For understanding the prevalent situation of swine disease in certain regions in Guangxi,we detected CSFV,PRRSV,PCV2,PRV,PPV,JEV and BVDV from 26 samples by RT-PCR or PCR in the investigation.The results showed that the swine in investigated regions were mixture infection,infected by three or more viruses in most cases,and the situation was complicated.

本研究采用RT-PCR方法从培养的鹅外周血淋巴细胞总RNA中成功扩增到鹅IFN-γ基因。将克隆在pMD18-T载体中的鹅IFN-γ基因插入原核表达载体pGEX-6P-1,得到重组质粒pGEX-6P-1-IFN-γ。重组质粒转化大肠杆菌BL21,经IPTG诱导后作SDS-PAGE分析,得到约43 ku融合表达蛋白特异条带。用GST亲和纯化柱对原核表达产物中目的蛋白进行纯化,得到1.2 mg/L的纯化蛋白。用100TCID50病毒量在鹅副黏病毒(GPMV)/鹅胚成纤维细胞系统上测定表达蛋白的抗病毒活性,观察不同处理组的细胞形态并用RT-PCR方法鉴定,发现表达蛋白稀释倍数小于104可抑制GPMV复制,按照Reed-Muench法计算表达的鹅IFN-γ抗病毒效价为2.0×103U/mL,表明表达蛋白具有良好的抗病毒活性。

猪瘟是一种由猪瘟病毒引起的高度接触性传染病,是目前影响我国养猪业最严重的传染病之一[1,2].近几年养猪业的不断发展,猪瘟在不少地方仍不断发生和流行,特别是由于非典型猪瘟和温和性猪瘟的出现,猪瘟的流行病学、临床表现、病理变化方面发生了很大变化,给猪瘟的诊断和防治工作带来了新的难题[3,5].

[Objective]The present study was aimed to investigate the presence and spread of H9N2 subtype influenza virus in wild bird in order to understand its trends of occurrence and to develop proper control measures for preventing its infection on human beings in Guangxi. [Method]The lung tissue was collected from wild birds of Guangxi, the virus was isolated and inoculated to 9-11 day chicken-Embryo. The HA and NA subtypes were identified using serological tests followed by amplification and sequencing of HA and NA genes . [Result]The isolates were found to be inhibited by H9 subtypes positive serum, and identified as N2 subtype by neuraminidase test. This isolate belonged to the H9N2 subtype of avian influenza virus and named as A/wild bird/Guangxi/H2/07. The sequence analysis results of HA and NA genes showed 82.4-99.0% and 83.1-99.9% homologies of this virus strain with H9 and N2 subtypes, respectively. [Conclusion]This study proved that the H9N2 subtype influenza virus exists in wild birds of Guangxi, and provided the reference for monitoring and control of Guangxi influenza epidemic situations.

The IFN-γ gene was inserted into the eukaryotic expression vector pcDNA3.1(+),and the eukaryotic expression plasmid pcDNA3.1(+)-IFN-γ was constructed and identified by sequencing.Electroporation instrument was set to 425 V of voltage and 25 μF of capacitor,then pulsed 2 times consecutively under room temperature.20 μg/mL plasmid pcDNA3.1(+)-IFN-γ was transformed into 1×106 cells/mL BHK-21 in 400 μL of volume.After consecutive selection by 600 μg/mL G418 for 14 days,resistant cell clones were selected,and the cell supernatants were identified by Western-blot test.The results indicated that goose IFN-γ was expressed in BHK-21 cells.

A H9N2 subtype strain of swine influenza virus(SIV) was isolated from swine in Guangxi by SPF chicken embryo.The SIV strain had a stable haemagglutiain(HA) values for 27 after there generations,and can be inhibited by H9 positive serum.The HA and NA genetic amplification showed that this strain had the height homology with the subtype H9N2 SIV,which named A/Swine/Guangxi/01/2009.

对具有流感临床症状的病马组织经处理后接种SPF鸡胚,分离到1株流感病毒.该病毒经鸡胚接种7代后,出现稳定的对鸡红细胞凝集效价,效价为25,能被H3亚型血清中和,与H1、H5、H7、H9亚型阳性血清无交叉反应；对HA基因及NA基因进行序列分析,结果显示:HA基因与马流感病毒H3亚型同源性最高,NA基因与马流感病毒N8亚型同源性最高,判断该病毒属于H3N8亚型马流感病毒,命名A/EQ/Guangxi/01/09.

Full-length cDNAs of polymerase genes PB2 and PB1 of avian influenza virus strain GS/GX/01/06 were amplified by RT-PCR.The cDNAs were cloned into pMD18-T vectors and sequenced.Sequence analysis showed that open reading frames of the PB2 and PB1 cDNAs contain 2 280 nt and 2 277 nt encoding 759 and 758 amino acids,respectively.Nucleotide sequence and amino acid identity rates of the polymerase genes PB2 and PB1 were over 95% between avian influenza virus strain QA/HK/G1/97 that belong to the subtype H9N2 AIV,there were in closer relation between them.

目前广西农村中小规模猪场多,这些猪场饲养管理技术水平落后、猪病综合防控体系不完善,导致疫病发生频繁,发病情况日趋复杂,多病原混合感染严重.这种多病原体的感染使病猪所表现的临床症状缺乏特征性,而是表现一系列的综合症候群,这增加了临床诊断的难度,也给疫病的控制带来困难,致使猪群发病率和死亡率增高.2010年2月广西北海某猪场发生了一起以体温升高,呼吸困难,局部皮肤发紫,众多脏器出血为主要特征的传染病,经流行病学调查、临床诊断、病理剖检和实验室检测,诊断为猪繁殖与呼吸综合征病毒变异株、猪瘟病毒和链球菌混合感染.现报道如下.

A pair of primers and a TaqMan probe were designed according to the CSFV 5′UTR sequences of CSFV taken from GenBank,for performing TaqMan fluorescent quantitative PCR.RNA of classical swine fever virus(CSFV) was used as template,and the reaction conditions of TaqMan fluorescence PCR were optimized based on conventional PCR.The TaqMan fluorescent quantitative PCR method for CSFV was developed,which showed high specificity and high sensitivity to CSFV(detection sensitivity of 5.03×10-2 μg/μL),as revealed by the sensitivity and specificity tests.Therefore,this method can be used for rapid detection of CSFV.

根据已发表的猪链球菌9型(SS9)荷兰5218分离株的基因核苷酸序列保守区域,设计一对特异性引物,以SS9钦州分离株抽提的DNA为模板,通过PCR方法扩增cps9H基因片段,并对其进行克隆、测序和分析.结果表明,4株SS9钦州分离株的cps9H目的基因长为389bp,与已知的7个国内外SS9毒株cps9H基因片段的核苷酸及推导的氨基酸序列比较,同源性分别为96.7%～100%和91.5%～100%.

A pair of primer was designed using the nucleotide sequence of Northeast white goose IFN-α gene obtained from GenBank(Accession No.AY524422).Total RNA was extracted from cultures of goose peripheral blood lymphocytes under stimulation of GPMV,and the cDNA was amplified using reverse transcription-polymerase chain reaction(RT-PCR),which was subsequently cloned into pMD18-T vector and sequenced.The results showed that the open reading frame(ORF) of Guangxi goose interferon-α(GoIFN-α) gene consisted of 576 bp,encoding 192 amino acid residues.Among them,there were seven cysteine residues and two potential glycosyl binding site(NDT and NHT).Compared with the sequences of other goose IFN-α gene in GenBank,the homologies of nucleotide and amino acid between Guangxi goose and white Northeast goose(AY524422) was found the highest(98.3 and 96.4%,respectively).However,these two indices among Guangxi goose and chicken,cow,swine,rat,dog and human,etc.were lower.These results indicated that the IFN had species specificity,the homologies of interferon among different animal species depend on their genetic relationships.

In the present study,strain W13 derived from egret was used and five pairs of primers were designed from genome sequence of paramyxovirus GPV-SF02 strain of goose(GenBank).Five fragments of L gene of avian paramyxovirus 1 strain W13 derived from egret,viz.,LA(1121 bp),LB(1481 bp),LC(1439 bp),LD(1476 bp) and LE(1608 bp) were amplified by RT-PCR.After comparative analysis using DNA Star software,five fragments were joined and 6760 bp of nucleotide sequence containing whole length of L gene was obtained.It was found that whole length of mRNA of L gene from strain W13 was 6704 bp,and the open reading frame contained 6615 basic groups and coded 2204 amino acids.The homology analysis of amino acid and nucleotide showed that L gene from strain W13 had more than 97.0% and 93.0% homology to strains SF02,ZJl,NA-1 derived from goose and Guangxi7 derived from chicken,respectively.The results revealed that strain W13 had closer relationship to strain SF02,ZJl,NA-1 and Guangxi7.

A pair of primer was designed using the nucleotide sequence of Northeast white goose IFN-α gene obtained from GenBank(Accession No.AY524422).Total RNA was extracted from cultures of goose peripheral blood lymphocytes under stimulation of GPMV,and the cDNA was amplified using reverse transcription-polymerase chain reaction(RT-PCR),which was subsequently cloned into pMD18-T vector and sequenced.The results showed that the open reading frame(ORF) of Guangxi goose interferon-α(GoIFN-α) gene consisted of 576 bp,encoding 192 amino acid residues.Among them,there were seven cysteine residues and two potential glycosyl binding site(NDT and NHT).Compared with the sequences of other goose IFN-α gene in GenBank,the homologies of nucleotide and amino acid between Guangxi goose and white Northeast goose(AY524422) was found the highest(98.3 and 96.4%,respectively).However,these two indices among Guangxi goose and chicken,cow,swine,rat,dog and human,etc.were lower.These results indicated that the IFN had species specificity,the homologies of interferon among different animal species depend on their genetic relationships.

One strain of Newcastle disease virus(NDV) was isolated from diseased pheasant in a pheasant farm in Guangxi in 2008,and the biological characteristic of the strain was determined.The results showed that the mean death time of chicken embryos for the isolated strain was 50.4 hours,and the intracerebral pathogenicity index to one-day-old chicks was 15.8.The homology of functional domains of F gene nucleotide sequence of the isolated strain with standard strains F48E9 and Lasota was 84.5% and 81.8%,respectively.The deduced amino acids sequence of cleavage site was 112G-R-R-Q-K-R-F117.These results showed the isolated strain was a strong virulent strain of NDV.

[Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus,two pairs of primers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region;the amplified products were ligated into pMD18-T vector and sequenced. [Result] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length,respectively;both showed the homologues of about 97.3% with GPV-SF02 strain,of 80.3%-97.5% with strains LaSota,F48E9 and JS,of just 84.8% with Miyadera strain. [Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain,belonging to genotype Ⅶ of APMV-1 strain.

With 10-days goose embryo,a strain of virus was isolated from the spleen and liver of 5 days old gosling died from gosling plague in Guangxi.It was determined by the real-time quantitative PCR method.The length of 589 nucleotides of VP3 were amplified,then the fragments were cloned and sequenced.Analysing the sequences of VP3 gene of the isolated strain,GDFSH strain,82-0308 strain,HLJ01 strain and HBZF07 strain,their homologies of nucleotides were 96.2% to 98%.The results showed that the isolated strain was goose parvovirus.

猪细胞巨化病毒病又称猪包涵体鼻炎,是由猪细胞巨化病毒(PCMV)引起的仔猪的一种常见传染病[1].在易感猪群中可引起胚胎和仔猪死亡,表现生长发育不良、仔猪鼻炎、肺炎及增重差等临床症状.PCMV主要是一种"机会性感染"病毒,多呈隐性感染,潜伏感染状态的猪在长途运输、寒冷、分娩等应激因素作用下,病毒可被激活并重新排毒.常规条件下饲养的大多数猪群均存在PCMV感染,PCMV抗体阳性率较高,但大多数为亚临床型,临床发病则很少见[2,3].

小鹅瘟是由鹅细小病毒 (Goose Parvovirus,GPV)引起的雏鹅和雏番鸭的一种急性或亚急性、败血性传染病.该病主要侵害3～20日龄小鹅,以渗出性肠炎、肝脏、肾脏、心脏等实质器官炎症为主要特征.该病传播快,发病率和死亡率高,是严重危害养鹅业的重要传染病.

According to the complete genome of foot-and-mouth disease virus (FMDV) type O, a pair of special primers was designed to amplify VP1 gene. The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG. Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight. Then the product was purified by GSTrap FF columns. The product was detected through Western-blot that showed the protein has antigenicity. It provided fundamental data and materials for further investigation on diagnosis method of FMDV.