPR), 1982, as well as from swine and birds. Oligonucleotide mapping revealed significant structural differences in the genes coding for neuraminidase of Hsw1N1 viruses. The experimental results indicate a high degree of the enzyme variability in this group of viruses.     

Influenza virus neuraminidase with hemagglutinin activity

Isolated intact influenza virus neuraminidase (NA) molecules of the N9 subtype have been found to possess hemagglutinin (HA) activity which, at equivalent protein concentration, was fourfold higher than that of isolated hemagglutinin molecules of the H3 subtype. The amino-terminal sequence of the N9 NA is the same as in neuraminidases of the eight other influenza A virus NA subtypes previously reported. Viruses possessing N9 NA therefore have two different HA activities and antibody to either HA or NA alone was incapable of inhibiting hemagglutination by the virus. However, antibody to the HA of an H1N9 virus neutralized its infectivity as effectively as it neutralized H1N1 or H1N2 viruses whose neuraminidases have no HA activity. (Antibodies to N9 NA did not neutralize the infectivity of viruses with N9 neuraminidase). 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid inhibited N9 NA activity but had no effect on the HA activity of the isolated N9 NA. One interpretation of this result would be that the HA and NA activities are located in separate sites. Pronase-released N9 NA heads form crystals suitable for X-ray diffraction studies and preliminary data to 2.9 A establish the space group as cubic, I432 with cell dimension a = 184 A. Data extend to beyond 1.9 A resolution, and these will be collected in the future.     

Influenza virus recombination. I. Matrix protein markers and segregation during mixed infections.

The M (membrane or matrix) proteins from three type A influenza viruses, A/BEL/42 (H0N1), A/Port Chalmers/73 (H3N2) and A/Shearwater/Aust/72 (Hav6Nav5) were isolated and digested with trypsin, and the tryptic peptides were mapped. The maps were almost identical, but a small number of clearcut differences could be seen. One peptide, in particular, on the maps of Port Chalmers and Shearwater virus M proteins, was absent from the BEL M protein map. This peptide, which contained eight amino acids, could be resolved from the others by one-dimensional electrophoresis and served as a convenient marker for the matrix proteins of viruses obtained during recombination experiments.Embryonated chicken eggs were mixedly infected with BEL and Port Chalmers viruses or with these two viruses plus the Shearwater virus. Yields from these mixed infections were cloned at limit dilution without antibody selection and the hemagglutinin and neuraminidase subunits of viruses obtained in this way were characterised antigenically. The M proteins of viruses obtained in the two-parental cross were isolated and identified by one-dimensional peptide mapping. Results showed (i) that during recombination the M protein segregated independently with respect to the hemagglutinin and neuraminidase and (ii) that no stable virus was obtained after cloning which had more than one kind of hemagglutinin or neuraminidase subunit. These results suggest that, while incorporation of the pieces of RNA into the influenza virus genome may occur randomly, a mechanism exists which ensures that each infectious virus particle contains one copy and only one of each piece of RNA. It is not known whether this mechanism involves coordinate packaging of the genome during virus maturation or a selection of those virus particles containing only one copy of each piece of RNA.     

Antigenic relationships of strains of the epidemic virus H1N1 isolated in November-December 1977 in the USSR to inluenza viruses of human and animal origin

The results of the study on the antigentic properties of influenza viruses causing an epidemic outbreak in November, 1977, in the USSR established their relationship with human influenza viruses H1N1 to the homologous titer, with H0N1 to 1/16 titer and the virus isolated from whales in the Pacific ocean (H0-H1Nav2) to 1/8 titer. The epidemic strains have been shown to possess neuraminidase of human influenza virus type 1.