Air-breathing has evolved independently serval times with a variety of air-breathing organs (ABOs) in fish. The physiology of the air-breathing in bimodal respiration fish has been well understood, while studies on molecular mechanisms of the character are very limited. In the present study, we first determined the gill indexes of 110 fish species including 25 and 85 kinds of bimodal respiration fishes and non-air-breathing fishes, respectively. Then combined with histological observations of gills and ABOs/non-ABOs in three bimodal respiration fishes and two non-air breathing fishes, we found that the bimodal respiration fish was always of a degeneration gill and a well-vascularized ABO. Meanwhile, a comparative transcriptome analysis of posterior intestines, namely a well vascularized ABO in Misgurnus anguillicaudatus and a non-ABO in Leptobotia elongata, was performed to expound molecular variations of the air-breathing character. A total of 5,003 orthologous genes were identified. Among them, 1,189 orthologous genes were differentially expressed, which were enriched in 14 KEGG pathways. More specially, the expressions of hemoglobin genes and various HIF/VEGF signaling pathway genes were obviously upregulated in the ABO of M. anguillicaudatus. Moreover, we found that HIF-1α, VEGFAa, and MAP2K1 were co-expressed dramatically higher in ABOs of bimodal respiration fishes than those of non-ABOs of non-air-breathing fishes. These results indicated that the HIF/VEGF pathway played an important role in ABO angiogenesis/formation to promote fish to do aerial respiration. This study will contribute to our understanding of molecular mechanisms of air-breathing in fish.

The release and sensation of sex pheromone play a role in the reproductive success of vertebrates including fish. Previous studies have shown that the weather loach Misgurnus anguillicaudatus perceives sex pheromones by olfaction to stimulate courtship behavior. It was speculated that weather loaches use smell to recognize intraspecific mates. However, the identification of loach pheromone receptor has not been reported. By comparative transcriptomic approach, we found that the olfactory receptor gene or114-1 was male-biasedly expressed in the olfactory epithelium of M. anguillicaudatus, M. bipartitus and the closely related species Paramisgurnus dabryanus. This sex-biased expression pattern implicated that or114-1 presumably encoded a sex pheromone receptor in loaches. M. bipartitus and P. dabryanus, like zebrafish, possess one or114-1 only. However, in M. anguillicaudatus, or114-1 has two members: Ma_or114-1a and Ma_or114-1b. Ma_or114-1a, not Ma_or114-1b, showed sex-differential expression in olfactory epithelium. Ma_or114-1b has base insertions that delayed the stop codon, causing the protein sequence length to be extended by 8 amino acids. Ma_or114-1a was subject to positive selection resulting in adaptive amino acid substitutions, which indicated that its ligand binding specificity has probably changed. This adaptive evolution might be driven by the combined effects of sexual selection and reinforcement of premating isolation between the sympatric loach species.

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.

Three H5N8 avian influenza viruses isolated from domestic geese in China in 2014 were characterized phylogenetically and biologically. Phylogenetic analysis of the complete genomic sequences of the three isolates from this study and those of 61 other H5N8 viruses retrieved from the GISAID platform indicated that, chronologically and geographically, all H5N8 viruses of the Asian H5N1 HA lineage of clade 2.3.4.4 are the direct descendents of the K1203 (H5N8)-like viruses first isolated in China in 2010. The three viruses from this study shared high sequence similarity in all eight gene segments with three other isolates from China in 2013, and two Korean isolates were distinct from the recently circulating reassortants causing outbreaks in Asia, Europe and the United States in 2014 and 2015. In vitro viral growth curves indicated that these H5N8 viruses replicated to high titers in CEF, DEF, MDCK and A549 cells but to significantly lower titers in Vero cells. Pathogenicity studies in vivo indicated that these viruses were all highly virulent to chickens and mallard ducks, while they varied from moderate to high virulence in mice. Additionally, hemagglutination assays using α-2,3-sialidase-treated goose red blood cells and solid-phase direct binding assays with different glycans demonstrated that the three viruses could bind to both avian-type SAα-2,3Gal and human-type SAα-2,6Gal receptors. Our findings confirmed the progenitor nature of the K1203-like viruses in generating recent prevalent clade 2.3.4.4 H5N8 reassortants, which have caused tremendous damage to the poultry industry and are a potential threat to public health.

[Objective] Based on the difference of hemagglutinin (HA) and neuraminidase (NA), avian influenza viruses (AIVs) are classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. According to the differences in pathogenicity, AIVs can be divided into highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV). H4 AIVs are low pathogenic influenza viruses which are generally produced asymptomatic infections in poultry. But H4 AIVs also has potential threats to both poultry and mammals. Strengthening investigation on H4 subtype avian influenza viruses is important for the study of evolution of AIVs. The objective of this experiment is to investigate the molecule characteristics and genetic evolution of H4 subtype avian influenza virus.[Method]One H4N8 subtype avian influenza virus, designated as A/duck/Nanjing/1102/2010 (H4N8) <br> (DK/NJ/1102), was isolated from a live poultry market in eastern China during epidemiological surveillance in 2010. The complete genome sequences of the strain was sequenced and analyzed. The virus was identified by HA/HI test and RT-PCR test. The gene was cloned into pGEM-Teasy vector for sequencing, respectively. BLAST the nucleotide identity in GeneBank. The genome sequences of H4 subtype influenza viruses available in GeneBank and some other reference sequences were downloaded for genetic analysis .[Result]The results showed that the HA gene of DK/NJ/1102 had the highest nucleotide sequence identity of 98.9% with A/duck/Mongolia/274/2007(H4N3), and the amino acid sequence at the cleavage region of the HA gene was “P-E-K-A-S-R-G”, which is typical for low pathogenicity AIVs. NA gene had the highest nucleotide sequence identity of 98.8% with a duck-origin virus A/Duck/Eastern China/n91/2009 (H3N8) isolated from eastern China, whereas PB1, PA and NP genes were all mostly related with H1 subtype avian influenza viruses. The M gene shared the greatest nucleotide sequence identities (over 99.9%) with A/wild duck/Korea/CSM4-12/2009(H5N1) while the NS gene was most closely related to H7N7 subtype AIV isolated from Korea in 2009 and there was no amino acids missing in NS1 protein at 80-84 sites.[Conclusion]These data indicated that the genome composition of the strain A/duck/ Nanjing/1102/2010(H4N8) was complicated, it may be a reassortant whose genes originated from different subtypes of AIV.

ABSTRACT PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus. IMPORTANCE Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode as many as 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly identified protein in segment 3 that acts to decrease the virulence of the 1918 H1N1 virus in mice by modulating host gene expression. Our study extends these functions of PA-X to H5N1 HPAIV. We demonstrated that loss of PA-X expression increases the virulence and replication of an H5N1 virus in mice and avian species and alters the host innate immune and cell death responses. Our report is the first to delineate the role of the novel PA-X protein in the pathogenesis of H5N1 viruses in avian species and promotes our understanding of H5N1 HPAIV.

流感病毒是现今世界流行最为广泛、传播最为迅速的人兽共患病毒之一,而气源性传播是流感病毒最有效的传播方式.流感病毒的相关基因,尤其是相关基因的关键氨基酸位点对其自身是否具有气源性传播能力具有决定性的作用.外界环境的改变会直接影响病毒气溶胶颗粒形成的数量和大小,进而影响传播效率.目前,经过长期的筛选和比较,将豚鼠和雪貂作为研究流感气源性传播的模式动物,根据不同目的或者单独使用或者协同实验.文章通过对病毒本身的生物学特性以及外界环境对气源性传播产生的影响进行了初步总结,对进行气源性传播的实验动物给出了综合评价,一方面为研究者提供较为全面的总结,另一方面为防治流感提供依据.

ABSTRACT H9N2 avian influenza virus has been prevalent in poultry in many parts of the world since the 1990s and occasionally crosses the host barrier, transmitting to mammals, including humans. In recent years, these viruses have contributed genes to H5N1 and H7N9 influenza viruses, threatening public health. To explore the molecular mechanism for the airborne transmission of H9N2 virus, we compared two genetically close strains isolated from chickens in 2001, A/chicken/Shanghai/7/2001(SH7) and A/chicken/Shanghai/14/2001 (SH14). SH7 is airborne transmissible between chickens, whereas SH14 is not. We used reverse genetics and gene swapping to derive recombinant SH7 (rSH7), rSH14, and a panel of reassortant viruses. Among the reassortant viruses, we identified segments HA and PA as governing the airborne transmission among chickens. In addition, the NP and NS genes also contributed to a lesser extent. Furthermore, the mutational analyses showed the transmissibility phenotype predominantly mapped to the HA and PA genes, with HA-K363 and PA-L672 being important for airborne transmissibility among chickens. In addition, the viral infectivity and acid stability are related to the airborne transmissibility. Importantly, airborne transmission studies of 18 arbitrarily chosen H9N2 viruses from our collections confirmed the importance of both 363K in HA and 672L in PA in determining their levels of transmissibility. Our finding elucidates the genetic contributions to H9N2 transmissibility in chickens and highlights the importance of their prevalence in poultry. IMPORTANCE Our study investigates the airborne transmissibility of H9N2 viruses in chickens and the subsequent epidemic. H9N2 virus is the donor for several prevalent reassortant influenza viruses, such as H7N9/2013 and the H5N1 viruses. Poultry as the reservoir hosts of influenza virus is closely associated with human society. Airborne transmission is an efficient pathway for influenza virus transmission among flocks and individuals. Exploring the mechanism of the airborne transmission of the H9N2 virus in chickens could provide essential data regarding prevention and control of influenza endemics and pandemics.

It is reported that the H5N2 highly pathogenic avian influenza virus A/chicken/Hebei/1102/2010 (HB10) is a natural reassortant between circulating H5N1 and endemic H9N2 influenza viruses. To evaluate the potential of its interspecies transmission, the wild-type HB10 was adapted in mice through serial lung passages. Increased virulence was detectable in 5 sequential lung passages in mice and a highly virulent mouse-adapted strain (HB10-MA) with a 50% mouse lethal dose of 10(2.5) 50% egg infectious dose was obtained in 15 passages. The virulence and the replication efficiency of HB10-MA in mice were significantly higher than those of HB10 while HB10-MA grew faster and to significantly higher titers than HB10 in MDCK and A549 cells. Only five amino acid mutations in four viral proteins (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) of HB10-MA virus were found when compared with those of HB10, indicating that they may be responsible for the adaptation of the novel reassortant H5N2 avian influenza virus in mice with increased virulence and replication efficiency. The results in this study provide helpful insights into the pathogenic potential of novel reassortant H5N2 viruses to mammals that deserves further attentions.

H5N1 subtype influenza A virus has evolved into many HA clades since late 1990s. Six circulating H5N1 influenza viruses clustered to a novel branch in clade 2.3.4 and could escape vaccine protection, indicating their antigenic drift. Eleven amino acids substitutions in three antigenic sites of the hemagglutinin of these isolates were found when compared with the hemagglutinin of the primary viruses in clade 2.3.4. On the backbone of the novel isolates A/chicken/Northern China/k0602/2010, we generated a panel of recombinant viruses with HA mutations of restoring the primary vaccine strain Re-5's amino acid and homologous antisera to determine the role of these substitutions. The results of cross-HI assay, micro-neutralization assay and the antigen map of the mutated recombinant viruses showed that three substitutions in antigenic site B, especially D205K, are the major contributors to the antigenic drift of the novel branch of clade 2.3.4. Our study highlights the importance of surveillance of antigenic drift of H5N1 viruses for the control and preparedness of pandemic threats.

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.

One H5N8 and three H5N5 highly pathogenic avian influenza (HPAI) viruses which derived their HA genes from the Asian H5N1 lineage were isolated from poultry during 2009–2010 in mainland China. Pathogenicity studies showed that these viruses were all highly virulent to chickens, while they varied from moderate to high virulence in mice and from mild to intermediate virulence in mallards. Phylogenetic analyses showed that these viruses were reassortants bearing the H5N1 backbone while acquiring PB1, NP and NA genes from unidentified non-H5N1 viruses, and had developed into three distinct genotypes (B–D). Molecular characterization indicated that all these viruses might resist to antiviral agents. Our findings highlight the emergence and development of HPAI H5 viruses of other NA subtypes in H5N1 endemic areas and their potential threat to poultry industry and public health.

ABSTRACT Here, we report the complete genomic sequence of a novel reassortant H4N2 influenza virus isolated from domestic ducks in the Jiangsu province of China in 2011. Phylogenetic analysis showed that all the viral genes except for hemagglutinin (HA) were highly homologous to the clade 2.3.4 H5N2 viruses. The data suggest that genetic reassortment occurred between H4 and H5N2 avian influenza viruses, which highlights the role of domestic poultry as a reassortment vessel in China.

We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009-2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.

ABSTRACT Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.

Here, we report the complete genome sequence of an H3N6 avian influenza virus (AIV) isolated from domestic ducks in Jiangsu province of eastern China in 2010. Phylogenetic analysis showed that the H3N6 virus is a natural recombinant virus whose genes were derived from H3N8, H4N6, H6N6, H7N7, and H11N2 AIVs. This analysis will help to understand the molecular characteristics and evolution of the H3N6 influenza virus in eastern China.

在疫苗学领域,概念性的进展不断增长,促进了该领域的大量密集型研究,其研究进展作为杠杆又可快速地推动生产进步,这也是疫苗学研究的主要目的,即保障动物个体的生存和健康。对于兽医疫苗学,尤其是禽类疫苗学,所产生的效果往往超出了增强动物健康的最初目的,该领域的目的和收益反映出群体免疫的概念,远较其它领域的推进速度更快。朝着这一目标,禽类疫苗学的发展超乎于其它领域,使我们更好地认识到疫苗及免疫系统的作用不应仅仅局限于使动物得以生存,更在于提高动物的生产率及如何改善以保证动物健康养殖所需的基础或必要条件。如果不能将提高生产性能和提高机体免疫力自然结合起来,就不能使得免疫学研究突出于家禽生产中的其他环节。正是这对矛盾体,刺激着更加敏感有效的策略研究,最大限度地发挥动物的生产性能,同时最大限度提高机体应对各种外界挑战的能力。

食品微生物检测是食品加工过程中确保食品安全的关键项目.在对生禽肉中沙门菌进行检测时,传统培养检测技术由于采集样品所经历的前处理和后加工方法缺乏统一的标准容易造成结果假阳(阴)性.而基于rRNA的检测技术与传统培养方法相比,可在很大程度上简化检测程序,提高检测结果的精确度,节约人工成本,减少实验室污染和失误的可能性.本文重点介绍了可用于环境和食品(包括家禽产品)中细菌检测的rRNA分子生物学技术.

In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007–2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.