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 recom...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.展开更多
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/...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.展开更多
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 antim...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.展开更多
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 an...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.展开更多
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 ...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.展开更多
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 Ki...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.展开更多
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 conditio...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.展开更多
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 ...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.展开更多
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...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.展开更多
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, generat...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.展开更多
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 gen...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.展开更多
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 ...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.展开更多
The mechanism of rabies virus (RABV) infection still needs to be further characterized. RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells. Herein, based on...The mechanism of rabies virus (RABV) infection still needs to be further characterized. RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells. Herein, based on a reverse genetic system of the Evelyn-Rokitnicki-Abelseth (rERA) strain, we generated a recombinant RABV rERA-N/mCherry strain that stably expresses an additional ERA nucleoprotein that fuses with the red fluorescent protein mCherry (N/mCherry). The rERA-N/mCherry strain retained growth property similar to the parent strain rERA in vitro. The N/mCherry expression showed genetic stability during passage into mouse neuroblastoma (NA) cells and did not change the virulence of the vector. The rERA-N/mCherry strain was then utilized as a visual viral model to study the RABV-cell binding and internalization. We directly observed the red self-fluorescence of rERA-N/mCherry particles binding to the cell surface, and further co-localizing with clathrin in the early stage of infection in NA cells by fluorescence microscopy. Our results showed that the rERA-N/mCherry strain uses clathrin-dependent endocytosis to enter cells, which is consistent with the well-known mechanism of RABV invasion. The recombinant RABV rERA-N/mCherry thus appears to have the potential to be an effective viral model to further explore the fundamental molecular mechanism of rabies neuropathogenesis.展开更多
In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic Peo...In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea(DPRK)were published.During the period 2013–2014,HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.Therefore,the monitoring and control of influenza virus in ducks must be given top priority,which are essential components to prevent and control HPAI.展开更多
In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of pri...In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of primers designed according to the 3' UTR of US8 gene and 5' end of the new getting sequence were used to amplify a 2,426 bp sequence toward the TRs region.Sequence analysis revealed that the both sequences contained an identical 990 bp open reading frame of DEV US1 gene.The two ORFs were in opposite transcription orientation.Sequence comparison of the nucleotide sequence and the deduced amino acid sequence of US1 gene showed relatively high identity to Mardivirus.Phylogenetic tree analysis showed that the eleven herpesviruses viruses were classified into three groups,and the duck enteritis virus was most closely related to Mardivirus.展开更多
The acidic leucine-rich nuclear phosphoprotein 32 kDa(ANP32)family con sists of evolutionarily con served proteins of 220-291 amino acids characterized by an N-terminal leucine-rich repeat domain(LRR)and a C-terminal ...The acidic leucine-rich nuclear phosphoprotein 32 kDa(ANP32)family con sists of evolutionarily con served proteins of 220-291 amino acids characterized by an N-terminal leucine-rich repeat domain(LRR)and a C-terminal low-complexity acidic region(LCAR).ANP32 family proteins regulate a variety of physiological functions,including chromatin remodeling apoptosis and nervous system development.Abnormal ANP32 expression is closely related to tumori-genesis.In recent years,the role of ANP32 family proteins in viral infections has received considerable attention due to their activity supporting influenza virus replication and restriction of virus cross-species transmission.Moreover,ANP32 proteins are closely related to the replication of HIV and nonsegmented negative-strand RNA viruses(NNSVs).In this review,the general physiological functions of ANP32 family proteins,as well as their roles in virus replication,are summarized in detail.展开更多
Infectious bursal disease(IBD),caused by IBD virus(IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe in...Infectious bursal disease(IBD),caused by IBD virus(IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe including Pakistan.While the disease is threatening the poulty industry,the nature of predominant strains of IBDV in Pakistan remained l-defined.In this study,an epidemiology survey was conducted in the main chicken-farming regions of Pakistan.The batch of Pakistan IBDVs genes simultaneously covering both VP1 and VP2 were amplified,sequenced,and analyzed.The unique segment-reassortant IBDVs(vv-A/Uniq-B),carrying segmentA from vvIBDV and segment B from one unique ancestor,were identifed as one important type of circulating strains in Pakistan.The data also discovered the characteristic molecular features of Pakistan IBDVs,which will contribute to scientific vaccine selection and effective prevention of the disease.展开更多
Infectious bursal disease virus（IBDV）is responsible for the highly contagious infectious bursal disease of chickens.Further understanding the gene-function is necessary to design the tailored vaccine.The amino acid ...Infectious bursal disease virus（IBDV）is responsible for the highly contagious infectious bursal disease of chickens.Further understanding the gene-function is necessary to design the tailored vaccine.The amino acid residue 279,located on strand P_F of VP2,is one of the three residues that have been reported to be involved in cell-tropism but with some inconsistency.In this study,to further clarify the amino acids involved in the cell tropism of IBDV,a series of mutations about residue 279were introduced into the VP2 of vv IBDV Gx strain.With the reverse genetic system,we found single mutation of D279N,double mutations of D279N/A284T or Q253H/D279N were not enough to adapt IBDV to chicken embryo fibroblast（CEF）cell.To evaluate whether residue 279 could influence the replication and virulence of IBDV,the virus r Gx HT-279 with three mutations（Q253H/D279N/A284T）was rescued and evaluated.Results showed that the mutation of residue 279 in VP2had no efficient effects on both the replication efficiency in vitro and the virulence to SPF chickens of IBDV.In summary,the results demonstrated that residue 279 of VP2 did not contribute efficiently to cell tropism,replication efficiency,and virulence of IBDV at least in some strains.These findings provided further information for understanding the gene function of IBDV.展开更多
To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defectiv...To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.展开更多
To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. T...To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1 a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1 a P 130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-la genome, we sequenced and analyzed the ORF5 gene of CH-la strain of different passages （P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130） and found that three mutations （C5Y, H38Q and L146Q） which may be related with the attenuation of CH-la. In addition, we also found a unique restriction enzyme site （TspEI） in the ORF5 gene of attenuated CH-la, which can be used as a genetic marker to distinguish original and attenuated CH- 1 a.展开更多
基金supported by the National Key Research and Development Program of China(2021YFD1800200)the Laboratory for Lingnan Modern Agriculture Project(NT2021007)the China Agriculture Research System of the MOF and MARA(CARS-41-G12)。
文摘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.
基金This work was supported by the National Key R&D Program of China(2016YFD0501602,2017YFD0500701,and 2016YFEO203200)the National Natural Science Foundation of China(3167131307)+1 种基金the China Agriculture Research System(CARS-41-G12)and Central Publicinterest Scientific Institution Basal Research Fund(1610302017001).
文摘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.
基金supported by grants from the National Key Research and Development Program of China (2016YFD0501608)the National Natural Science Foundation of China (31470893)+1 种基金the Special Fund for Agro-scientific Research in the Public Interest,China (201403054)the National High Technology Research and Development Program of China (2011AA10A210)
文摘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.
基金supported by the Special Foundation for State Basic Research Program of China(2013FY113300-8)the National Key R&D Program of China(2016YFD0500800)
文摘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.
基金supported by grants from the National Basic Research Program of China (973 Program,2005CB523200)the National High-Tech Research and Development Program of China (863 Program,2006AA10A20 4)+1 种基金the National Key Technology R&D Program (2006BAD 06A04/18/01/03)the National Natural Science Foundation of China (30470072)
文摘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.
基金supported by the National Key R&D Program of China(2016YFD0500800)。
文摘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.
基金supported by grants from the National Natural Science Foundation of China(No. 31101801)
文摘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.
基金supported by grants from the National Key R&D Program of China (2016YFD0500705, 2017YFD0500105, and 2017YFC1200502)the Fundamental Research Funds for the Central Publicinterest Research Institutes (Y2017LM08)。
文摘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.
基金supported by the National Basic Research Program of China (2005CB523202)
文摘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.
基金supported by the Natural Science Foundation of Heilongjiang Province,China(JQ2019C005)the National Natural Science Foundation of China(31702265 and 32172847)。
文摘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.
基金supported by the National Natural Science Foundation of China(31302109)
文摘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.
基金supported by the National Key R&D Program of China(2016YFD0500800)the International Science&Technology Cooperation Program of China(2014DFR31260)
文摘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.
基金supported by the National Natural Science Fundation of China (31800138)the National Key Research and Development Program of China (2016YFD0500403)
文摘The mechanism of rabies virus (RABV) infection still needs to be further characterized. RABV particle with self-fluorescent is a powerful viral model to visualize the viral infection process in cells. Herein, based on a reverse genetic system of the Evelyn-Rokitnicki-Abelseth (rERA) strain, we generated a recombinant RABV rERA-N/mCherry strain that stably expresses an additional ERA nucleoprotein that fuses with the red fluorescent protein mCherry (N/mCherry). The rERA-N/mCherry strain retained growth property similar to the parent strain rERA in vitro. The N/mCherry expression showed genetic stability during passage into mouse neuroblastoma (NA) cells and did not change the virulence of the vector. The rERA-N/mCherry strain was then utilized as a visual viral model to study the RABV-cell binding and internalization. We directly observed the red self-fluorescence of rERA-N/mCherry particles binding to the cell surface, and further co-localizing with clathrin in the early stage of infection in NA cells by fluorescence microscopy. Our results showed that the rERA-N/mCherry strain uses clathrin-dependent endocytosis to enter cells, which is consistent with the well-known mechanism of RABV invasion. The recombinant RABV rERA-N/mCherry thus appears to have the potential to be an effective viral model to further explore the fundamental molecular mechanism of rabies neuropathogenesis.
基金supported by the China Agriculture Research System of MOF and MARA(CARS-41)。
文摘In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea(DPRK)were published.During the period 2013–2014,HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.Therefore,the monitoring and control of influenza virus in ducks must be given top priority,which are essential components to prevent and control HPAI.
基金Founding of Heilongjiang province education department major program(10541Z004)Heilongjiang province key scientific and technological program(GB04B504)
文摘In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of primers designed according to the 3' UTR of US8 gene and 5' end of the new getting sequence were used to amplify a 2,426 bp sequence toward the TRs region.Sequence analysis revealed that the both sequences contained an identical 990 bp open reading frame of DEV US1 gene.The two ORFs were in opposite transcription orientation.Sequence comparison of the nucleotide sequence and the deduced amino acid sequence of US1 gene showed relatively high identity to Mardivirus.Phylogenetic tree analysis showed that the eleven herpesviruses viruses were classified into three groups,and the duck enteritis virus was most closely related to Mardivirus.
基金supported by grants from the Natural Science Foundation of China to HL Chen and XJ Wang(31521005)HL Zhang(32002275)a Natural Science Foundation of Heilongjiang Province grant to HL Zhang(YQ2020C021).
文摘The acidic leucine-rich nuclear phosphoprotein 32 kDa(ANP32)family con sists of evolutionarily con served proteins of 220-291 amino acids characterized by an N-terminal leucine-rich repeat domain(LRR)and a C-terminal low-complexity acidic region(LCAR).ANP32 family proteins regulate a variety of physiological functions,including chromatin remodeling apoptosis and nervous system development.Abnormal ANP32 expression is closely related to tumori-genesis.In recent years,the role of ANP32 family proteins in viral infections has received considerable attention due to their activity supporting influenza virus replication and restriction of virus cross-species transmission.Moreover,ANP32 proteins are closely related to the replication of HIV and nonsegmented negative-strand RNA viruses(NNSVs).In this review,the general physiological functions of ANP32 family proteins,as well as their roles in virus replication,are summarized in detail.
基金This work was supported by the National Key Research and Development Program of China(2016YFE0203200,2017YFD0500704)the Heilongjiang Province Foundation for the National Key Research and Development Program of China(GX18B011)+1 种基金the National Natural Science Foundation of China(31430087)the earmarked fund for the China Agriculture Research System(CARS-41-G15).
文摘Infectious bursal disease(IBD),caused by IBD virus(IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe including Pakistan.While the disease is threatening the poulty industry,the nature of predominant strains of IBDV in Pakistan remained l-defined.In this study,an epidemiology survey was conducted in the main chicken-farming regions of Pakistan.The batch of Pakistan IBDVs genes simultaneously covering both VP1 and VP2 were amplified,sequenced,and analyzed.The unique segment-reassortant IBDVs(vv-A/Uniq-B),carrying segmentA from vvIBDV and segment B from one unique ancestor,were identifed as one important type of circulating strains in Pakistan.The data also discovered the characteristic molecular features of Pakistan IBDVs,which will contribute to scientific vaccine selection and effective prevention of the disease.
基金supported by a grant from National Natural Science Foundation of China(31430087)the Scientific and Technological Research Project of Harbin,China(2014AB3AN058)+2 种基金the Special Fund for Scientific and Technological Innovative Talents of Harbin,China(2014RFQYJ129)China-France Cai-Yuanpei Program(2011008007)the Modern Agro-industry Technology Research System of China(nycytx-42-G3-01)
文摘Infectious bursal disease virus（IBDV）is responsible for the highly contagious infectious bursal disease of chickens.Further understanding the gene-function is necessary to design the tailored vaccine.The amino acid residue 279,located on strand P_F of VP2,is one of the three residues that have been reported to be involved in cell-tropism but with some inconsistency.In this study,to further clarify the amino acids involved in the cell tropism of IBDV,a series of mutations about residue 279were introduced into the VP2 of vv IBDV Gx strain.With the reverse genetic system,we found single mutation of D279N,double mutations of D279N/A284T or Q253H/D279N were not enough to adapt IBDV to chicken embryo fibroblast（CEF）cell.To evaluate whether residue 279 could influence the replication and virulence of IBDV,the virus r Gx HT-279 with three mutations（Q253H/D279N/A284T）was rescued and evaluated.Results showed that the mutation of residue 279 in VP2had no efficient effects on both the replication efficiency in vitro and the virulence to SPF chickens of IBDV.In summary,the results demonstrated that residue 279 of VP2 did not contribute efficiently to cell tropism,replication efficiency,and virulence of IBDV at least in some strains.These findings provided further information for understanding the gene function of IBDV.
基金supported by the Chinese National S&T Plan(2004BA519A55)Scientific Research Program of State Key Laboratory of Veterinary Biotechnology(NKLVBP200818)
文摘To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.
基金supported by the National High Technology Research and Development Program of China(2011AA10A213)the Key Technology R&D Program of Harbin, China(2010AA6AN083)the Excellent Youth Foundation of Heilongjiang Province of China(JC201020)
文摘To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1 a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1 a P 130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-la genome, we sequenced and analyzed the ORF5 gene of CH-la strain of different passages （P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130） and found that three mutations （C5Y, H38Q and L146Q） which may be related with the attenuation of CH-la. In addition, we also found a unique restriction enzyme site （TspEI） in the ORF5 gene of attenuated CH-la, which can be used as a genetic marker to distinguish original and attenuated CH- 1 a.
