Functional Characterization of CsF3Ha and Its Promoter in Response to Visible Light and Plant Growth Regulators in the Tea Plant.

Flavanone 3-hydroxylase (F3H) catalyzes trihydroxyflavanone formation into dihydroflavonols in the anthocyanin biosynthesis pathway, serving as precursors for anthocyanin synthesis. To investigate the CsF3Ha promoter's regulation in the 'Zijuan' tea plant, we cloned the CsF3Ha gene from this plant. It was up-regulated under various visible light conditions (blue, red, and ultraviolet (UV)) and using plant growth regulators (PGRs), including abscisic acid (ABA), gibberellic acid (GA3), salicylic acid (SA), ethephon, and methyl jasmonate (MeJA). The 1691 bp promoter sequence was cloned. The full-length promoter P1 (1691 bp) and its two deletion derivatives, P2 (890 bp) and P3 (467 bp), were fused with the ß-glucuronidase (GUS) reporter gene, and were introduced into tobacco via Agrobacterium-mediated transformation. GUS staining, activity analysis, and relative expression showed that visible light and PGRs responded to promoter fragments. The anthocyanin content analysis revealed a significant increase due to visible light and PGRs. These findings suggest that diverse treatments indirectly enhance anthocyanin accumulation in 'Zijuan' tea plant leaves, establishing a foundation for further research on CsF3Ha promoter activity and its regulatory role in anthocyanin accumulation.

Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China.

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

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.

The molecular determinants of antigenic drift in a novel avian influenza A (H9N2) variant virus.

BACKGROUND: In early 2020, a novel H9N2 AIV immune escape variant emerged in South China and rapidly spread throughout mainland China. The effectiveness of the current H9N2 vaccine is being challenged by emerging immune escape strains. Assessing key amino acid substitutions that contribute to antigenic drift and immune escape in the HA gene of circulating strains is critical for understanding virus evolution and in selecting more effective vaccine components. METHODS: In this study, a representative immune escape strain, A/chicken/Fujian/11/2020 (FJ/20), differed from current H9N2 vaccine strain, A/chicken/Anhui/LH99/2017 (AH/17) by 18 amino acids in the head domain in HA protein. To investigate the molecular determinants of antigenic drift of FJ/20, a panel of mutants were generated by reverse genetics including specific amino acids changes in the HA genes of FJ/20 and AH/17. The antigenic effect of the substitutions was evaluated by hemagglutination inhibition (HI) assay and antigenic cartography. RESULTS: Fujian-like H9N2 viruses had changed antigenicity significantly, having mutated into an antigenically distinct sub-clade. Relative to the titers of the vaccine virus AH/17, the escape strain FJ/20 saw a 16-fold reduction in HI titer against antiserum elicited by AH/17. Our results showed that seven residue substitutions (D127S, G135D, N145T, R146Q, D179T, R182T and T183N) near the HA receptor binding sites were critical for converting the antigenicity of both AH/17 and FJ/20. Especially, the combined mutations 127D, 135G, 145N, and 146R could be a major factor of antigenic drift in the current immune escape variant FJ/20. The avian influenza A (H9N2) variant virus need further ongoing epidemiological surveillance. CONCLUSIONS: In this study, we evaluated the relative contributions of different combinations of amino acid substitutions in the HA globular head domain of the immune escape strain FJ/20 and the vaccine strain AH/17. Our study provides more insights into the molecular mechanism of the antigenic drift of the H9N2 AIV immune escape strain. This work identified important markers for understanding H9N2 AIV evolution as well as for improving vaccine development and control strategies in poultry.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

OBJECTIVE: The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly. MATERIALS AND METHODS: Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses. RESULTS: This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%-98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor. CONCLUSION: This study described the features of recent H9N2 viruses spreading in Java's poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

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.

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.

Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses.

As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade 2.3.4.4 variants in or around the recognized antigenic epitopes A-E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single-site or multi-site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade 2.3.4.4) as the HA backbone to study their antigenic variations, respectively. The hemagglutination-inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A.

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

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.

[Development and verification of reference nucleic acid materials of H9N2 influenza viruses by real-time RT-PCR].

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 109 copies/µL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017.

In 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required. Research highlights HPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017. Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and Europe Nonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks. Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

Development and verification of reference nucleic acid materials of H9N2 influenza viruses by real-time RT-PCR / 生物工程学报

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 10⁹ copies/μL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails.

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails.

Molecular genotyping of measles viruses in Yancheng area in 2016 and analysis of the genetic characterization of hemagglutinin genes / 中华实验和临床病毒学杂志

Objective@#To analyze the genotypes and the genetic evolution of the hemagglutinin genes of measles viruses in the city of Yancheng in 2016.@*Methods@#Using a set of primers and probes for screening positive for measles viruses, specimens of throat swab were detected using the method of real time RT-PCR. The sequences of the nucleoprotein and hemagglutinin genes of measles viruses were amplified through one step RT-PCR method and the PCR products were sequenced. The sequences of nucleotide and amino acid of the nucleoprotein and hemagglutinin genes of measles viruses were analyzed and the evolutional trees were generated using bioinformatics software.@*Results@#The genotypes of measles viruses in the Yancheng area in 2016 included subgenotype H1a and genotype D8. Phylogenetic trees analysis showed that the five representative strains of subgenotype H1a in Yancheng area and Jiangxi representative strain (KJ136545) clustered into independent evolutionary branches, belonged to the clade of H1a -1 evolutionary genes. The seven representative strains of genotype D8 in Yancheng area were clustered with the American representative strain in 2009 (JN635404), belonged to the D8-3-2 small clade genes. Compared with vaccine strain of Shanghai S191, the amino acid site in 240thof the five representative strains of subgenotype H1a in Yancheng area mutated from serine to asparagine, leading to a loss of the N-glycosylation site NLS238-240. The seven representative strains of genotype D8 in Yancheng area had no change in N-glycosylation.@*Conclusions@#In 2016, the prevalent strains of measles viruses in Yancheng area were mainly Chinese H1a dominant subgenotype and D8 imported genotype. In addition to a loss of the N-glycosylation site NLS238-240in 240thof the five representative strains of subgenotype H1a, most of the major neutralizing antigen sites of hemagglutinin gene of measles viruses in Yancheng area did not mutate. The Chinese vaccine of Shanghai S191 can effectively prevent infection caused by subgenotype H1a and subgenotype D8 strains.

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.

Variation of HA gene of influenza A H1N1 influenza virus isolated from 2009 to 2014 in Chongqing city / 重庆医学

Objective In order to analyze the variation of HA genes of influenza viruses (H1N1) by being compared with the vaccine strain A/California/07/2009(H1N1) recommended by WHO ,influenza viruses (H1N1) isolated from 2009 to 2014 were selected to do this study .Methods According to the different isolating time and place ,47 strains of H1N1 collected from 2011 to 2014 were selected .Then the 47 strains′ nucleotide sequence of HA genes which were sequenced in the study and other 25 se‐quences of HA genes which were sequenced in 2009 were collected .Nucleotide and amino acid sequences were analyzed by using molecular biology software ,and the phylogenetic trees were drawn .Results A total of 72 strains isolated from 2009 to 2014 were closely related to the vaccine strain A/California/07/2009(H1N1) ,the nucleotide variance and amino acid variance between the 72 strains were 0-2 .7% and 0-3 .1% respectively .Compared with the vaccine strain A/California/07/2009(H1N1) ,the nucleotide variance and amino acid sequence variance were 0 .4% -2 .4% and 0 .9% -3 .1% respectively .The amino acids sequence indicated that ,although the variance was increased by years ,the H1N1 viruses were still showed characteristics of low pathogenic influenza viruses .It was also found that there were 9 strains lost their potential glycosylation site at HA protein site 481 in 2009 ,while in 2013 there were 6 strains got new potential glycosylation sites at HA protein site 162 .Conclusion The vaccines (H1N1) recom‐mended by WHO was still protective to people in Chongqing .But as time goes by ,antigen drift may occur in some new antigenic drift strains and the routine monitoring of influenza viruses should be continued .

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.

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.