[Show abstract][Hide abstract] ABSTRACT: New reassortant H5N8 highly pathogenic avian influenza viruses were isolated from waterfowl in Southern China. Blast analysis demonstrated that the PB2 gene in these viruses were most closely related to A/wild duck/Shangdong/628/2011 (H5N1), while their NP genes were both more closely related to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8). However, the HA, NA, PB1, PA, M, and NS genes had the highest identity with A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenetic analysis revealed that their HA genes belonged to the same GsGd H5 clade 126.96.36.199 detected in China in 2010. Therefore, we supposed that these H5N8 viruses might be novel reassortant viruses that have a H5N8 backbone while acquiring PB2 and NP genes from H5N1 viruses. This study is useful for better understanding the genetic and antigenic evolution of H5 avian influenza viruses in Southern China.
Frontiers in Microbiology 11/2015; 6:1170. DOI:10.3389/fmicb.2015.01170 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three human cases of H10N8 virus infections were initially reported in China in late 2013 and early 2014, two of which were fatal. This was the first time the H10N8 subtype has been detected in humans, and the pathogenicity of this virus remains under characterized. We first assessed its pathogenicity by infecting BALB/c mice with two H10N8 isolates, A/Jiangxi-Donghu/346-1/2013 and A/Chicken/Jiangxi/102/2013. The human isolate (H346-1) demonstrated stronger capability of replication and induced higher cytokine response in vivo than the chicken isolate (C102). In addition, H346-1 was fatal to mice, while all mice (N = 14) in C102-infected group survived during the infection course without weight loss. We hypothesized that the 627K mutation in the PB2 gene (PB2-K627) in H346-1 was associated with high pathogenicity in mice. Taken together, this study based on mouse model provides some insight into understanding the pathogenicity of the emerging viruses in mammals.
The American journal of tropical medicine and hygiene 09/2015; DOI:10.4269/ajtmh.15-0064 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fourteen influenza A(H7N9) viruses were isolated from poultry or the environment in live poultry markets in Guangdong Province, China during 2014-2015. Phylogenetic analysis showed that all viruses were descended from viruses of the second wave of influenza A(H7N9) virus infections during 2013. These viruses can be divided into 2 branches.
[Show abstract][Hide abstract] ABSTRACT: Pathogen-associated molecular patterns (PAMPs) play important role in inflammation which means response of the host to stimuli. NOD-like receptor protein 3 (NLRP3) inflammasome is involved in the onset and development of inflammation. NLRP3, as one of the most important inflammasome sensors, has significant effect on the regulation of inflammasome activation to avoid the consequences of over activation. Up to date, there are no detailed tissue specific expression and distribution data about NLPR3 in chicken. Here, NLRP3 of Chinese yellow chicken was cloned and sequence analyzed, the polyclonal antibody was produced by purified protein of recombinant prokaryotic expression. Relative expression levels and tissue distribution of NLRP3 were investigated by real-time quantitative PCR and immunohistochemical analysis, respectively. The results showed that NLRP3 gene is highly variable between mammalian and avian. The nucleotide homology of NLRP3 between yellow chicken and Bos taurus, Hainan black goat, Sus scrofa, Callithrix jacchus, Homo sapiens, Macaca mulatta, Mus musculus and Rattus norvegicus were 54.2 %, 53.9 %, 53.7 %, 55.4 %, 54.3 %, 54.5 %, 53.5 % and 53.7 %. NLRP3 expressed in all detected tissues and higher in the trachea are lung than in other tissues. Cytoplasmic expression of NLRP3 was detected in ciliated epithelial cells, basal cells and cells in lamina propria of trachea, alveolar epithelial cells, cardiac muscle cells, cerebral cortex neurons, epithelial reticular cells of the spleen, and lymphocytes of medulla in stannius follicle, liver cells and the renal tubule epithelial cells. The results will help to elucidate the role of NLRP3 of different tissues in inflammatory diseases of chicken and provide a basis for further investigations in the function and evolution of NLRP3 in different species, which would be helpful for further research on avian inflammatory diseases.
Veterinary Research Communications 08/2015; 39(3). DOI:10.1007/s11259-015-9641-6 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptor 2 (TLR2), an important pattern recognition receptor, activates proinflammatory pathways in response to various pathogens. It has been reported in humans and chicken, but not in geese, an important waterfowl species in China. Since some vaccines stimulate robust immune responsesl in chicken but not in geeeses we speculated that their immune systems are different.
In this study, we cloned the goose TLR2-1 gene using rapid amplification of cDNA ends (RACE)and showed that geese TLR2-1 encoded a 793-amino-acid protein, containing a signal secretion peptide, an extracellular leucine-rich repeat domain, a transmembrane domain and a Toll/interleukin-1 receptor signaling domain deduced from amino acid sequence. TLR2-1 shared 38.4%-93.5% homology with its homologues in other species. Tissue expression of geese TLR2-1 varied markedly, and was higher in kidney, cloacal bursa, skin and brain compared to other organs/tissues. HEK293 cells transfected with plasmids carrying goose TLR2-1 and NF-κB-luciferase responded significantly to stimulation with Mycoplasma fermentans lipopeptide. Furthermore, geese infected with Mycoplasma gallisepticum (MG) and Salmonella enteritidis (SE) showed significant upregulation of TLR2-1 in both in vivo and in vitro.
Geese TLR2-1 is a functional homologue of TLR2 present in other species and plays an important role in bacterial recognition in geese.
BMC Veterinary Research 05/2015; 11(1):108. DOI:10.1186/s12917-015-0420-y · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively.
The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues.
The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.
[Show abstract][Hide abstract] ABSTRACT: H5N1 influenza viruses with high lethality are a continuing threat to humans and poultry. Recently, H5N1 high-pathogenicity avian influenza virus (HPAIV) has been shown to transmit through aerosols between ferrets in lab experiments by acquiring some mutation. This is another deeply aggravated threat of H5N1 HPAIV to humans. To further explore the molecular determinant of H5N1 HPAIV virulence in a mammalian model, we compared the virulence of A/Duck/Guangdong/212/2004 (DK212) and A/Quail/Guangdong/90/2004 (QL90). Though they were genetically similar, they had different pathogenicity in mice, as well as their 16 reassortants. The results indicated that a swap of the PB2 gene could dramatically decrease the virulence of rgDK212 in mice (1896-fold) but increase the virulence of rgQL90 in mice (60-fold). Furthermore, the polymerase activity assays showed that swapping PB2 genes between these two viruses significantly changed the activity of polymerase complexes in 293T cells. The mutation Ser715Asn in PB2 sharply attenuated the virulence of rgDK212 in mice (2710-fold). PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice and 715 Ser in PB2 plays an important role in determining high virulence of DK212 in mice.
Frontiers in Microbiology 02/2015; 6:73. DOI:10.3389/fmicb.2015.00073 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some highly pathogenic H5N1, H7N9 and H10N8 isolated from China carried six internal genes from H9N2 AIVs, and the key amino acids at 627 in PB2 of these viruses had mutated to K. To investigate the mechanism of increased pathogenicity for H9N2 AIV PB2 627K, we analyzed the difference in mouse lung proteins expression response to PB2 K627E. By iTRAQ method, we found that the mutated K627E contributed to a set of differentially expressed lung proteins, including five upregulated proteins and nine downregulated proteins at 12 h post infection; ten upregulated proteins and twenty five downregulated proteins at 72 h post infection. These proteins were chiefly involved within the cytoskeleton and motor proteins, antiviral proteins, regulation of glucocorticoids signal-associated proteins, pro- and anti-inflammatory proteins. Alteration of moesin, FKBP4, Hsp70, ezrin and sp-A may play important roles in increasing virulence and decreasing lungs antiviral response. Further, three upregulated proteins (moesin, ezrin and sp-A) caused by PB2 K627E were also confirmed in A549 cells. Moreover, overexpression of sp-A in A549 inhibited virus replication and downregulation promoted virus replication. In this study, sp-A as a potential virulence determinant associated H9N2 AIV PB2 E627K mutation was identified using comparative proteomics.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-β, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.
[Show abstract][Hide abstract] ABSTRACT: BackgroundH9N2 avian influenza virus (AIV) becomes the focus for its ability of transmission to mammals and as a donor to provide internal genes to form the new epidemic lethal influenza viruses. Residue 627 in PB2 has been proven the virulence factor of H9N2 avian influenza virus in mice, but the detailed data for inflammation difference between H9N2 virus strains with site 627 mutation is still unclear. The inflammasome NLRP3 is recently reported as the cellular machinery responsible for activation of inflammatory processes and plays an important role during the development of inflammation caused by influenza virus infection.Methods
In this study, we investigated the expression pattern of NLRP3 and its related cytokines of IL-1ß and TNF-¿ in BALB/c mice infected by H9N2 AIV strains with only a site 627 difference at both mRNA and protein levels at different time points.ResultsThe results showed that the expression level of NLRP3, IL-1ß and TNF-¿ changed in the lung and brain of BALB/c mice after infection by VK627 and rVK627E. The immunohistological results showed that the positive cells of NLRP3, IL-1ß and TNF-¿ altered the positive levels of original cells in tissues and infiltrated inflammatory cells which caused by H9N2 infection.Conclusions
Our results provided the basic data at differences in expression pattern of NLRP3 and its related cytokines in BALB/c mice infected by H9N2 influenza viruses with only a site 627 difference. This implied that NLRP3 inflammasome plays a role in host response to influenza virus infection and determines the outcome of clinical manifestation and pathological injury. This will explain the variable of pathological presentation in tissues and enhance research on inflammation process of the AIV H9N2 infection.
[Show abstract][Hide abstract] ABSTRACT: A novel H10N8 influenza A virus has been detected in three humans in China since December 2013. Although this virus was hypothesized to be a novel reassortant among influenza viruses from wild birds and domestic poultry, its evolutionary path leading to human infection is unknown. Sporadic surveillance at the live poultry market (LPM) suspected to be the source of infection for the first H10N8 patient has shown a gradual increase in influenza virus prevalence culminating with a predominance of H10N8 viruses. Influenza viruses detected in the LPM up to 8 months prior to human infection contributed genetic components to the zoonotic virus. These H10N8 viruses have continued to evolve within this LPM subsequent to the human infection, and continuous assessments of these H10N8 viruses will be necessary. Serological surveillance showed that the virus appears to have been present throughout the LPM system in Nanchang, China. Reduction of the influenza virus burden in LPMs is essential in preventing future emergence of novel influenza viruses with zoonotic and pandemic potential.
[Show abstract][Hide abstract] ABSTRACT: H5N1 highly pathogenic avian influenza virus (HPAIV) of clade 2.3.2 has been circulating in waterfowl in Southern China since 2003. Our previous studies showed that certain H5N1 HPAIV isolates within clade 2.3.2 from Southern China had high pathogenicity in different birds. Guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts. However, few studies have reported pathogenicity and transmissibility of H5N1 HPAIVs of this clade in guinea pigs. In this study, we selected an H5N1 HPAIV isolate, A/duck/Guangdong/357/2008, to investigate the pathogenicity and transmissibility of the virus in guinea pigs. The virus had high pathogenicity in mice; additionally, it only replicated in some tissues of the guinea pigs without production of clinical signs, but was transmissible among guinea pigs. Interestingly, virus isolates from co-caged guinea pigs had the D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs but did not demonstrate enhanced the transmissibility among guinea pigs. These findings indicate the transmission of the H5N1 virus between mammals could induce virus mutations, and the mutant viruses might have higher pathogenicity in mammals without higher transmissibility. Therefore, the continued evaluation of the pathogenicity and transmissibility of avian influenza virus (AIVs) in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains.
Frontiers in Microbiology 11/2014; 5:642. DOI:10.3389/fmicb.2014.00642 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Considered an epicenter of pandemic influenza virus generation, southern China has recently seen an increasing number of human H7N9 infections. However, it is not the only threat. On 30 November 2013, a human H10N8 infection case was first described in China. The origin and genetic diversity of this novel virus is similar to that of H7N9 virus. As H10N8 avian influenza virus (AIV) was first identified from a duck in Guangdong Province during 2012 and there is also evidence of H10N8 infected dogs in this region, we sought to examine archived sera from animal workers to see if there was evidence of subclinical human infections before the first human H10N8 cases.Methods
We studied archived serum samples (cross-sectional study, convenience sample) collected between May and September 2013 from 710 animal workers and 107 non-animal exposed volunteers living in five cities of Guangdong Province. Study participants¿ sera were tested by horse red blood cells (RBCs) hemagglutination inhibition (HI) and microneutralization (MN) assays according to World Health Organization guidelines. The A/Jiangxi-Donghu/346-1/2013(H10N8) virus was used. Sera which have an HI assay ¿1:20 were further tested with the MN assay. Questionnaire data were examined for risk factor associations with positive serological assays. Risk factor analyses failed to identify specific factors associated with probable H10N8 infections.ResultsAmong the 827 sera, only 21 animal workers had an HI titer ¿1:20 (18 had an HI titer of 1:20 and 3 had an HI titer of 1:40). None of these 21 subjects reported experiencing any influenza symptoms during the three months before enrollment. Among the three subjects with HI titers of 1:40, two had MN antibody titers of 1:40, and one had a MN antibody titer of 1:80 (probable H10N8 infections).Conclusions
Study data suggest that animal workers may have been infected with the H10N8 virus before the first recognized H10N8 human infection cases. It seems prudent to continue surveillance for H10N8 viruses among animal workers.
BMC Medicine 10/2014; 12(1):205. DOI:10.1186/PREACCEPT-1724095651325168 · 7.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The retroviral integrase plays an essential role in the integration of reverse-transcribed retroviral cDNA into the host cell genome, and serves as an important target for anti-viral therapeutics. In this study, we identified the COP9 signalosome subunit 6 (CSN6) as a novel avian leukosis virus (ALV) integrase binding protein. Co-immunoprecipitation and GST pull-down assays showed that CSN6 bound to ALV integrase likely through direct interaction of CSN6 to the catalytic core of the integrase. We further demonstrated CSN6 inhibited integrase activity in vitro; knockdown of CSN6 in DF-1 promoted ALV production. These results indicated that CSN6 may be a negative regulator of ALV replication by binding to and inhibiting integrase. Our findings provided the insight into the integrase-based host defense system and may have implications in the development of integrase-based anti-viral strategies.
Biochemical and Biophysical Research Communications 10/2014; 453(3). DOI:10.1016/j.bbrc.2014.09.116 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the aetiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs. 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.
Research in Veterinary Science 10/2014; 97(2). DOI:10.1016/j.rvsc.2014.04.014 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Haemophilus parasuis infection is responsible for important economic losses to the pig industry. The increasing emergence of resistance to multiple antibiotics is of concern and to study the role of the acrRAB operon in H. parasuis drug resistance, acrB or acrR mutants were generated from H. parasuis serovar 4 clinical strains. The susceptibilities of the clinical strains and their acrB/acrR mutants to a number of antibiotics were determined. The acrB mutants were more susceptible to novobiocin, erythromycin, clarithromycin, and azithromycin. In the acrR mutant of H. parasuis, acrB was up-regulated, as determined by quantitative reverse transcriptase polymerase chain reaction. The results of this study indicated that the efflux pump AcrB may play a role in multidrug resistance of H. parasuis.
The Veterinary Journal 10/2014; 202(1). DOI:10.1016/j.tvjl.2014.05.045 · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The H9N2 avian influenza virus is a pandemic threat which has repeatedly caused infection in humans and shows enhanced replication and transmission in mice. Previous reports showed that host factors, the interferon-inducible transmembrane (IFITM) protein, can block the replication of pathogens and affect their pathogenesis. BALB/c mice are routine laboratory animals used in influenza virus research, but the effects of H9N2 influenza virus on tissue distribution and expression pattern of IFITM in these mice are unknown. Here, we investigated the expression patterns and tissue distribution of IFITM1 and IFITM3 in BALB/c mice by infection with H9N2 AIV strains with only a PB2 residue 627 difference. The results showed that the expression patterns of ITITM1 and IFITM3 differ in various tissues of BALB/c mice at different time points after infection. IFITM1 and IFITM3 showed cell- and tissue-specific distribution in the lung, heart, liver, spleen, kidney and brain. Notably, the epithelial and neuronal cells all expressed the proteins of IFITM1 and IFITM3. Our results provide the first look at differences in IFITM1 and IFITM3 expression patterns in BALB/c mice infected by H9N2 influenza viruses. This will enhance research on the interaction between AIV and host and further will elucidate the pathogenesis of influenza virus infection based on the interferon-inducible transmembrane (IFITM) protein.
Medical Microbiology and Immunology 09/2014; 204(4). DOI:10.1007/s00430-014-0361-2 · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human infection with a novel influenza A(H10N8) virus was first described in China in December 2013. However, the origin and genetic diversity of this virus is still poorly understood. We performed a phylogenetic analysis and coalescent analysis of two viruses from the first case of influenza A(H10N8) (A/Jiangxi-Donghu/346-1/2013 and A/Jiangxi-Donghu/346-2/2013 and a novel A(H10N8) virus (A/chicken/Jiangxi/102/2013) isolated from a live poultry market that the patient had visited. The haemagglutinin (HA), neuraminidase (NA), PA subunit of the virus polymerase complex, nucleoprotein (NP), M and nonstructural protein (NS) genes of the three virus strains shared the same genetic origins. The origins of their HA and NA genes were similar: originally from wild birds to ducks, and then to chickens. The PA, NP, M, and NS genes were similar to those of chicken influenza A(H9N2) viruses. Coalescent analyses showed that the reassortment of these genes from A(H9N2) to A(H10N8) might have occurred at least twice. However, the PB1 and PB2 genes of the chicken A(H10N8) virus most likely originated from H7-like viruses of ducks, while those of the viruses from the case most likely stemmed from A(H9N2) viruses circulating in chickens. The oseltamivir-resistance mutation, R292K (R291K in A(H10N8) numbering) in the NA protein, occurred after four days of oseltamivir treatment. It seems that A(H10N8) viruses might have become established among poultry and their genetic diversity might be much higher than what we have observed.
Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 06/2014; 19(25):20841. DOI:10.2807/1560-7917.ES2014.19.25.20841 · 5.72 Impact Factor