Analysis of the Codon Usage Pattern of HA and NA Genes of H7N9 Influenza A Virus.

Novel H7N9 influenza virus transmitted from birds to human and, since March 2013, it has caused five epidemic waves in China. Although the evolution of H7N9 viruses has been investigated, the evolutionary changes associated with codon usage are still unclear. Herein, the codon usage pattern of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), was studied to understand the evolutionary changes in relation to host, epidemic wave, and pathogenicity. Both genes displayed a low codon usage bias, with HA higher than NA. The codon usage was driven by mutation pressure and natural selection, although the main contributing factor was natural selection. Additionally, the codon adaptation index (CAI) and deoptimization (RCDI) illustrated the strong adaptability of H7N9 to Gallus gallus. Similarity index (SiD) analysis showed that Homo sapiens posed a stronger selection pressure than Gallus gallus. Thus, we assume that this may be related to the gradual adaptability of the virus to human. In addition, the host strong selection pressure was validated based on CpG dinucleotide content. In conclusion, this study analyzed the usage of codons of two genes of H7N9 and expanded our understanding of H7N9 host specificity. This aids into the development of control measures against H7N9 influenza virus.

Ongoing genetic evolution of H9N2 avian influenza viruses in Iranian industrial poultry farms.

Despite the use of wide-scale vaccination programmes against the H9N2 virus, enzootic outbreaks of H9N2 avian influenza (AI) have often occurred and caused serious nationwide economic losses, particularly in broiler chickens. In this study, the haemagglutinin (HA) and neuraminidase (NA) genes of nine recent H9N2s and a common vaccine strain were fully sequenced and compared with other representative Iranian viruses. Phylogenetic analysis revealed that all Iranian viruses were grouped into the G1 sub-lineage with different clusters in which recent isolates (2014-2017) formed a distinct cluster compared to the vaccine group (1998-2004). All Iranian H9N2s exhibited low pathogenicity AI connecting peptide feature with an R/KSSR motif. Amino acid 226, located in the 220 loop of the receptor binding site, was leucine among the recent Iranian viruses, a characteristic of human influenza viruses. With an overall gradual increase in the genetic diversity of H9N2s, Bayesian skyline plots of Iranian HA and NA genes depicted a fluctuation and a relative stable situation, respectively. It is recommended to apply constant surveillance to assess any increase in viral human adaptation and evolutionary changes in circulating field H9N2s. Moreover, antigenic characterisation of the prevailing H9N2 viruses seems to be necessary for evaluating the possible antigenic drift from the vaccine strain.

Genetic changes in influenza A(H3N2) viruses circulating during 2011 to 2013 in northern India (Lucknow).

Genetic variability in the hemagglutinin (HA1) and the neuraminidase (NA) genes of influenza viruses results in the emergence of new strains which differ in pathogenicity and severity. The present study was undertaken for genotypic characterization of the HA1 and NA genes of the influenza A(H3N2) strains, detected during the 2011-2013. A total of fifty five influenza A(H3N2) positive samples [2011 (n = 20), 2012 (n = 4) and 2013 (n = 31)] were studied. The 824 bp segment of HA1 gene and 931 bp segment of NA gene were amplified and sequenced by Big-Dye terminator kit on ABI3130, Genetic analyzer. Molecular and phylogenetic analysis was done by MEGA 5.05 software and PhyML program (v3.0). Mutations were determined by comparing the deduced amino acid sequences of study strains with that of 2009-2013 vaccine strains. The studied influenza A(H3N2) strains showed 98.1-99.6% similarity in HA1 and NA amino acid sequences with the influenza A/Victoria/361/2011 vaccine strain. Four mutations in the HA1 amino acid sequences (T128A, R142G, L157S and N278K) and three unique mutations in the NA amino acid sequences [D251V, S315G and V313A] were found. These mutations were observed only in strains from the year 2013 (cluster II). None of the strains showed the presence of mutations, N294S and R292K, markers of oseltamivir resistance. In conclusion, Lucknow strains have accumulated the significant number of mutations in the antigenic sites of the HA and the NA coding sequences and continue to be evolving from the 2013 vaccine strain [A/Victoria/361/2011], however, mutations specific for oseltamivir resistance were not detected.

Naked neck gene and intermittent thermal manipulations during embryogenesis improve posthatch performance and thermotolerance in slow-growing chickens under tropical climates.

Many studies have shown that thermal manipulations during the incubation (TMI) and naked neck gene (Na) positively affect heat-stressed broilers' thermotolerance, hatching process, and posthatch performance. Their combination could increase the beneficial effect on broilers reared under natural tropical climatic conditions. The aim of this study was to investigate the effects of the Na gene and TMI on hatching and posthatch performance of slow-growing broilers under tropical climates. The study included 1,200 hatching eggs from 2 different crosses: 1) females and males, both with a normal or fully feathered neck (na na group), and 2) females (with a normal neck) and males (bare neck) (Na na group), incubated in similar conditions until d 7. Thereafter, they were assigned to 3 subgroups for each cross: the control group (C) was incubated at standard incubation conditions (37.8°C, 60% RH). The TMI-1 group was subjected to TMI-1 (T = 38.5°C, RH = 65%, E10-18, 6 h/d) and TMI-2 group to TMI-2 (T = 39.5°C, RH = 65%, E7-16, 12 h/d). Between 450 and 504 h of incubation, eggs were checked for hatching events. During the posthatch phase, chicks from each incubation subgroups (Na na-C, Na na-TMI-1, Na na-TMI-2, na na-C, na na-TMI-1, na na-TMI-2) were raised for 12 wk at a tropical natural ambient temperature. Hatchability, hatching time, chick's temperature, final body weight (FBW), and feed conversion ratio (FCR) were determined. The results revealed that the Na gene reduced (P ˂ 0.05) hatchability. The control group had the highest mortality rate compared to TMI-1 and TMI-2 groups. There was an interaction between genotype and TMI on incubation duration, hatching weight, chick quality, FBW, and FCR (P ˂ 0.05). In conclusion, the Na gene influenced the effects of thermal manipulation. TMI-1 combined with Na gene improved the productive performances of broilers in a tropical climate.

Molecular Characterization of Influenza A/H3N2 Virus Isolated from Indonesian Hajj and Umrah Pilgrims 2013 to 2014.

The Hajj and Umrah are the annual mass gatherings of Muslims in Saudi Arabia and increase the transmission risk of acute respiratory infection. This study describes influenza infection among pilgrims upon arrival in Indonesia and the genetic characterization of imported influenza A/H3N2 virus. In total, 251 swab samples with influenza-like illness were tested using real-time RT-PCR for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and influenza viruses. Complete sequences of influenza A/H3N2 HA and NA genes were obtained using DNA sequencing and plotted to amino acid and antigenicity changes. Phylogenetic analysis was performed using a neighbour-joining method including the WHO vaccine strains and influenza A/H3N2 as references. The real-time RT-PCR test detected 100 (39.5%) samples positive with influenza with no positivity of MERS-CoV. Mutations in the HA gene were mainly located within the antigenic sites A, B, and D, while for the NA gene, no mutations related to oseltamivir resistance were observed. Phylogenetic analysis revealed that these viruses grouped together with clades 3C.2 and 3C.3; however, they were not closely grouped with the WHO-recommended vaccine (clades 3C.1). Sequences obtained from Hajj and Umrah pilgrims were also not grouped together with viruses from Middle East countries but clustered according to years of collection. This implies that the influenza A/H3N2 virus mutates continually across time.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

BACKGROUND: Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. RESULTS: An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. CONCLUSIONS: The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Molecular evolution of influenza A/H3N2 viruses HA and NA genes circulating in Shenzhen in 2017 / 中华疾病控制杂志

Objective To investigate the molecular evolution of the Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 viruses in Shenzhen in the first half of 2017, so as to provide scientific basis for predicating influenza epidemic and variation. Methods A total of 40 influenza A/H3N2 viruses strains were selected and the molecular phylogenetic trees were constructed by bioinformatics software DNAStar, MEGA 7.0, etc. Then, the genetic characteristics and variation of HA and NA genes along with corresponding amino acids were analyzed. Results The homology of Shenzhen isolates reached 97.8%-100.0%, which located in the human-derived branch of Asia and North America. Compared with the vaccine strains A/Switzerland/9715293/2013(H3N2) and A/Hong Kong 14801/2014(H3N2) recommended by world Health Oraganication (WHO), there was a higher sequence similarity. Compared with the vaccine strain, HA and NA proteins had a number of amino acid sites replaced, of which HA 6 antigen sites and 2 receptor binding sites change; NA had a mutation of D151N/G located in enzyme activity sites. Potential N-glycosylation sites for HA and NA also changed. Conclusions The influenza A/H3N2 viruses in Shenzhen in the first half of 2017 has not yet formed a new subtype in the epidemic. Currently, the recommended vaccine strains still have some protective effects on the population. The replacement mutation of multiple amino acids sites of HA and NA suggests that the dynamic monitoring of molecular level of influenza A/H3N2 viruses need to be strengthened.

Phylogenetic analysis of influenza B viruses in Qingdao from 2011 to 2018 / 中华微生物学和免疫学杂志

Objective To investigate the molecular epidemiological characteristics of hemaggluti-nin (HA) and neuraminidase (NA) of influenza B viruses (IBV) isolated in Qingdao from 2011 to 2018. Methods A total of 12236 samples of influenza-like cases in Qingdao from 2011 to 2018 were collected to extract viral RNAs. All samples were screened for influenza A viruses ( IAV) and IBV by one-step multiplex real-time RT-PCR. Lineages of IBV were identified. One hundred and eighty-two strains of IBV were select-ed to amplify HA and NA genes by RT-PCR and then analyzed by sequencing. Phylogenetic analysis and variation analysis of genes and amino acids were carried out. Results IBV was detected almost every year in Qingdao from 2011 to 2018. The positive rate was only slightly lower than that of IAV ( 4. 99％ vs 6. 21％). B/Victoria linkage had two prominent epidemic years (2011-2012, 2015-2016), while B/Yama-gata linkage had three (2013-2014, 2014-2015, 2017-2018). Most of the infected people were children un-der 10 years old, and the people infected with the two lineages had similar age characteristics. Phylogenetic analysis of HA genes showed clusters in Victoria clades of 1A and 1B and Yamagata clades of 2 and 3. IBV of Yamagata lineage had more amino acid mutation sites than those of Victoria lineage in HA genes with grea-ter genetic diversity. The B/Yamagata strains had 12 amino acid mutations and the B/Victoria strains had seven in four major epitopes. In the receptor binding sites, two amino acid mutations were detected in the B/Yamagata strains and three in the B/Victoria strains. In Qingdao, 26 strains of IBV were intra-lineage reas-sortments, mostly of the B/Victoria lineage, and 23 strains were inter-lineage reassortments, mostly between HA-B/Yamagata and NA-B/Victoria strains. A possible resistant strain to NA inhibitor was found. Conclu-sions The significance of IBV in seasonal influenza should not be neglected. Amino acid substitution, in-sertion/deletion and gene reassortment were the main strategies for the natural evolution of IBV. Influenza surveillance was of great importance and influenza vaccine strains needed to be updated in time.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

Analysis of NA gene variation of influenza A(H1N1) virus isolated in Chongqing from 2011 to 2016 / 中华实验和临床病毒学杂志

Objective@#To research the gene characteristics of neuraminidase(NA) gene of influenza A(H1N1) virus of Chongqing from 2011 to 2016.@*Methods@#Forty-three H1N1 influenza virus strains isolated from 2011 to 2016 were chosen according to the different time and places of isolation. NA genetic sections were amplified by RT-PCR and sequenced, and then the sequences of nucleic and amino acid were analyzed.@*Results@#Phylogenetic analysis indicated that the relationship between H1N1 viruses from 2011 to 2016 and the vaccine strain was close to each other. Compared with the vaccine strain A/California/07/2009(H1N1), the homologies in nucleotide and amino acid sequences for NA gene were 97.9%-99.4% and 96.8%-98.9%, respectively. Most of the strains had the same eight potential glycosylation sites as the vaccine strains, and some strains got new potential glycosylation sites at NA protein site 42, while some lost their potential glycosylation sites at NA protein site 386.@*Conclusions@#The changes of H1N1 NA gene had increased year by year, but H1N1 viruses in Chongqing area were still sensitive to Daffy. And monitoring the drug resistant viruses should be strengthened, in order to provide measures against H1N1 viruses.

Detection on the pandemic( H1N1 ) 2009 virus of an influenza epidemic situation and study on the characteristic of HA and NA gene / 中华微生物学和免疫学杂志

Objective To identify the pathogen of an influenza epidemic situation and analyze the genetic characteristic of hemagglutinin( HA ) gene and neuraminidase(NA) gene of this pathogen. Methods Real-time RT-PCR was used to dectect nucleic acid of the pandemic( H1N1 ) 2009 virus from oropharyngeal swabs of initial influenza-like illness in epidemic. The viruses were was inoculated and isolated with embryonated eggs. And the HA gene and NA gene were sequenced to analyze their characteristic. Results The influenza epidemic situation was caused by the pandemic( H1N1 ) 2009 virus. The HA and NA sequences data showed that the virus had the high homology with reference virus, and the NA sequences had not the H274Y mutation. Conclusion In this study, the pandemic( H1N1 ) 2009 virus were similar with the vaccine-like virus and the isolated virus of China, and sensitive to oseltamivir.