旨在了解国内鸡群马立克病病毒(MDV)和禽网状内皮增生病病毒(REV)的混合感染及自然重组状况,探讨两种病原的基因重组对MDV病原学特性的潜在影响及危害.利用PCR扩增、序列测定及间接免疫荧光试验(IFA),对来自河南商品蛋鸡群的17个MDV分离株进行研究.结果表明,其中两个毒株HNGS206和HNXZ103的病毒基因组中存在REV-LTR的重组插入.进一步的IFA结果显示,HNGS206和HNXZ103并非MDV与REV的自然重组流行毒株,这两个毒株基因组中LTR的插入发生于临床病例鸡混合感染后的病毒分离及传代培养过程中.结果提示,虽然目前国内鸡群中MDV和REV的混合感染较为普遍,但MDV与REV自然重组毒株的流行状况仍需要进一步的流行病学监测.

为评估市售商品化猪乙型脑炎(JE)弱毒活疫苗的质量以及实验室条件下疫苗的保存稳定性,采用TCID50效价测定和RT-PCR检测病毒的保守基因(E基因和C/prM基因)2种方法对2012年市场销售的主要国产疫苗产品进行了质量评估,同时将供试疫苗产品置-30℃保存3、6、9、12个月后分别取样测定乙脑病毒(JEV) TCID50效价.结果表明,市售的猪乙脑同类疫苗中不同品牌产品之间活病毒含量差异较大,最高可达104.33TCID50/头份,但部分产品含量极低,甚至难以检测到活病毒;E基因和C/prM基因的RT-PCR扩增产物丰度的差异与TCID50测定结果基本一致.多数疫苗产品在有效期内病毒效价基本稳定,同一厂家生产的疫苗保存期越短,其中相对活病毒越多,效价越高.

To investigate the molecular characteristics of Marek′s disease virus ( MDV ) prevalent in chicken flocks in Henan Province ,liver tissues were sampled from the suspecious cases in the flocks suffered from MD from 2012 to 2014.We amplified the MDV 132 bp repeat sequence from these tissues by polymerase chain reaction (PCR) and simultaneously,the meq,gE and gI genes were amplified,cloned,sequenced and then compared with the reference MDV strains and/or isolates.The main amplicons of the 132 bp repeat PCR products were the same size as 2 copies of the 132 bp repeats,similar to that of the positive control (GX0101).The sequences of meq,gE and gI genes cloned from clinical tissues were relatively conserved , with the homology among 98 .5%-100%, 98.8%-100%and 98 .0%-100%respectively compared to the reference MDV isolates and/or strains .Based on the sequences of meq,gE and gI genes,the phylogenetic analysis were performed and the results showed that com-pared to the strains or isolates from USA ,Australia,Japan and India ,the MDVs prevalent recently in Henan Prov-ince were genetically closer to the previously reported Henan MDV isolates .Together with the isolates from the other regions in China ,the Chinese isolates formed an independent branch in the phylogenetic tree .This study provides more detailed information regarding the field MDVs circulating in Henan Province and may be meaningful for the further control of MD .

[目的]了解马立克氏病病毒(Marek's disease virus,MDV)河南分离株Meq基因的分子特性,为中原地区MDV流行情况提供较详细的信息,为更好地防控MD奠定基础.[方法]应用PCR方法分别扩增了17株MDV河南省野毒分离株的Meq基因片段,克隆测序后,将各毒株Meq基因序列与vv+ MDV 648A株、vvMDV RBIB株、vMDV GA株、mMDV疫苗株CVI988株和814株的Meq基因序列进行比对分析.[结果]17株分离株中除HNGS201和HNLC503的Meq基因扩增产物较预期稍大外,其余15个的片段长度为1 020 bp,与预期长度相符;MDV河南分离株之间及其与参考毒株Meq基因的核苷酸同源性为99.0％～100％,部分毒株Meq基因的氨基酸序列有10个位点的氨基酸存在规律性突变;MDV河南分离株和参考株共组成3个进化分支,其中强毒株GA、RB1B和648A位于一个分支,分离株HNGS201、HNLH304、HNLC503与mMDV毒株CVI988和814位于同一分支,其余14株分离株位于另一个较大的分支.[结论]河南省可能流行着不同毒力的MDV,一些mMDV毒株的Meq蛋白存在59个或60个氨基酸的插入.

The microRNA (miRNA) mdv1-miR-M4, a functional miR-155 ortholog encoded by oncogenic Marek's disease virus (MDV), has previously been suggested to be involved in MDV pathogenesis. Using the technique of bacterial artificial chromosome mutagenesis, we have presently evaluated the potential role of mdv1-miR-M4 in the oncogenesis of the very virulent (vv) MDV strain GX0101. Unexpectedly, deletions of the Meq-cluster or mdv1-miR-M4 alone from the viral genome strongly decreased rather than abolished its oncogenicity. Compared to GX0101, mortalities of mutants GXΔmiR-M4 and GXΔMeq-miRs were reduced from 100% to 18% and 4%, coupled with the gross tumor incidence reduction from 28% to 22% and 8%, respectively. Our data suggests that the mdv1-miR-M4 is possibly an important regulator in the development of Marek's disease (MD) lymphomas but is not essential for the oncogenicity of vvMDV. In addition, some of the other Meq-clustered miRNAs may also play potentially critical roles in vvMDV induction of lymphomas.

Marek's disease is a highly contagious, oncogenic, and immunosuppressive avian viral disease. Surveillance of newly registered Marek's disease virus (MDV) isolates is meaningful for revealing the potential factors involved in increased virulence. Presently, we have focused on the molecular characteristics of all available MDVs from China, including 17 new Henan isolates. Based on Meq, gE, and gI genes, we found that most Chinese isolates contain conserved amino acid point mutations in Meq, such as E(77), A(115), A(139), R(176), and A(217), compared to USA virulent MDVs. However, the 59-aa or 60-aa insertions are only found in a few mild MDVs rather than virulent MDVs in China. Further phylogenetic analysis has demonstrated that a different genotype of MDV has been prevalent in China, and for virulent MDVs, their recent evolution has possibly been geographically restricted. Our study has provided more detailed information regarding the field MDVs circulating in China.

To investigate the features of Japanese encephalitis virus(JEV) currently pravalent and the genetic stability in pig farm in Henan province,the genetic stability of E gene of JEV isolate CSF.XZ-2D passaged in BHK-21 cells(baby hamster kidney cell line) was studied.The results showed that the E gene became stable after 25 passages in BHK-21 cells.After serial subcultivation,two stable amino acid(aa) mutations happened at the sites E271(E→V) and E278(V→L).No mutations were observed at part of the virulence correlated aa sites,such as E107,E138,E176,E177 and E315,same to those of vaccine strain SA14-14-2.However,mutations at the other two virulence correlated aa sites of E279(K→M→K) and E312(K→R→K→R) happened repeatedly.Whether these mutations are correlated with the virulence variation and adaptability of JEV to host cells needs to be further studied.

In this paper ,the genetic stability of E gene of JEV isolates BSF .ZZ-1 and BSF .ZZ-3 passaged in BHK-21 cells(Baby hamster kidney cell line ) was studied.The results showed that the E gene is becoming stable after 60 passages in BHK-21 cells.After serial sucultivation,stable amino acid(aa) mutation happened at the sites E21(F→L),E200(T→A),E244(G→E),E279(N→S) and E426(G→D) in isolate BSF.ZZ-1 and sites E244 (G→E),E255(F→L),E285(M→L),E368(L→S) and E97(N→S) in isolate BSF.ZZ-3.No mutations were ob-served at part of the virulence correlated aa sites ,such as E107,E138,E176,E177 and E315,similar to those of vaccine strain SA14-14-2.However,mutations at the virulence correlated aa sites of E279(M→K→M)of BSF.ZZ-3 happened repeatedly .Whether these mutations are correlated with the virulence variation and adaptability of JEV to host cells needs to be further studied .

To determine and evaluation the qualities of commercial live attenuated vaccines ofJapanese encephalitis(JE),batches of commercial vaccines produced in 2009-2011 were determined by the virus titer determination of 50% tissue culture infective dose(TCID50) and reverse transcriptase-polymerase chain reaction(RT-PCR).The results showed that presently large differences of the virus titers were existed among three brand vaccines produced by different commercial companies.Single-dose of some vaccines archived the highest virus titer of 103.80 TCID50 while some others were nearly undetectable.The results detected by RT-PCR were in accordant with the outcome of TCID50 determination.The study provides useful information for helping the farmers to choose suitable vaccine products for control of JE in swine populations.

In 2011-2012,the tumorigenic and immunosuppresive disease were broken out in the commercial layer chickens in large areas of Henan province.The clinical symptoms and macroscopic pathological inspections suggested that it might be a case of Marek′s disease(MD).In order to identity the cause of the disease,three pairs of primers specific to the MDV meq,gB and pp38 genes were designed according to the genomic sequence of MDV strain GA.Meanwhile,polymerase chain reaction(PCR) was carried out on liver and spleen samples of those suspected cases.The results showed that three different sizes of PCR products of MDV-specific genes were successfully amplified from the randomly tested five cases collected in each region.It indicated that MDV was widely spread in Henan Provice and led to the outbreak of MD in the commercial layer chickens.The study provided useful information for the rapid diagnosis and efficient control of MD in the future.

In order to learn more about the complete genome and molecular evolution regular of Japanese encephalitis virus(JEV) strains isolated from swine,the full-length genome sequences of three JEV isolates,namely as BSF.ZZ-1,BSF.ZZ-3 and CSF.XZ-2D isolated from swine were analyzed in this paper.The results show that the length of the complete genomes of BSF.ZZ-1,BSF.ZZ-3 and CSF.XZ-2D was 10 977,10 977,10 976 nt,respectively.There is an insert base of G at the 10 701 nt site in the genomes of both BSF.ZZ-1 and BSF.ZZ-3.Compared to the virulent strain SA14,the homologies of nucleotide and amino acid sequences of BSF.ZZ-1,CSF.XZ-2D and BSF.ZZ-3 are 99.4% and 99.0%,respectively.Compared to the isolates HW(HW1),HWe(HW2) and SH0601 from swine,the homologies of the present three isolates are all more than 98.0%.Phylogenetic analysises with other 30 reference isolates covering all five JEV genotypes indicated that BSF.ZZ-1,CSF.XZ-2D and BSF.ZZ-3 located in a same evolutionary branch,belonging to the genotype III.

【Objective】 The objective of this experiment is to develop an immunochromatographic strip for rapid detection of neomycin(NEO).【Method】 The monoclonal antibodies(mAb) against neomycin were generated and labeled with colloidal gold particles.Then the immunochromatographic strips for detection of NEO were developed and identified.【Result】 Ka of NEO monoclonal antibody was 3.75×1010 mol·L-1.The detection limit of the strip was 50 ng·mL-1 by naked eyes.The cross-reactivity with other antibiotics,such as gentamicin,streptomycin,tobramycin,sisomicin,ractopamine,salbutamal,tylosin,chloramphenic,was not detected.In parallel experiments,the test strip gave the same results as HPLC-MS-MS did.【Conclusion】 An immunochromatographic strip test method was established and the strip can be used to detect NEO residues rapidly with high specificity,sensitivity and simplicity in different samples.