Identification of two novel linear epitopes on the p30 protein of African swine fever virus

African swine fever(ASF) is a hemorrhagic disease caused by African swine fever virus(ASFV), which belongs to the Asfarviridae family. ASF has become prevalent in Africa since it was first reported in Kenya in 1921(Rowlands et al. 2008;Costard et al. 2009). In 1957, it was introduced to Portugal in Europe, after which ASFV rapidly spread to other European countries and has already been eradicated in several countries except Sardinia(Mur et al. 2016). In 2007, ASF was introduced to Georgia and has continued its spread to other countries in Europe(Smietanka et al. 2016;Kolbasov et al. 2018;Garigliany et al. 2019;Linden et al. 2019). In China, an ASF outbreak was first reported on August 3, 2018 in a pig farm in Shenyang, Liaoning Province(Zhou et al. 2018). Since then, the dramatic spread of ASF throughout China has caused huge economic losses to the swine industry(Jiang et al. 2021).

Prevalence and genetic characterization of hepatitis E virus in domestic Bactrian camels in northern China:Identification of a novel HEV8 subtype

Hepatitis E virus(HEV)infection is associated with epidemic and sporadic viral hepatitis worldwide.It is a small,quasi-enveloped,single-stranded positive-sense RNA virus(Emerson and Purcell 2003).Its genome varies from 6.6 to 7.3 kb in length,containing three open reading frames(ORFs)named ORF1,ORF2,and ORF3.They encode non-structural polyprotein,viral capsid protein,and a small phosphoprotein that is essential for viral release,respectively.

Effects of dissolved oxygen on intestinal bacterial community and immunity of Atlantic salmon Salmo salar

Dissolved oxygen(DO)is one of most important factors which affect wide range physiologic features of including immune responses and intestinal bacterial community.However,the underlying mechanisms remain enigmatic.To address this question,the intestinal bacterial community compositions and the immune features of Atlantic salmon(Salmo salar)grown in recirculating aquaculture systems(RAS)were characterized.Fish were reared under different DO saturation levels,e.g.,200% saturation named high group(H),100%saturation named control group(CK),and 60%saturation named lower group(L).Large variations in the operational taxonomic units(OTUs)frequency distribution for the intestinal bacterial community of Atlantic salmon were observed.The intestinal bacterial community of all groups was dominated mainly by three phyla,e.g.,Proteobacteria.Firmicutes,and Bacteroidetes.Interestingly,Acinetobacter baumannii,an opportunistic pathogen of salmon was increased significantly in L group.We further monitored the immunity features of fish under different DO levels.The results show that leucocyte number,cortisol level,the expressions of interleukin-1β(IL-1β),Toll-like receptor 4(TLR4),and nucleotide-binding oligomerization domain like protein 2(NOD2)were higher at significant levels in the L group than those in the other two groups.TLR4 and NOD2 are usually related with the bacterial infections;therefore,it is reasonable to believe that the stronger immune responses observed in the L group might be related with the higher abundance of A.baumannii in the inte stine of Atlantic salmon.Overall,these findings demonstrated that low DO level may induce stronger immunity responses in Atlantic salmon.

Epidemiological Investigation and Analysis of Major Viral Diseases of Pig Farms in Guangxi from 2019 to 2022

In order to understand the epidemic characteristics of major viral infectious diseases of pig farms in Guangxi from 2019 to 2022,the PCR and RT-PCR methods were applied to detect the pathogens of 1046 samples suspected CSFV(swine fever virus),PRRSV(porcine reproductive and respiratory syndrome virus),PCV-2(porcine circovirus virus type 2),PCV-3(porcine circovirus virus type 3),PRV(pseudorabies virus),TGEV(transmissible gastroeneritis of pigs virus),PEDV(porcine epidemic diarrhea virus)and PoRV(porcine rotavirus)infections in recent four years,and epidemiological analysis was carried out.The results showed that positive rates of CSFV,PRRSV,PCV-2,PCV-3,PRV,TGEV,PEDV and PoRV were 3.91%(21/537),20.46%(143/699),29.90%(116/388),16.10%(81/503),27.75%(121/436),2.10%(8/381),28.50%(61/214)and 13.74%(18/131).The infection rates of PCV-2 and PEDV were decreasing year by year,while the infection rate of TGEV was increasing year by year.In single infection,the infection rates of PRV and PEDV were higher,with an average infection rate of 13.53%and 18.22%.In the mixed infection,the infection rate of PRRSV and PCV-2 was the highest,with an average infection rate of 11.34%.The second mixed infection rate was PEDV and PoRV,and the average infection rate was 8.05%.The infection rate of PCV-3 and PEDV was higher in spring,with 18.80%and 40.00%respectively.The infection rate of PRRSV,PCV-2,PRV,TGEV and PoRV was higher in winter,with 24.40%,40.57%,37.30%,3.23%and 19.44%respectively.The results showed that PRRSV,PCV-2,PRV and PEDV were the main viral diseases with serious damage to pig farms in Guangxi.It was necessary to strengthen pathogen monitoring and improve comprehensive prevention and control ability,in order to reduce the incidence of disease and economic losses.

Protective efficacy of an H5/H7 trivalent inactivated vaccine(H5-Re13,H5-Re14, and H7-Re4 strains) in chickens, ducks, and geese against newly detected H5N1, H5N6, H5N8, and H7N9 viruses

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains(H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin(HA) and neuraminidase(NA) genes of an H5 N6 virus that bears the clade 2.3.4.4 h HA gene, H5-Re14 contains the HA and NA genes of an H5 N8 virus that bears the clade 2.3.4.4 b HA gene, and H7-Re4 contains the HA and NA genes of H7 N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses(an H5 N1 virus, an H5 N6 virus, and an H5 N8 virus) bearing the clade 2.3.4.4 b HA gene, an H5 N6 virus bearing the clade 2.3.4.4 h HA gene, and an H7 N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.3.4.4 h or 2.3.4.4 b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7 N9 viruses that are currently circulating in nature.

Immune mechanisms of Concanavalin A model of autoimmune hepatitis

As a chronic inflammatory disease of the liver,the pathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been elucidated,with prognosis and diagnosis remaining unsatisfied.Currently the only viable treatments of AIH are immunosuppressant application and liver transplantation.It is considered that lack of good animal AIH models is the main reason for the shortage of a simple and efficient cure.The Concanavalin A (Con A) model is a typical and well established model for investigating T-cell and macrophage dependent liver injury in mice,which closely mimics the pathogenesis mechanisms and pathological changes of patients,and is regarded as the best experimental model for AIH research so far.In this paper we eluci-dated the pathogenic mechanisms of AIH and the evolution of relative animal models.We go on to further focus on Con A-induced liver injury from the point of immunological mechanisms and the change of cytokine levels.Finally,we manifested the clinical significance of the AIH animal models and the challenges they would meet during their future development.

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5N6) (DK/GZ/S4184/17) (a clade 2.3.4.4d virus), A/chicken/Liaoning/SD007/2017(H5N1) (CK/LN/SD007/17) (a clade 2.3.2.1d virus), and A/chicken/ Guangxi/SD098/2017(H7N9) (CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.

Comparison of the protective effects of resveratrol and pterostilbene against intestinal damage and redox imbalance in weanling piglets

Background: Evidence indicates that early weaning predisposes piglets to intestinal oxidative stress and increases the risk of intestinal dysfunction;however, there are minimal satisfactory treatment strategies for these conditions.This study investigated the potential of resveratrol and its analog, pterostilbene, as antioxidant protectants for regulating intestinal morphology, barrier function, and redox status among weanling piglets.Methods: A total of 144 piglets were selected at 21 days of age and randomly allocated into one of four treatment groups, each of which included six replicates. Piglets in a sow-reared control group were suckling normally between ages 21 and 28 days, while those in weaned groups were fed a basal diet, supplemented with either 300 mg/kg of resveratrol or with 300 mg/kg of pterostilbene. Parameters associated with intestinal injury and redox status were analyzed at the end of the feeding trial.Results: Early weaning disrupted the intestinal function of young piglets, with evidence of increased diamine oxidase activity and D-lactate content in the plasma, shorter villi, an imbalance between cell proliferation and apoptosis, an impaired antioxidant defense system, and severe oxidative damage in the jejunum relative to suckling piglets. Feeding piglets with a resveratrol-supplemented diet partially increased villus height(P = 0.056) and tended to diminish apoptotic cell numbers(P = 0.084) in the jejunum compared with those fed a basal diet. Similarly, these beneficial effects were observed in the pterostilbene-fed piglets. Pterostilbene improved the feed efficiency of weanling piglets between the ages of 21 and 28 days;it also resulted in diminished plasma diamine oxidase activity and D-lactate content relative to untreated weaned piglets(P < 0.05). Notably, pterostilbene restored jejunal antioxidant capacity, an effect that was nearly absent in the resveratrol-fed piglets. Pterostilbene reduced the malondialdehyde and 8-hydroxy-2′-deoxyguanosine contents of jejunal mucosa possibly through its regulatory role in facilitating the nuclear translocation of nuclear factor erythroid-2-related factor 2 and the expression levels of NAD(P)H quinone dehydrogenase 1 and superoxide dismutase 2(P < 0.05).Conclusions: The results indicate that pterostilbene may be more effective than its parent compound in alleviating early weaning-induced intestinal damage and redox imbalance among young piglets.

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

Somatostatin （SS） is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus （PRRSV）. A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets

Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China＇s pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses （3×103-3×105 TCID50）.The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever （41.0-41.9oC） and high morbidity and mortality （60-100%）,the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection （DPI）.The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.

Construction of Salmonella Pullorum ghost by co-expression of lysis gene E and the antimicrobial peptide SMAP29 and evaluation of its immune efficacy in specific-pathogen-free chicks

In this study, a safety enhanced Salmonella Pullorum （S. Pullorum） ghost was constructed using an antimicrobial peptide gene, and evaluated for its potential as a Pullorum disease （PD） vaccine candidate. The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S. Pullorum ghosts. No viable bacteria were detectable either in the fermentation culture after induction of gene E- and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products. Specific-pathogen- free （SPF） chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality, clinical symptoms or signs of PD such as anorexia, depression and diarrhea were observed. On challenge with a virulent S. Pullorum strain at 4 wk post-immunization, a comparatively higher level of protection was observed in the S. Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups. In addition, immunization with the S. Pullorum ghosts induced a potent systemic IgG response and was associated with significantly increased levels of cytokine IFN-y and IL-4 and relative percentages of CD4＋ and CD8＋ T lymphocytes. Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts, and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.

Development of a cELISA for effective detection of the antibody against H7 subtype of avian influenza virus

H7 avian influenza viruses(AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs(H7 N2, H7 N3, and H7 N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA. Until March 2013, human infections with H7 N9 AIVs were reported in China. Since then, H7 N9 AIVs have continued to circulate in both humans and birds. Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic. In this study, a competitive enzyme-linked immunosorbent assay(cELISA)method for the detection of antibody against H7 AIVs was established. The optimal concentration of antigen coating was 5 μg mL^(-1), serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000. To determine the cut-off value of cELISA, percent inhibition(PI) was determined by using receiver operating characteristic(ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples(n=546). When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively. This method could detect the antibodies against H7 Nx(N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus(NDV), Infectious bronchitis virus(IBV) and Infectious bursal disease virus(IBDV), exhibiting good specificity. This method showed a coincidence rate of 98.56% with hemagglutinin inhibition(HI) test. And the repeatability experiment revealed that the coefficients of variation(CV) of intra-and inter-batch repetition were all less than 12%. The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment.Until now,traditional RT-PCR,fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV,but these methods require high-level instruments and experimental conditions,not suitable for the rapid detection in field and farms.In order to develop a rapid,sensitive and practical method to detect and identify AIV subtypes,4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established.Using this method,the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein,without cross reaction with other susceptible avian viruses.In addition,the detection limit of the common H1,H5,H7,and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit),which was 10 times more sensitive than that using the routine RT-PCR.Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR.All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast,convenient and practical method for the clinic test and epidemiological investigation of AIV.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Construction and Virulence of Filamentous Hemagglutinin Protein B1 Mutant of Pasteurella multocida in Chickens

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two filamentous hemagglutinin genes, fhaB1 and JhaB2, are the potential virulence factors. In this study, an inactivationfhaB1 mutant ofP. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of thefhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation offhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. ThefhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90% convalescent chicken serum. ThefhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum （P〈0.007）. These results confirmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus(FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease(FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements(VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.