The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsyn-onymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in vi-ruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected posi-tions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.     

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province,Central China, 2013–2017

H9N2 subtype avian influenza virus(AIV) is an influenza A virus that is widely spread throughout Asia, where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens. To better understand the epidemicity and genetics of H9 subtype AIVs, we conducted active surveillance in live poultry markets(LPMs) in Hubei Province from 2013 to 2017. A total of 4798 samples were collected from apparent healthy poultry and environment. Realtime RT-PCR revealed that the positivity rate of influenza A was 26.6%(1275/4798), of which the H9 subtype accounted for 50.3%(641/1275) of the positive samples. Of the 132 H9N2 viral strains isolated, 48 representative strains were subjected to evolutionary analysis and genotyping. Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%–100% nucleotide homology, clustered with genotype 57, and had high homology with human H9N2 viruses isolated from2013 to 2017 in China. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to change over time. Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein, and all isolates had I155 T and Q226 L mutations. Moreover, 44 strains had A558 V mutations in the PB2 protein and four had E627 V mutations, along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017. These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes, indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin

Previous studies have indicated that two monoclonal antibodies(mAbs; A1-10 and H1-84) of the hemagglutinin(HA)antigen on the H1 N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue(Guo et al. in Immunobiology 220:941–946, 2015). However,characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2(LVLWGIHHP191–199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1 N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.     

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.     

The origin of a novel kind of reassortant (H1N2) of influenza A virus

Genetic analysis of three H1N2 viruses indicated that only HA genes of H1N2 viruses were similar to that of A/Guangdong/6/91(H1N1) virus (PR8-like strain), while the other seven genes of them were similar to those of H3N2 virus circulating in man in 1995. Therefore, it could be considered that the H1N2 viruses were derived from reassortment between PR8-like strain and H3N2 virus circulating in man in 1995. However, the genomes of H1N2 viruses were very similar to each other. So the H1N2 viruses isolated in 1998 were not derived from new reassortment between PR8-like strain and H3N2 virus circulating in man in 1998, but derived from the evolution of H1N2 virus found in 1995.     

Proteotyping：A New Approach Studying Influenza Virus Evolution at the Protein Level

Phylogenetic methods have been widely used to detect the evolution of influenza viruses.However,previous phylogenetic studies of influenza viruses do not make full use of the genetic information at the protein level and therefore cannot distinguish the subtle differences among viral genes.Proteotyping is a new approach to study influenza virus evolution.It aimed at mining the potential genetic information of the viral gene at the protein level by visualizing unique amino acid signatures(proteotypes).Neuraminidase gene fragments of some H5N1 avian influenza viruses were used as an example to illustrate how the proteotyping method worked.Bayesian analysis confirmed that the NA gene tree was mainly divided into three lineages.The NA proteotype analysis further suggested there might be multiple proteotypes within these three lineages and even within single genotypes.At the same time,some proteotypes might even involve more than one genotype.In particular,it also discovered some amino acids of viruses of some genotypes might co-reassort.All these results proved this approach could provide additional information in contrast to results from standard phylogenetic tree analysis.     

Development and optimized pairing of mouse monoclonal antibodies for detecting hemagglutinin in novel H7 subtype influenza viruses

The H7 subtype avian influenza threatens public health with respect to poultry and humans. Thus, a specific and sensitive diagnostic test is essential for the management of H7 subtype influenza infections. In this study, five mouse monoclonal antibodies(mAbs) against hemagglutinin(HA) of influenza A/Anhui/1/2013(H7N9) were produced and characterized by the Western blot, immunofluorescence, and hemagglutination inhibition assays. All five specific mAbs reacted with the HA protein of H7N9 but not with that of H1 N1, H3 N2, or H5 N1. With the combination arrays of capture and detection antibodies, the matched pair m Abs(1 C4-coated and 2 D7-labeled) were selected and employed in a double-antibody sandwich ELISA(DASELISA). Detection limits of the sandwich ELISA were 0.45 ng mL~(–1) for the HA protein derived from A/Anhui/1/2013(H7N9);or 1 and 2 HA units/50 μL for A/Anhui/1/2013(H7N9) and A/GD/17 SF003/2016(H7N9), respectively. These anti-HA mAbs against subtype H7 and the novel DAS-ELISA provide a valuable approach for specific detection of the H7 subtype influenza virus and quantification of its HA protein, especially for the novel epidemic H7N9.     

Molecular Characterization of Two Silenced y-type Genes for Glu-B1 in Triticum aestivum ssp. yunnanese and ssp. tibetanum

The high molecular weight glutenin subunits （HMW-GSs） are a major class of common wheat storage proteins. The breadmaking quality of common wheat flour is influenced by the composition of HMW-GSs. In the present study, two unexpressed 1 By genes from Triticum aesitvum L.ssp.yunnanese AS332 and T. aesitvum ssp.tibetanurn AS908 were respectively cloned and characterized. The results indicated that both of the silenced 1By genes in AS332 and AS908 were 1Byg. In contrast to previously reported mechanisms for silenced genes lAx and lay, which was due to the insertion of transposon elements or the presence of premature stop codon via base substitution of C→T transition in trinucleotides CAA or CAG, the silence of 1By9 genes was caused by premature stop codons via the deletion of base A in trinucleotide CA.A, which lead to frameshift mutation and indirectly produced several premature stop codons （TAG） downstream of the coding sequence.     

Insights from avian influenza surveillance of chickens and ducks before and after exposure to live poultry markets

<正>Dear Editor,Live poultry markets are the major trading system of poultry in China and many other countries, and are also thought to be important for the generation and spread of avian influenza viruses and other avian pathogens (Zhang et al., 2013; Shi et al., 2017; Quan et al., 2018). Surveillance studies indicate that the H7 avian influenza viruses were maintained and circulated for years in the New York live poultry markets(Suarez et al., 1999). H9N2 and H5N1 influenza viruses have been detected in poultry in China since 1994 and 1996, respectively, and vaccines have been developed and used in poultry to control these two subtypes of viruses (MOA,     

Labeling of influenza viruses with synthetic fluorescent and biotin-labeled lipids

Direct labeling of virus particles is a powerful tool for the visualization of virus–cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid(FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fluorescent(fluo-Ad-DOPE) or biotin-labeled(biot-CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin(HA) receptor specificity, neuraminidase(NA) activity, or replicative ability in vitro. Our data clearly show that neither construct significantly affected influenza virus infectivity or viral affinity to sialyl receptors. Neither construct influenced the NA activities of the influenza viruses tested, except the A/Puerto Rico/8/34(H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze influenza virus infection in vitro.     

Structural analysis and molecular modeling of two antitrichosanthin IgE clones from phage antibody library

Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS).In this work,the Vε and Vκ genes of the two clones were sequenced and their putative germline gene usages were studied.On the basis of the known 3D structure of Trichosanthin and antibody,molecular modeling was carried out to study the antigen-antibody interaction.The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed,and the reaction forces between TCS and two Fab fragments were also analyzed respectively.     

Preliminary analysis of the mitochondrial genome evolutionary pattern in primates

Since the birth of molecular evolutionary analysis,primates have been a central focus of study and mitochondrial DNA is well suited to these endeavors because of its unique features.Surprisingly,to date no comprehensive evaluation of the nucleotide substitution patterns has been conducted on the mitochondrial genome of primates.Here,we analyzed the evolutionary patterns and evaluated selection and recombination in the mitochondrial genomes of 44 Primates species downloaded from GenBank.The results revealed that a strong rate heterogeneity occurred among sites and genes in all comparisons.Likewise,an obvious decline in primate nucleotide diversity was noted in the subunit rRNAs and tRNAs as compared to the protein-coding genes.Within 13 protein-coding genes,the pattern of nonsynonymous divergence was similar to that of overall nucleotide divergence,while synonymous changes differed only for individual genes,indicating that the rate heterogeneity may result from the rate of change at nonsynonymous sites.Codon usage analysis revealed that there was intermediate codon usage bias in primate protein-coding genes,and supported the idea that GC mutation pressure might determine codon usage and that positive selection is not the driving force for the codon usage bias.Neutrality tests using site-specific positive selection from a Bayesian framework indicated no sites were under positive selection for any gene,consistent with near neutrality.Recombination tests based on the pairwise homoplasy test statistic supported complete linkage even for much older divergent primate species.Thus,with the exception of rate heterogeneity among mitochondrial genes,evaluating the validity assumed complete linkage and selective neutrality in primates prior to phylogenetic or phylogeographic analysis seems unnecessary.     

The epitope study on the SARS-CoV nucleocapsid protein

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this prot     

Recent Advances in Cloning and Characterization of Disease Resistance Genes in Rice

Rice diseases caused by fungi, bacteria and viruses are one of the major constraints for sustainable rice （Oryza sativa L.） production worldwide. The use of resistant cultivars is considered the most economical and effective method to control rice diseases. In the last decade, a dozen resistance genes against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas oryzae pv. oryzae have been cloned. Approximately half of them encode nuclear binding site （NBS） and leucine rich repeat （LRR）-containing proteins, the most common type of cloned plant resistance genes. Interestingly, four of them encode novel proteins which have not been identified in other plant species, suggesting that unique mechanisms might be involved in rice defense responses. This review summarizes the recent advances in cloning and characterization of disease resistance genes in rice and presents future perspectives for in-depth molecular analysis of the function and evolution of rice resistance genes and their interaction with avirulence genes in pathogens.     

Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.