Influenza A virus subtype H5N2

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Pathogenesis and Transmission of Novel Highly Pathogenic Avian Influenza H5N2 and H5N8 Viruses in Ferrets and Mice

Pathogenesis and Transmission of Novel Highly Pathogenic Avian Influenza H5N2 and H5N8 Viruses in Ferrets and Mice

Avian-adapted influenza viruses bind preferentially to the ␣ -2,3-linked sialic acid receptors abundant in the gastrointestinal tract of birds, while human influenza viruses preferentially bind to ␣ -2,6-linked sialic acid receptors on cells found in the upper re- spiratory tract (40). The distribution of influenza virus receptors in the ferret respiratory tract resembles the airways of humans which may contribute to the utility of this animal model (41, 42). However, several differences between the ferret and human respi- ratory tract have been identified which could influence the out- come of infection. For example, the presence of Sda epitopes that carry ␣ -2,3-NeuAc in the ferret respiratory tract is hypothesized to reduce potential binding sites for avian influenza viruses (43, 44). The HA of H5Nx viruses possess the key residues Gln226 and Gly228 (H3 numbering) required for 2,3-linked sialic acid bind- ing, indicating the virus has not adapted toward human-type re- ceptor specificity. Interestingly, the novel H5N8 and H5N2 vi- ruses circulating in United States have a Thr-to-Ala substitution in HA at position 160, which results in a loss of a glycosylation motif at asparagine residue 158. The removal of this glycosylation site has previously been shown to be critical for H5 subtype influ- enza viruses to gain enhanced receptor binding affinity to human- like receptors and transmission in mammalian hosts (45, 46). The weak binding of the H5Nx viruses to human-like receptors (30, 47) along with reduced replication efficiency at temperatures found in the upper respiratory tract of humans likely contribute to the poor transmission of the H5Nx viruses. Others have reported similar transmission results for clade 2.3.4.4 HPAI H5N8 viruses in ferrets (4) and guinea pigs (47). However, an H5N2 virus iso- lated in China (A/duck/Eastern China/1112/2011) that had high nucleotide identity with H5N8 viruses circulating in South Korea FIG 4 Replication kinetics of influenza viruses in polarized human airway epithelial cells. Calu-3 cells grown on transwells were infected apically in triplicate at an MOI of 0.01 with Pin/WA/40964 (H5N2), Gyr/WA/41088-6 (H5N8), Bris/59 (H1N1), or Th/16 (H5N1). The cells were incubated at 37°C (A) or 33°C (B), and culture supernatants were collected at 2, 16, 24, 48, and 72 h p.i. for virus titer determination by standard plaque assay. Asterisks indicate the statistical significance between Th/16 and other tested H5Nx viruses (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).
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Emergence and Evolution of Avian H5N2 Influenza Viruses in Chickens in Taiwan

Emergence and Evolution of Avian H5N2 Influenza Viruses in Chickens in Taiwan

Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available informa- tion suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multi- ple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the preva- lence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003.
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Circulation of the low pathogenic avian influenza subtype H5N2 virus in ducks at a live bird market in Ibadan, Nigeria

Circulation of the low pathogenic avian influenza subtype H5N2 virus in ducks at a live bird market in Ibadan, Nigeria

extraction. This was followed by one-step real-time re- verse transcription-polymerase chain reaction (RT-PCR). Primers and fluorescence probes were obtained from the WHO Influenza Collaborating Laboratory, Center for Disease Control (CDC) in Atlanta, USA. The system combines superscript III reverse transcriptase (RT) and platinum Taq DNA Polymerase in a single enzyme mix: both cDNA synthesis and PCR are performed in a single reaction. Gene specific primers and probes designed for the generic matrix gene (F: 5′-AGATGAGTCTTCTA ACCGAGGTCG-3′ R: 5′-TGCAAAGACACTTTCCAG TCTCTG-3′, FAM- 5′- TCAGGCCCCCTCAAAGCC GA-3′) were used. The master mix was prepared by dis- pensing 5.5 ul nuclease free water; 0.5 ul of forward, reverse primers, and probe; 0.5 ul superscript Tag poly- merase; and 12.5 ul PCR master mix enzyme (2×) for a single reaction multiplied by the number of samples tested. The Applied Biosystems fast real-time PCR system 7,500 thermocycler, and software (Applied Biosystems, Foster City, CA, USA) were used with the following cycling con- ditions: 50°C for 30 minutes, 95°C for 15 minutes, followed by 40 cycles of 95°C for 10 seconds, and 60°C for 30 sec- onds and 5–60 seconds [7]. Samples that were positive for the M-gene, which codes for the matrix protein common to all influenza A viruses, were subtyped under the same conditions with H5 (F: 5′- TTATTCAACAGTGGCGAG- 3′, R: 5′- CCAKAAAGATAGACCAGC-3′, P: 5′- CC CTAGCACTGGCAATCATG-3′) and H1 (F: 5′-GTG CTA TAA ACA CCA GCC TYC CA-3′, R: 5′-CGG GAT ATT CCT TAA TCC TGT RGC 3′, P: 5′- CA GAA TAT ACA “T”CC RGT CAC AAT TGG ARA A 3′).
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Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy

The inability of LPAI vaccines to effectively protect against infection with antigeni- cally drifted viruses or newly emerging viruses underlines the need for the develop- ment of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. In this study, we demonstrate that immunization of mice with the cHA H9/H5N2 vaccine induced broad cross-reactive antibody responses that protected immunized hosts against homologous and heterologous lethal challenges with H5N8 and maH5N2 viruses (Fig. 7 to 9). In contrast, all H9N2-vaccinated mice succumbed to death by 13 dpi (Fig. 9B and C). It should be noted that the chimeric H9/H5N2 vaccine groups also showed about 15% weight loss for up to 7 days following maH5N2 virus challenge (Fig. 9C), although most mice recovered after 7 dpi. These results might be explained by the small amino acid variations between the HA2 regions of viruses of the same H5 subtype. Although the HA2 regions have been considered to be conserved within the same groups of viruses (47), our amino acid comparison results show that there is 96% (8/220) amino acid homology between the HA2 regions of H5N8 and maH5N2 viruses, even though they were clustered in the same HA group, group I (Fig. 1C). For comparison, ⬍ 65% identity between H9 and H5 viruses was observed. There- fore, the different protection efficacies between strains might be explained by se- quence variations in HA2 regions, which were previously considered to be conserved within the same subtype.
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Newly Emergent Highly Pathogenic H5N9 Subtype Avian Influenza A Virus

Newly Emergent Highly Pathogenic H5N9 Subtype Avian Influenza A Virus

H5N9 virus is an infrequently isolated subtype among influ- enza A viruses. Until 2013, most of the isolated H5N9 viruses were of low pathogenicity, with the exception of the pathogenic virus A/turkey/Ontario/7732/1966 (H5N9) (22) distributed in North America and Europe (Fig. 7A). In Asia, only low-pathogenicity H5N9 viruses have been isolated, at Aomori in Japan in 2008. The host range of H5N9 viruses developed gradually from turkey (On- tario, 1966) to mallards, northern pintails, emus, and occasionally chickens (Fig. 7B). However, to date, the highly pathogenic H5N9 subtype avian influenza virus has not been isolated in Eurasia. In our study, the novel H5N9 virus carrying HA protein with the PQRERRRKR/GL motif, which is characteristic of HPAIV, was first isolated from chickens with coexistent AIV subtypes in LBMs in China. This implies that a Eurasian HPAI H5N9 virus was pos- sibly revealed and prompted vigilance in the poultry industry. More interestingly, the novel H5N9 virus belongs to clade 2.3.2.1 (Fig. 2) and matches the inactivated vaccine against clade 2.3.2.1 FIG 5 Virulence of YH1 virus in mice. (A) Body temperature. Groups of five
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Comparison of Two Multiplex Methods for Detection of Respiratory Viruses: FilmArray RP and xTAG RVP

Comparison of Two Multiplex Methods for Detection of Respiratory Viruses: FilmArray RP and xTAG RVP

Multiplex respiratory virus PCR. The FilmArray RP detects the following agents: influenza A virus, influenza A virus subtype H1, influenza A virus subtype H3, influenza A virus subtype H1N1 swine-origin variant, influenza B virus, respiratory syncytial virus, human metapneumovirus, coronavirus NL63, corona- virus OC43, coronavirus 229E, coronavirus HKU1, adenovirus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, boca- virus, rhinovirus/enterovirus, Bordetella pertussis, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. The xTAG RVP detects influenza A virus, influenza A virus subtype H1, influenza A virus subtype H3, influenza B virus, respiratory syncytial virus, human metapneumovirus, adenovirus, parainfluenza 1 virus, parainfluenza 2 virus, parainfluenza 3 virus, and rhinovirus/enterovirus. Both assays include internal controls for amplification and extraction and were per- formed according to the manufacturer’s instructions, following training by the respective companies. The FilmArray RP pouch contains dried reagents for all the steps needed for extraction, PCR amplification, and detection of the respi- ratory viruses listed above. As shown in Fig. 1, the pouch is rehydrated under negative pressure with 1 ml molecular reagent-grade water in the reagent port. Two hundred fifty microliters of sample is diluted into 0.5 ml sample buffer, of which 300 ␮l is injected into the sample port. The pouch is then placed in the FilmArray RP instrument and identified by bar code, and the assay is started. Results are available in about an hour. For the xTAG RVP, nucleic acid extrac- tion with the addition of an extraction control was done with a Roche MagNA Pure compact instrument using a 200-␮l sample eluted in 50 ␮l, of which 5 ␮l was used for the assay. The remaining extracted nucleic acid and aliquots of the original frozen specimen were stored at ⫺ 70°C for further testing. The xTAG is currently FDA cleared only for nasopharyngeal swabs and extraction with the bioMe ´rieux EasyMag, the bioMe ´rieux MiniMag, and the Qiagen QIAamp MiniElute.
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Antiviral Susceptibility of Avian and Swine Influenza Virus of the N1 Neuraminidase Subtype

Antiviral Susceptibility of Avian and Swine Influenza Virus of the N1 Neuraminidase Subtype

Analysis of the NA sequences of the isolates that had re- duced oseltamivir susceptibility revealed a number of muta- tions outside the catalytic and framework residues. Structural analysis suggests that these mutations may indirectly affect the stability of the active site residues and their interaction with oseltamivir. It has been documented that residues far away from an enzymatic active site can affect the enzymatic activity indirectly, through “energy channels” throughout the protein (2, 38), and this may be the case for the NA enzyme of influ- enza virus as well. Interestingly, a number of residue changes, such as V267I and N307D, appeared in two of three of the avian isolates with reduced oseltamivir susceptibility. One iso- late carried a V321I substitution. Residues V267 and N307 are near a hydrophobic patch that may be important for stabilizing active site residues H274 and E276 (Fig. 3B). Residue V267 resides in the hydrophobic patch, and mutation to a bulkier isoleucine may distort this region. Residue N307 forms a hy- drogen bond with E311, and mutation of N307 to negatively charged aspartate would likely disrupt this hydrogen bond and cause repulsion. Repulsion of E311 would disrupt hydrophobic patch residues, which again might affect the stabilization of active site residues H274 and E276. Another strain of avian influenza virus with reduced susceptibility had NA mutations K262R and V321I. Residue K262 lies on the surface of NA and is unlikely to affect oseltamivir binding upon mutation to the similar residue arginine. Taken together, these data suggest that residues outside the NA active site, such as V267I, N307D, and V321I, may distort hydrophobic pockets and indirectly affect the NA catalytic and framework residues. These residues may be new potential markers for reduced susceptibility to oseltamivir. It is also possible that two or more mutations could be acting in concert. Analysis of 188 sequences of avian influ- enza viruses of N1 NA subtype available in the public domain revealed that changes in the NA residues determined in our study apparently occur among the wild waterbird species but at a relatively low frequency. Thus, these NA mutations do not appear to have a selective advantage and probably would not be maintained in wild birds. It is also possible that a combina- tion of mutations located outside the NA active site act to- gether and is the basis for the reduced susceptibility to oselta- mivir in some isolates. One report identified three NA mutations in avian H5N1 viruses (K150N, I222L, and S246N) that had decreased susceptibility to oseltamivir (4).
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Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

NP readout. The replication of influenza viruses was also assessed by measuring the production of NP by ELISA with the following procedure: at various time points postinoculation of the MDCK cells, the cell culture plates were washed once with Oxoid PBS (product no. BR0014G; Fisher Scientific, Landsmeer, The Netherlands), fixed by adding 200 ␮ l acetone (80% in water) to each well, and stored at ⫺ 20°C. After removal of ace- tone, the wells were washed trice with PBS-0.05% Tween 20 (Merck Mil- lipore, Amsterdam, The Netherlands) before adding the broadly reactive (13, 14) influenza A virus NP-specific mouse monoclonal antibody HB65 (European Veterinary Laboratory [EVL], Woerden, The Netherlands). Approximately 100 ng of antibody HB65 was added per well in a volume of 200 ␮ l PBS containing 2% skim milk powder (PBS-SMP) (product no. 70166; Sigma). After incubation for 1 h at room temperature, the plates were washed three times with PBS-0.05% Tween 20, and 3 ng of a horse- radish peroxidase-labeled goat anti-mouse IgG antibody preparation (product no. 626520; Invitrogen) was added in 100 ␮ l PBS-SMP and incubated for 1 h at room temperature. Following three wash steps with PBS-0.05% Tween 20, ready-to-use 3,3=,5,5=-tetramethylbenzidine (TMB) (product no. T0440; Sigma; 100 ␮ l/well) was added, and the plates were incubated at room temperature for 30 min before adding 100 ␮ l stop solution (1N H 2 SO 4 ) (product no. 320501; Sigma). The optical densities
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A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

FIG. 2. Generation of nine-segment influenza viruses carrying both subtype H1 and H3 HAs. (A) Generation of PB1-HA(HK)-PB1 and PB2-HA(HK)-PB2 constructs. The A/HK/1/68 HA ORF was amplified from a pCAGGS-HK HA plasmid (27) by PCR and used to replace the GFP ORF of PB1-GFP-PB1 and PB2-GFP-PB2 constructs in Fig. 1A, generating the PB1-HA(HK)-PB1 and PB2-HA(HK)-PB2 constructs. (B) Genome structure of ⫺ PB1(ps) ⫹ HK HA virus. Similar to ⫺ PB1(ps) ⫹ GFP virus in Fig. 1B, the virus contained a chimeric NA-PB1mut-NA segment instead of a wild-type PB1, seven A/PR/8/34 segments (PB2, PA, HA, NP, NA, M, and NS), and a ninth PB1-HA(HK)-PB1 chimeric segment. (C) Genome structure of ⫺ PB2(ps) ⫹ HK HA virus. The chimeric PB2-HA(HK)-PB2 segment was used to replace the PB2-GFP-PB2 of the ⫺ PB2(ps) ⫹ GFP virus in Fig. 1C, generating the ⫺ PB2(ps) ⫹ HK HA virus. (D) Growth curves of viruses in 10-day-old embryonated chicken eggs at 37°C. The error bars represent standard deviations. (E) Western blot to detect the A/PR/8/34 and A/HK/1/68 HAs in purified virions. Viruses [rA/PR/8/34, X31, ⫺ PB2(ps) ⫹ HK HA and ⫺ PB1(ps) ⫹ HK HA] were grown in eggs at 37°C and purified through a 30% sucrose cushion. A Western blot was performed to detect the presence of NP and HA proteins using specific mouse monoclonal antibodies: PY102 for A/PR/8/34 HA0 and HA1 (26), HT103 for A/PR/8/34 NP (21), 66A6 for A/HK/1/68 HA0 and HA1, and 12D1 for A/HK/1/68 HA0 and HA2 (27). (F) Western blot to detect the A/PR/8/34 and A/HK/1/68 HAs in virus-infected MDCK cells. MDCK monolayers were infected by viruses [rA/PR/8/34, X31, ⫺ PB1(ps) ⫹ HK HA, and ⫺ PB2(ps) ⫹ HK HA] at an MOI of 10 to 0.0001. One day postinfection, the cells were washed with PBS and harvested using 2 ⫻ protein loading buffer (100 mM Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 20% glycerol, 5% ␤ -mercaptoethanol, 0.2% bromophenol blue) and run on a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The A/PR/8/34 HA0, NP, and A/HK/1/68 HA0 were detected by monoclonal antibodies PY102, HT103, and 66A6, respectively (21, 26, 27). (G) H1/H3 sandwich ELISA to determine whether both subtype H1 and H3 HA proteins were incorporated into the same particles of the ⫺ PB1(ps) ⫹ HK HA and ⫺ PB2(ps) ⫹ HK HA viruses (see Materials and Methods). OD, optical density. The error bars represent standard deviations. (H) Analysis of the vRNA genome packaging efficiency of the recombinant viruses. Four recombinant viruses [rA/PR/8/34, X31, ⫺ PB1(ps) ⫹ HK HA, and ⫺ PB2(ps) ⫹ HK HA] were grown in eggs at 37°C, and purified viral RNA was separated (0.5 ␮ g/lane) on a 2.8% acrylamide gel and visualized by silver staining. The rRNA band was confirmed based on size and previously reported findings. The identity of an additional band marked with “?” is unknown.
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Sequence Analysis and Phylogenetic Study of Hemagglutinin Gene of H9N2 Subtype of Avian Influenza Virus Isolated during 1998-2002 in Iran

Sequence Analysis and Phylogenetic Study of Hemagglutinin Gene of H9N2 Subtype of Avian Influenza Virus Isolated during 1998-2002 in Iran

Sequence analysis and phylogenetic study of hemagglutinin (HA) gene of H9N2 subtype of avian influenza virus isolates (outbreaks of 1998-2002) in Tehran province (Iran) were studied. Two sets of forward and reverse primers in highly conserved regions, based on sequences of HA gene in Genbank, were designed. PCR products of a 430-bp fragment of 16 isolates were sequenced and then were aligned with the reported sequences in Genbank. Nucleotide sequence comparisons of HA gene from Iranian isolates showed 97-99% identity within the group, and 98% homology with the two isolates [A/Parakeet/Narita/92A/98 (H9N2)] and [A/Parakeet/Chiba/1/97 (H9N2)] from Pakistani parakeets imported to Japan. On the basis of phylogenetic evidence, it is proposed that the emergence of H9N2 avian influenza infection in Iran originated in Pakistan, and it was due to low quarantine measures in the international boundaries. Due to the high percentage of H9N2 homology isolates of Iran with other isolates, namley A/quail/HongKong/G1, in Genbank and based on published reports for high similarity with infecting human H5N1 isolates, it seems that the potential of Iranian avian influenza isolates to infect human should be considered. Iran. Biomed. J. 8 (4): 167-172, 2004
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Application of Subtype Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

Application of Subtype Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

When the influenza A virus H2 strains recur in the future, it becomes impossible to distinguish H1 strains from H2 strains with C179. However, the similarity of the DNA sequences of the genes for the H1 and H2 stem regions is so high (7) that it is hard to raise monoclonal antibodies which recognize H1 stem regions but not H2 stem regions by immunizing mice with viral proteins. Therefore, we are now planning to raise H1 strain-specific monoclonal antibodies by immunizing mice with the DNA coding for the stem region of H1 z HA. On the other hand, we have produced monoclonal antibodies against influ- enza type B virus which react to all type B strains. Applications for anti-B monoclonal antibodies in newly developed methods are under investigation, and it should facilitate the clearer detection and identification of viruses in clinical specimens.
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Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene

Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n ⴝ 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occa- sional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.
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Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High Resolution Melting Analysis

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High Resolution Melting Analysis

Most of the methods currently available for influenza A virus subtyping use subtype-specific primers to amplify the HA or the NA gene, which allows the detection of only one specific pathogen at a time (36). For sample screening and subtype identification in an outbreak, multiplexing or multiple reac- tions followed by post-PCR electrophoresis to separate ampli- cons of different sizes are usually required. Although real-time RT-PCR has successfully been applied to the detection of influenza A virus, a TaqMan probe or a hybridization probe is usually needed, and the use of these probes can increase the expense on a cost-per-sample basis (8, 25). Alternatively, anal- ysis of M-gene sequence variants forms a basis for HA subtyp- ing. The principle of this type of assay is based on phylogenetic analysis, which reveals the preferential association between the influenza A virus HA and M genes and the coevolution of these genes (24, 32). This notion allows the design of an assay for analysis of the M-gene nucleotide components and the differentiation of influenza A virus HA subtypes. For instance, restriction fragment length polymorphism analysis of the M- gene PCR product has been applied to the subtyping of human influenza A viruses (6, 17). A real-time LightCycler hybridiza- FIG. 3. HRM plots and derivative plots for influenza A virus subtyping. (A to C) High-resolution melting and derivative plots were obtained by heteroduplex formation (HD) between the PCR products of the indicated influenza A virus subtype and the reference strain of H1N1 (A/Taiwan/421/2006) or H5N1 (A/HongKong/156/97). The melting (A) and derivative (A and C) plots reveal the HRM profile for each virus subtype. The derivative plots for five to six measurements of each influenza A virus subtype reveal the minimal interassay variability (B). Note the consistent derivative plot pattern for each virus subtype. (D) Tenfold serial dilutions of H3N2 plasmid DNAs (from 2.8 ⫻ 10 8 to 28 copies) were
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Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

The extraction of viral RNA was conducted from a virus containing allantoic fluid using a spin column purifica- tion kit (Koma Biotech. Inc., Korea). The influenza sub- type was first screened using H5, H7, H9, N1, N2 and N3 specific primers (Table 1). Amplification of internal genes was performed with gene-specific primers for the six viral genes: PB2, PB1, PA, HA, NP, NA, M and NS (Table 1) using a Koma one step RT PCR kit (Koma Bio- tech. Inc., Korea). PCR amplicons were subjected to electrophoresis in a 1.5% agarose gel. Specific bands of expected sizes were excised and purified using a QIA- quick gel extraction kit (Qiagen, Germany). Purified RT- PCR products were sequenced directly in both forward and reverse directions (Macrogen, Korea). Different gene sequences were assembled by trimming primer-linker. Sequence data of the H9N2 Egyptian strain isolated in the current study are available in the GenBank database (Accession No: JQ611701 - JQ611708).
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Cocirculation of Two Distinct Lineages of Equine Influenza Virus Subtype H3N8

Cocirculation of Two Distinct Lineages of Equine Influenza Virus Subtype H3N8

encoding amino acids 84 to 256. This region of HA was chosen because it contains all amino acids which have been shown to make up the receptor binding pocket of the molecule, and it also contains all amino acids composing antigenic regions A, B, and D. These regions have been demonstrated in previous studies to be extremely variable among equine influenza virus subtype H3N8 strains (1, 12), and they are presumably under strong immune system-mediated selective pressure in the horse. A comparison of the deduced amino acid sequences generated in this study with those of the most recently isolated Swedish virus (BOL/96 [11]) (Fig. 1) revealed complete ho- mology of samples CS1 to CS9 with BOL/96 with the exception of position 190. Position 190 forms the membrane-distal limi- tation of the receptor binding site in the human H3N2 influ- enza virus strain (14) and is thus significant in determining both the affinity and the specificity of receptor binding of this virus. To exclude the possibility that the CS1 to CS9 sequences originated from the same virus due to laboratory contamina- tion, we determined the entire sequences of the HA proteins of CS1, CS5, and CS9. The complete sequence of CS10 HA was also determined. All sequences showed heterogeneity: CS1 and CS5 differed at positions in the HA1 region, CS9 differed from CS1 only in the HA2 region (alanine to leucine change at position 42), and CS10 differed from CS1 at a number of positions. Amino acid differences in HA1 are shown in Fig. 2A. On the basis of this sequence heterogeneity we conclude that samples CS1 to CS10 are of different origins. CS10 shows extreme divergence from the BOL/96 strain, with substantial differences at antigenic regions B and C and also at the recep- tor-binding site. Comparisons with sequences of previously reported isolates of equine influenza virus show that CS10 has more likeness to European-lineage equine influenza virus than to American-lineage virus, to which BOL/96 belongs. A se- quence alignment of CS10 HA1 with HA1 of the most recently isolated Swedish strain of the European lineage (AVE/93) (Fig. 2B) shows the close relatedness of these two strains at the amino acid level.
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Factors associated with clinical outcome in 25 patients with avian influenza A (H7N9) infection in Guangzhou, China

Factors associated with clinical outcome in 25 patients with avian influenza A (H7N9) infection in Guangzhou, China

Guangzhou is located in the southern portion of Guangdong Province, in the center of the Pearl River Delta. On January 11, the first case of A (H7N9) was identified, and by April 8, 2014, 25 cases had been reported. Fourteen patients (56 %) had died. This death rate was much higher than that of other areas, which ranged from 10 % in Hangzhou city [9] to 39.4 % in Shanghai [10]. This is of particular concern, as Guangzhou is a highly developed city in China with an advanced medical infrastructure, and the HA and NA sequences of the H7N9 viral isolates from Guangzhou patients are identical to those of H7N9 virus strains recently isolated from human patients in other areas (A/Anhui/1/2013, A/Shanghai/1/2013). The present retrospective study on A (H7N9) patients in Guangzhou was conducted to better describe the clinical characteris- tics of human A (H7N9) patients and evaluate the influen- tial factors associated with the clinical outcomes for the management of high risk patients.
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Pooling Nasopharyngeal/Throat Swab Specimens To Increase Testing Capacity for Influenza Viruses by PCR

Pooling Nasopharyngeal/Throat Swab Specimens To Increase Testing Capacity for Influenza Viruses by PCR

Reverse transcription and DNA amplification. Detection of the 2009 H1N1 virus and seasonal influenza viruses A and B was performed using the CDC rRT-PCR swine flu panel (24) and the CDC rRT-PCR flu panel, respectively. The CDC rRT-PCR swine flu panel included primers and TaqMan probes for seasonal influenza viruses (InfA), universal swine in- fluenza virus A (swInfA), the swine influenza virus H1N1 (swH1) subtype, and RNase P (RP). The InfA primer and probe set was designed to detect all influenza A viruses, that for swInfA to detect all swine influenza A viruses, and that for swH1 to specifically detect the 2009 H1N1 virus. The RP primer and probe are designed to detect the human RNase P gene, and they serve as an internal positive control for human nucleic acid. PCR was performed according to the CDC protocol (24) using the SuperScript III platinum one-step quantitative RT-PCR kit (Invitrogen Corporation, Carlsbad, CA) on the Applied Biosystems (ABI) 7500 Fast real-time PCR system (Applied Biosystems, Inc., Foster City, CA).
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Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I

Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I

tion of C-terminal regulatory domain by Riplet (RING finger pro- tein leading to RIG-I activation) and the subsequent complex for- mation between RIG-I and IPS-1 (beta interferon promoter stimulator 1, also known as MAVS, VISA, or Cardif) on the sur- face of the mitochondria (13). The ensuing signal cascade causes the nuclear translocation of IRF3, IRF7, ATF-2/c-Jun, and NF- ␬ B, which activate the IFN- ␤ promoter and promote the expression of IFN (14). Finally, hundreds of interferon-stimulated genes (ISGs), such as those encoding PKR, 2=,5=-OAS, Mx1, and ADAR1, among others, are produced and exert their antiviral function via multiple mechanisms (15). Of note, RIG-I has been found not only in humans and mammals but also in a wide variety of other animal species, such as zebrafish, amphibians, and birds (16, 17). Interestingly, Galliformes (e.g., chickens) appear to have lost through evolution the gene that encodes RIG-I, although it is maintained in other avian species such as in Anseriformes (ducks and geese) (18, 19). This difference has been suggested as a possi- ble explanation for the increased susceptibility of chickens com- pared to that of ducks to highly pathogenic influenza viruses (18). To survive in host cells, influenza A viruses have evolved vari- ous strategies to circumvent the IFN response. The NS1 protein, a nonstructural protein generated during the early stages of virus infection, is a well-characterized IFN inhibitor (20). NS1 can limit IFN induction by various strategies, including (i) sequestration of viral dsRNA away from host-encoded RNA sensors (21), (ii) for- mation of complexes with RIG-I (9, 10, 22), (iii) suppression of TRIM25- and/or Riplet-mediated RIG-I ubiquitination (16, 22), and (iv) blocking of cellular pre-mRNA processing and transport and thus decreasing synthesis of antiviral proteins involved in in- nate immunity (23, 24). Apart from NS1, other influenza virus proteins also possess IFN-antagonizing capabilities. PB1-F2 can act on MAVS (25, 26) and/or interfere with the RIG-I/MAVS pro- tein complex (27), thereby inhibiting IFN production. In addi- tion, the PB2 protein (28, 29) from human influenza A virus alone or in the context of the three viral polymerase subunits has been shown to interact with MAVS and impair host antiviral responses mediated by IFN (30).
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Insights into Avian Influenza Virus Pathogenicity: the Hemagglutinin Precursor HA0 of Subtype H16 Has an Alpha-Helix Structure in Its Cleavage Site with Inefficient HA1/HA2 Cleavage

Insights into Avian Influenza Virus Pathogenicity: the Hemagglutinin Precursor HA0 of Subtype H16 Has an Alpha-Helix Structure in Its Cleavage Site with Inefficient HA1/HA2 Cleavage

2/99 (H16N3) was cloned and expressed in a baculovirus expres- sion system, as described previously (51). H16HA was crystallized at pH 7.0, and its structure was solved by MR to a high resolution of 1.7 Å using the H2 structure (PDB accession number 2WR0) as a search model (Table 1). A mutant with V327G of H16HA (HA16-V327G), representing some natural isolates of H16 sub- type, has also been solved at a resolution of 2.0 Å (Table 1). Each of the two structures has only one molecule in the asymmetric unit. The crystal structure shows that H16HA exists as a classical ho- motrimer with two distinct domains: a stem domain and a glob- ular head domain (Fig. 1A). The membrane-proximal stem do- main is composed of HA2 and two segments of HA1, residues 1 to 55 and 275 to 329. The C-terminal half of HA2 from three HA molecules forms a triple-stranded ␣ -helical coiled coil as the ho- motrimer core surrounded by the N-terminal half of the HA2 and HA1 segments. This region also contains the cleavage site where host enzymes normally cut HA0 into a disulfide bond-linked HA1 and HA2 complex. On top of the stem domain, the membrane- distal globular domain contains the receptor binding subdomain and the vestigial esterase subdomain. There are eight potential N-linked glycosylation sites, i.e., N20, N21, N46, N169, N170, N291, N474, and N483, but only two of them (N169 and N483) are visible in our structure. For glycosylation site N169, two N- acetylglucosamine (NAG) sugar monomers are observed, and only one NAG sugar monomer is observed in the N483 glycosyla- FIG 1 Overall structure of H16H0 and HA grouping based on 3D structures. (A) Overview of the H16HA0 trimer, represented as a ribbon diagram. For clarity, each monomer has been colored differently (A, green; B, blue; C, cyan). Carbohydrates observed in the electron density maps are colored orange. (B) Phyloge- netic tree of the 17 HA subtypes. The 17 HA subtypes can be divided phylogenetically into two groups on the basis of their full-length sequences: group 1 (blue) and group 2 (red). H16, highlighted in green, belongs to group 1. (C to E) Superimpositions of the long ␣ helix (residues 76 to 126) of HA2 reveal the displacements of the HA1 globular subdomain in seven HA subtype structures. The 190 helix (residues 188 to 195) in the receptor binding domain is used as a representative of the globular subdomain of each HA subtype. H1 is colored light blue, H2 is wheat, H3 is lemon, H5 is light orange, H7 is yellow, H9 is pink, H14 is pale yellow, and H16 is green. When we use H16 as a reference, the group 1 members rotate downward with negative-angle degrees (from ⫺ 10.82° to ⫺ 19.28°), while the group 2 members rotate upward with positive-angle degrees (from 4.08° to 10.78°).
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Isolation and Genetic Characterization of H5N2 Influenza Viruses from Pigs in Korea

Isolation and Genetic Characterization of H5N2 Influenza Viruses from Pigs in Korea

Neither virus had an E-to-K mutation at position 627 of the PB2 protein, which was responsible for the high virulence of A/Hong Kong/483/97 in a mammalian host model (16). The avian-like PB2/627E present in the swine-like PB2 is common for all Korean swine influenza isolates and does not represent a K-to-E mutation in the PB2 genes of the isolates in this study. Neither of their M2 proteins had the necessary mutations at residue 31 (Ser to Asn) to confer amantadine resistance (17, 20), nor did they have glutamic acid at position 92 of the NS1 molecule for escaping host antiviral cytokine responses (46). To determine whether the pig-passaged H5 isolates had un- dergone further substitutions or mammalian-like adaptations, viruses recovered from experimentally inoculated pigs (includ- ing from the contact swine of Sw/Kor/C13/08) and ferrets were sequenced. Analysis showed that the sequences of viruses ob- tained from pigs bore the same sequences as the swine farm isolates. Interestingly, viruses recovered from ferrets incurred additional sequence differences indicative of continuous ge- netic evolution (Table 5). In summary, the molecular analysis of the viral proteins of the swine H5N2 viruses clearly showed that they maintained a low-pathogenicity form, and no muta- tions related to virulence, drug resistance, or a shift in host preference were acquired.
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