Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.     

C型口蹄疫病毒VP1基因的原核表达及重组蛋白的纯化

利用PCR技术扩增获得C型口蹄疫病毒VP1基因,将其克隆到原核表达载体pET-30α(+)上,构建重组表达质粒pET-VP1。质粒pET-VP1转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导VP1基因的表达,收集不同时间的菌液进行SDS-PAGE、Western blot分析,结果得到分子量约为33 ku的目的条带,表达产物占菌体总蛋白的35%,且该目的条带能被C型FMDV阳性血清识别,说明VP1基因在大肠杆菌中得到高效表达。融合表达蛋白经镍柱纯化,重组蛋白纯度达90%以上。     

以乳酸杆菌为载体的口蹄疫病毒VP1基因DNA疫苗猪体免疫试验

给猪口服和肌肉注射以乳酸杆菌为载体的口蹄疫病毒VP1基因DNA疫苗（L．acido SFMD-1），以正向间接血凝试验（IHA）和MTT方法分别检测了免疫后FMDV VP1抗体的动态变化和特异性T细胞增殖反应情况，并与商用FMD油佐剂疫苗、裸质粒FMDV VP1基因DNA疫苗诱导的特异性免疫抗体水平进行了比较。结果显示，口服组猪在免疫后第21d抗体效价达到了1：2^7.7，而肌肉注射组猪为1：2^3.3。加强免疫后2周，口服免疫组抗体水平下降到1：2^5.3，到第3周快速上升到1：2^8；与此相对应，肌肉途径免疫组猪抗体效价缓慢地从1：2^5.3上升到1：2^6.7。口服途径和肌肉注射途径的刺激指数（SI）分别为1．93和2．00，2种免疫途径都可以诱导特异性T细胞增殖反应。证实，该疫苗能够在猪体诱发VP1特异性T细胞和B细胞反应，以乳酸杆菌为载体是DNA免疫和预防猪口蹄疫的一种极有前景的方法。     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...     

负载口蹄疫病毒VPl-VP4融合蛋白质的树突状细胞对T细胞的活化效应

为研究负载口蹄疫病毒VPl－VP4融合蛋白质的树突状细胞对淋巴结T细胞的活化效应,通过构建pET32a-VPl－VP4原核表达系统制备VPl－VP4融合蛋白。将纯化VPl－VP4融合蛋白负载骨髓源树突状细胞（BMDC）后与淋巴结T细胞共培养,用ELISA检测不同时间点的共培养上清液中IFN－γ的含量。结果表明,负载FMDVVPl－VP4融合蛋白后的BMDC可通过溶酶体－MHC－Ⅱ类分子途径有效地激活淋巴结T细胞,从而启动Thl细胞免疫应答,分泌大量IFN－γ。     

口蹄疫病毒Akesu/O/58株结构蛋白基因的克隆和细胞受体结合位点初探

将Akesu/O/58口蹄疫病毒分离株牛舌皮毒适应乳鼠,通过RT-PCR法分别获得了该病毒结构蛋白基因vp1和p1.结果表明:vp1和p1基因分别为639 bp和2 208 bp,与Akesu/O/58细胞适应株FMDV的vp1和p1基因核苷酸序列的同源性分别为83.9%和84.7%,氨基酸序列的同源性为89.7%和95.1%.本试验分离株与OHK99、O1K、Taiwan97病毒株的细胞受体结合位点均为RGD(Arg-Gly-Asp),而Akesu/O/58细胞适应株的细胞受体结合位点为SGD(Ser-Gly-Asp).     

Efficacy of a novel in ovo-attenuated live vaccine and recombinant vaccine against a very virulent infectious bursal disease virus in chickens

Infectious bursal disease (IBD) causes severe economic damage to the poultry industry worldwide. To prevent IBD virus (IBDV) infection, live virus vaccines have been widely used in chickens having wide-ranging levels of maternally derived antibodies. But, the risks of infection with other pathogens because of lesions related to atrophy of the bursa of Fabricius in vaccinated chickens are a concern. To resolve the problems, a recombinant turkey herpesvirus (HVT) vaccine expressing IBDV-VP2 protein (rHVT-IBD) has been developed. However, the induction of neutralizing antibodies by rHVT-IBD against a virulent IBDV might be delayed compared with that by the live IBD vaccine, leading to the high risks of IBDV infection for young chickens. To find the best selection of IBDV vaccine for the onset of immunity, we examine the protective efficacy of a novel in ovo-attenuated live IBDV (IBD-CA) vaccine and the rHVT-IBD vaccine in young chickens challenged with a very virulent IBDV (vvIBDV) strain. We show that the protective efficacy of IBD-CA vaccine was higher than that of the rHVT-IBD vaccine in 14-day-old chickens challenged with the vvIBDV strain, leading to the risk of IBDV infection for young chickens when vaccinated with rHVT-IBD. Our results suggest that farmers should select the best vaccines to maximize vaccine efficacy in consideration of the vaccine characteristics, prevalence levels of IBDV in the areas, and initial MDA levels of the chickens since the attenuated live and recombinant vaccines play a role in the different vaccine efficacies.     

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens,and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site ¹¹²RRRKGF¹¹⁷ and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗对禽流感H9亚型流行株攻毒的保护作用

为了监测鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗(LaSota株+M41株+SS/94株)对H9亚型禽流感病毒流行毒株的免疫保护效果,采用H9亚型禽流感病毒SS/94株及2009—2010年现地分离的3株H9亚型禽流感病毒对已免疫上述三联灭活苗的SPF鸡进行攻毒试验。结果显示,试验鸡以0.3 mL/只的剂量免疫三联灭活苗后21 d,其H9亚型禽流感病毒的HI抗体效价可达8～11log2,此抗体水平可抵抗2×106EID50的H9亚型禽流感病毒SS/94株、BLCN09株、WDZ09株、YT10株的攻击,攻毒保护率均达90%(9/10)以上。可见,以SS/94株作为禽流感疫苗抗原制备的三联灭活苗具有良好的免疫原性,能使免疫鸡抵抗2009—2010年期间现地分离的多株H9亚型禽流感病毒的攻击。     

The duration of the foot-and-mouth disease virus carrier state in African buffalo (i) in the individual animal and (ii) in a free-living herd

The maintenance of a virus depends on a number of factors, including the duration of infectivity and the size of the available host population. In this work, foot-and-mouth disease virus was shown to persist in individual African buffalo (Syncerus caffer) for up to at least five years; thus, the duration of infectivity is more than adequate to cover the normal periods between calving peaks. In a small isolated free-living population which varied from 30 to 100 buffalo, two immunological types of foot-and-mouth disease virus were maintained for at least 24 years and through several generations.     

Invasion and spread of single glycoprotein deleted mutants of Aujeszky''s disease virus (ADV) in the trigeminal nervous pathway of pigs after intranasal inoculation

The purpose of the study was to evaluate the role which non-essential envelope glycoproteins play in the neuroinvasion and neural spread of ADV. The invasion and spread in the trigeminal nervous pathway with the Ka strain of ADV and its single deletion mutants Ka gI⁻, Ka gp63⁻ and Ka gIII⁻ were examined after intranasal inoculation in neonatal pigs by virus isolation and immunocytochemistry. Evaluation was performed in the nasal mucosa, trigeminal ganglion (1st neuronal level), pons-medulla (2nd neuronal level) and thalamus-cerebellum (3rd neuronal level). The Ka gIII⁻ mutant invaded up to the 3rd neuronal level of the trigeminal pathway and spread in a similar way to the parental Ka strain. The Ka gp63⁻ mutant invaded up to the 3rd neuronal level but the spread of this mutant was impaired at all the neuronal levels. The Ka gI⁻ mutant was least neuroinvasive and reached only up to the 2nd neuronal level. The results showed that glycoproteins gI and gp63 play a role in the invasion and spread of ADV in the nervous system. However, the gI glycoprotein appears to be the most important for neuroinvasion and neural spread of ADV in pigs. Therefore, gI deleted vaccines may be considered to be safer with respect to the neuroinvasion than vaccines carrying single deletions of other non-essential envelope glycoproteins.     

表达传染性法氏囊vp2基因的重组马立克病疫苗对SPF鸡和商品鸡的免疫保护试验

国内外很多学者都在进行着传染性法氏囊(Infectious bursal disease,IBD)各种基因工程疫苗的研制,IBDV-vp2基因在多种载体中得到表达并取得了较好的保护效力。本实验室已构建了表达IBDV-vp2基因的重组马立克氏病病毒。本研究对该重组马立克氏病病毒疫苗的遗传稳定性以及对SPF鸡和含有母源抗体的商品鸡的免疫保护作用进行了评价。结果表明,103 PFU和104 PFU剂量的重组病毒免疫后对SPF鸡抗传染性法氏囊标准强毒的保护率分别为53%和73%。104 PFU剂量的重组病毒免疫后对含有母源抗体的商品鸡抗传染性法氏囊标准强毒的保护率为87%,结果显示重组疫苗具有一定的应用前景。