血红素加氧酶1对猪瘟病毒复制的影响

本实验研究了猪血红素加氧酶1对猪瘟病毒(CSFV)在PK-15细胞中复制的影响,本研究应用siRNA干扰方法抑制PK-15细胞中血红素加氧酶1表达后再接种CSFV,细胞中CSFV基因组增殖与对照组相比显著下降;感染病毒细胞中和培养上清中CSFV滴度也显著降低。本实验首次报道了干扰细胞中HO-1表达对CSFV细胞中增殖的影响,提出了CSFV与血红素加氧酶1新的相互作用关系。     

姜黄素对猪瘟病毒复制的抑制作用

为了研究姜黄素(curcumin)及其衍生物在猪肾上皮细胞(PK-15)上对猪瘟病毒(CSFV)复制的影响,通过蛋白免疫印迹、实时荧光定量PCR和共聚焦检测等方法,鉴定姜黄素的抗病毒作用及其机制。结果:Western blot检测发现CSFV非结构蛋白N^pro含量显著下降(P<0.05);激光共聚焦结果发现,姜黄素处理后病毒进入细胞依赖的纤维状肌动蛋白(F-actin)重排受阻;Western blot和RT-qPCR检测显示,姜黄素可抑制热休克蛋白70(HSP70)表达,显著下调环氧化酶2(COX-2)和上调过氧化物酶体增殖物激活受体(PPAR-γ)(P<0.05)。研究表明:姜黄素可减少CSFV进入PK-15细胞,抑制CSFV引起的细胞应激从而抑制病毒复制,在一定程度上保护PK-15细胞免受病毒感染,为姜黄素的抗病毒作用提供了参考。     

抗病毒蛋白Viperin抑制猪瘟病毒在PK-15细胞上的复制

Viperin是一种抗病毒蛋白,具有广谱的抗病毒功能。为了研究猪源抗病毒蛋白Viperin对猪瘟病毒(CSFV)的抗病毒作用及机制,通过构建细胞系过表达或通过siRNA转染抑制Viperin表达,采用荧光定量RTPCR和病毒滴度测定检测CSFV增殖水平的变化;检测培养上清和细胞中病毒含量的变化趋势,评价其对病毒释放的影响;进而通过共聚焦试验和免疫共沉淀(co-immunoprecipitation,Co-IP)试验检测Viperin与病毒E2蛋白的共定位与相互作用。与对照细胞相比,过表达Viperin蛋白可以显著抑制CSFV在PK-15细胞上的复制,在感染后24、48、72h,病毒基因水平和病毒滴度分别下降68.75%、83.61%、77.27%和68.75%、87.5%、80.39%。通过RNA干扰技术抑制Viperin在PK-Vi细胞中的表达后CSFV复制显著恢复(仍低于对照组)。培养上清和细胞中病毒含量均同等程度受到抑制,表明Viperin表达对病毒的释放没有影响。共聚焦试验证明Viperin蛋白与E2蛋白在细胞内存在共定位现象。免疫共沉淀试验证明Viperin蛋白与E2蛋白存在相互作用。该研究证实Viperin具有抗CSFV作用,该作用可能是通过与病毒E2蛋白相互作用实现的,这为CSFV与宿主免疫反应相互作用研究提供了理论基础。     

猪瘟病毒N~(pro)蛋白及细胞内环境改变对其IRES活性的影响

位于猪瘟病毒(CSFV)基因组5'端非翻译区(UTR)的内部核糖体进入位点(IRES)在调控CSFV基因组的翻译中发挥重要的作用,为探索CSFV自身蛋白以及细胞内环境改变对IRES活性的影响,本研究构建了包含CSFV IRES在内的荧光素酶报告基因质粒,采用编码CSFV非结构蛋白N~(pro)和NS5A蛋白表达重组质粒转染细胞,或通过葡萄糖剥夺、氨基酸剥夺、氧化应激、血清饥饿等条件分别处理细胞,结果显示N~(pro)蛋白比之前报道的NS5A蛋白具有更显著的IRES抑制效果,并且抑制程度呈浓度依赖性;氨基酸剥夺、氧化应激及血清饥饿时可以显著降低CSFV IRES的活性,而葡萄糖剥夺对其活性无显著影响。本研究明确了CSFV非结构蛋白N~(pro)以及细胞内环境的改变可以调控IRES的活性,为进一步阐明CSFV复制增殖的分子机制提供新的实验依据。     

猪瘟病毒E2蛋白配体表位的筛选与鉴定

为进一步精确定位猪瘟病毒(CSFV)E2蛋白配体表位信息,利用DNASIS(Ver.2.5)软件分析设计合成7条多肽,主要覆盖E2蛋白,分别命名为SE21、SE22、SE231、SE232、SE241、SE242、SE03,同时设计合成1条无关多肽CP,均标记FITC。将PK-15细胞作为靶细胞,通过多肽与PK-15细胞结合试验、多肽抑制CSFV感染PK-15细胞试验和CSFV阻断多肽与PK-15细胞结合试验,筛选和鉴定E2蛋白配体表位。结果表明,多肽SE241、SE242与PK-15细胞结合的平均荧光强度分别为74.43±4.59和83.60±3.11,显著高于其他多肽,抑制CSFV感染PK-15细胞的感染抑制率也显著高于其他多肽,同时这2条多肽与PK-15细胞的结合被CSFV有效阻断。证实SE241、SE242多肽为CSFV与靶细胞PK-15细胞结合的配体表位,并能抑制CSFV感染PK-15细胞。本试验为CSFV新型多肽疫苗或免疫佐剂的研制提供依据。     

猪源含缬酪肽蛋白(VCP)的真核表达及其活性鉴定

含缬酪肽蛋白(VCP)作为细胞中的重要活性蛋白,参与多种细胞过程,也在许多病毒的感染过程中发挥重要作用。为了探究VCP对猪瘟病毒(CSFV)、日本脑炎病毒(JEV)和伪狂犬病病毒(PRV)复制的影响,试验扩增了猪源VCP的基因片段,并将其连接到pEGFP-C1载体中。经双酶切鉴定结合基因测序结果,成功构建真核表达重组载体pEGFP-VCP。通过间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)证实,重组质粒EGFP-VCP能在猪肾细胞(PK-15)中成功表达。而在表达pEGFP-VCP的细胞中分别感染CSFV、JEV和PRV后,通过Western blot、实时荧光定量PCR(RT-qPCR)、半数细胞感染量(TCID₅₀)以及IFA试验,检测到过表达VCP能够明显促进CSFV、JEV以及PRV病毒蛋白的表达,提高胞内病毒核酸的含量,并且使胞外病毒滴度增加,极大地促进了3种病毒在PK-15细胞中的复制,为深入研究VCP促进多种病毒感染宿主细胞的分子机制奠定了理论基础。     

猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

为探究宿主蛋白Beclin1在猪瘟病毒(classical swine fever virus, CSFV)非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制,本研究在感染CSFV及表达NS5A蛋白的ST细胞中,利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况;利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系;通过在ST细胞中过表达或敲低Beclin1,研究其对CSFV复制的影响。结果表明,ST细胞感染猪瘟病毒或外源表达NS5A蛋白,Beclin1转录和蛋白表达水平均显著升高,且PI3K/Akt通路相关因子表达水平与之呈正相关。此外,CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后,作者发现在细胞中过表达Beclin1,对CSFV复制起到明显促进作用;反之,利用siRNA敲低Beclin1后,抑制PI3K/Akt通路活化,CSFV增殖表现出明显抑制效应。以上结果表明,Beclin1蛋白对CSFV复制具有促进作用,其机制是通过与NS5A的相互作用调控PI3K/Ak...     

猪瘟病毒荧光定量PCR的建立及对病毒体外复制动态的研究

为了了解猪瘟病毒(CSFV)在PK-15细胞上的增殖规律。根据GenBank中CSFV Shimen毒株的NS5A基因保守区域序列,设计1对特异性引物,以CSFV总RNA为反转录模板,经优化反应条件,建立了基于实时荧光定量PCR技术的CSFV检测方法,并对其进行了敏感性、特异性、重复性试验,结果显示,该方法重复性好、特异性强,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1 TCID_(50 )/mL。利用所建立的方法研究CSFV在PK-15细胞上清中的增殖动态,并与TCID_(50)方法绘制的复制动态曲线进行比较。结果显示,两种方法测定的CSFV在PK-15细胞上的复制动态具有一定的平行关系,该方法为研究猪瘟病毒的致病机理及利用体外细胞培养毒生产疫苗提供了试验依据。     

猪瘟病毒感染细胞中分子伴侣Jiv基因的表达检测与分析

Jiv蛋白是与猪瘟病毒(CSFV)细胞内复制密切相关的伴侣分子,为探究Jiv在CSFV感染细胞内的表达规律,应用实时荧光定量RT-PCR分别对3个猪源细胞株(SUVEC、PK-15、ST)在感染CSFV后不同时间Jiv和CSFV基因表达进行检测,分析两者的相关性。结果显示,3种猪源细胞株的Jiv和CSFV的基因在0~72h的表达逐步上调,72h~96h出现下降,二者变化趋势基本一致;此外,Jiv基因在0~48h上调幅度较其他时间段更为显著,ST细胞在不同时间段的Jiv表达均高于同时段的其他两种细胞,与ST细胞中CSFV复制趋势一致。结果表明,在CSFV感染细胞中Jiv基因的表达与CSFV复制呈正相关,即CSFV复制水平越高,Jiv基因表达水平也越高。本研究初步探明细胞Jiv蛋白在CSFV复制中的调节作用,为进一步明确Jiv蛋白参与CSFV复制的机制提供了新的资料。     

宿主膜联蛋白A2与伪狂犬病病毒US3蛋白互作及其对细胞凋亡的影响

US3蛋白激酶是伪狂犬病病毒（PRV）的一个重要毒力因子,研究显示US3参与了细胞骨架重排、病毒复制和传播、细胞凋亡和干扰素信号通路等多个生物学过程,鉴定新的和US3互作的宿主蛋白对于认识其生物学功能具有重要意义。本研究首先利用免疫共沉淀和pull-down技术验证了US3和宿主膜联蛋白A2（ANXA2）的相互作用;然后利用RNAi技术,通过荧光定量PCR、病毒滴度测定和免疫印迹分析,发现敲低ANXA2的表达显著抑制了PRV在PK-15细胞上的增殖;进一步发现过表达ANXA2可以抑制PRV或者凋亡刺激剂诱导的细胞凋亡,提示ANXA2具有负调控细胞凋亡的作用。本研究进一步完善了可以和US3蛋白互作的宿主蛋白成员,加深了人们对ANXA2生物学功能的理解。     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.