PCV2通过NF-κB/NLRP3信号通路调控体外培养PAMs分泌IL-1β

为探讨猪圆环病毒2型（porcine circovirus 2,PCV2）诱导猪肺泡巨噬细胞（porcine alveolar macrophages,PAMs）产生白细胞介素-1β（interleukin-1β,IL-1β）的分子机制,试验选取2头PCV2和PRRSV抗原、抗体均为阴性的6周龄普通仔猪,无菌分离PAMs,以体外培养的PAMs为研究对象,采用ELISA方法检测PAMs培养上清液中IL-1β的生成,采用实时荧光定量PCR方法检测PAMs中NLRP3和凋亡相关点样蛋白（apoptosis-associated speck-like protein containing a CARD,ASC）的mRNA表达水平,分别用小干扰RNA（siRNA）方法和核因子-kappa B（NF-κB）抑制试验分析NLRP3和NF-κB对PCV2诱导PAMs产生IL-1β的调控作用。结果显示,PCV2感染PAMs后能够显著或极显著增加IL-1β、NLRP3（1 h除外）和ASC（1、3 h除外）的生成（P<0.05;P<0.01）。siRNA能使58.3%的NLRP3基因沉默,且NLRP3沉默后PCV2诱导PAMs产生IL-1β的水平显著下降（P<0.05）。NF-κB被抑制后PCV2诱导PAMs产生IL-1β的水平也明显下降。结果表明,PCV2通过NF-κB/NLRP3信号通路调控体外培养PAMs分泌IL-1β。     

猪圆环病毒2型广东分离株ORF2基因的克隆和序列分析

为了解广东部分地区猪圆环病毒2型(porcine circovirus type 2,PCV2)流行毒株的基因型和遗传变异情况,本研究根据GenBank中PCV2的核苷酸序列设计针对ORF2基因的1对特异性引物,从分离于广东省的8株PCV2分离株BL、HD、Li、GD01、GD02、HD01、HD02和HD03株的细胞培养物中扩增ORF2基因,扩增片段克隆到pMD18-T上,获得重组质粒,并对其进行测序。序列分析结果表明,8株PCV2分离株ORF2基因与其他PCV2 ORF2基因核苷酸序列同源性为89.7%~100%,氨基酸序列同源性为87.2%~99.6%,进化分析结果表明,其中7株分离株处于同一分支,属于PCV2b-1A/1B,HD01株属于PCV2b-1C。     

Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n=8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n=9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n=9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n=9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n=9), pigs infected with VR-2385 (n=9), and pigs infected with NC16845b (n=9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.     

表达PRRSV ORF5和PCV2 ORF2基因的重组鸡痘病毒的构建及鉴定

分别将猪圆环病毒2型的ORF2基因和猪繁殖与呼吸综合征病毒的ORF5基因,插入到鸡痘病毒转移载体pUTA2-16-LacZ单一启动子和复合启动子下游,构建重组鸡痘病毒转移质粒pUTAL-ORF5-ORF2。将该重组质粒与鸡痘病毒282-E4株共转染鸡胚成纤维细胞,进行同源重组。通过3次溴脱氧尿嘧啶核苷(BrdU)加压筛选,经RT-PCR、IFA和Western blot鉴定,表明重组鸡痘病毒中的目的基因在鸡胚成纤维细胞中得到表达,获得一株携带有目的基因ORF2和ORF5的重组鸡痘病毒rFPV-ORF2-ORF5。     

Antigenic subtyping and epitopes' competition analysis of porcine circovirus type 2 using monoclonal antibodies

Porcine circovirus type 2 (PCV2) strains have been classified into two major genotypes (PCV2a and PCV2b) and 8 genetic clusters: PCV2b-1A to PCV2b-1C and PCV2a-2A to PCV2a-2E. To date, no studies have been performed to antigenically subtype PCV2 strains enclosing eight PCV2 clusters. The present study aimed to antigenically subtype PCV2 and perform epitopes' competition analysis using monoclonal antibodies (mAbs). Fourteen PCV2 strains representative for eight clusters were tested with 20 mAbs (fifteen of them were generated against PCV2a strain Stoon-1010 and 5 of them against PCV2b strain 1147) in immunoperoxidase monolayer assays. Four mAbs reacted to all 14 PCV2 strains and one mAb reacted with all strains except for a PCV2a-2C strain. One mAb reacted with all PCV2a strains, except for a PCV2a-2C strain and one mAb reacted with all PCV2b strains, except for a PCV2b-1C strain. Nine mAbs reacted with the strains of PCV2b-1A/1B, PCV2a-2A and PCV2a-2E. Three mAbs only reacted with the strains of PCV2a-2A and PCV2a-2E. One mAb reacted specifically with the strains of PCV2b-1A/1B. This suggests that discrete antigenic differences exist between different PCV2 genetic clusters and that these clusters can be discriminated by the use of a panel of universal and cluster-specific mAbs. Six mAbs were selected for cross-competition analysis by a competitive ELISA using PCV2 strain Stoon-1010. Six overlapping epitopes were identified on the PCV2 capsid protein. The universal mAbs recognized larger epitopes than the cluster-specific mAbs. These findings are helpful in the development of diagnostic tests and new generation vaccines against PCV2.     

Porcine reproductive and respiratory syndrome virus (PRRSV) influences infection dynamics of porcine circovirus type 2 (PCV2) subtypes PCV2a and PCV2b by prolonging PCV2 viremia and shedding

The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine circovirus type 2 (PCV2) subtypes a (PCV2a) or b (PCV2b) viremia and shedding characteristics in oral, nasal and fecal samples in experimentally infected pigs. Twenty-three, 2- to 6-week-old pigs were randomly divided into five groups: negative control (n=3), PCV2a-I (n=5), PCV2a-PRRSV-CoI (n=5), PCV2b-I (n=5), and PCV2b-PRRSV-CoI (n=5). Blood, oral, nasal and fecal swabs were collected in regular intervals from day post inoculation (dpi) 0 until dpi 70 and tested by quantitative real-time PCR for the presence and amount of PCV2 DNA and by ELISA for the presence of PCV2-specific antibodies. The results indicate that there were significantly (P<0.05) higher loads of PCV2a and PCV2b DNA in serum, oral swabs, nasal swabs and fecal swabs and a higher prevalence of detectable PCV2 antigen in tissues of pigs concurrently infected with PCV2 and PRRSV compared to pigs singularly infected with PCV2 further confirming that PRRSV enhances replication of PCV2. Moreover, PRRSV infection significantly prolonged the presence of PCV2 DNA in serum and increased the amount of PCV2 DNA in oral and nasal secretions and fecal excretions in the later stages of infection between dpi 28 and 70. Shedding patterns were similar between groups infected with PCV2a and PCV2b, indicating that there was no subtype-specific interaction with the PRRSV isolate used in this study. The results from this study highlight the interaction between PRRSV and PCV2 and the importance of controlling PRRSV infection in order to reduce PCV2 virus loads in pig populations.     

猪宿主蛋白G3BP1对猪圆环病毒2型在PK15细胞上复制的影响

猪宿主蛋白G3BP1在宿主抵抗病毒感染过程中起着重要作用,然而还未有相关报道G3BP1与猪圆环病毒2型(PCV2)感染过程中的联系。本文构建了G3BP1真核表达载体和干扰RNA,实现在PK15细胞上G3BP1的过表达及敲低。结果发现,PCV2感染细胞24 h后病毒复制出现显著差异,感染48 h相比于感染24 h病毒复制差异更显著,表明G3BP1能显著促进PCV2复制。过表达或敲低G3BP1后,用干扰素刺激DNA(ISD)刺激12 h, G3BP1能促进ISD诱导IFN-βmRNA的表达;感染PCV2 12 h后,G3BP1也能正调控IFN-βmRNA的转录。本文揭示了宿主蛋白G3BP1显著促进PCV2的复制,很可能是通过上调IFN-β表达引起的。     

Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha

Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-alpha production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-alpha antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-alpha generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs.     

Identification and functional analysis of the novel ORF6 protein of porcine circovirus type 2 <Emphasis Type="Italic">in vitro</Emphasis>

In the present study, the function of a novel ORF6 gene in the PCV2 genome was determined and functionally analyzed in vitro. ORF6 expression was demonstrated by indirect immunofluorescence in PCV2-infected cells. The antibody against ORF6 was detected in PCV2-infected pigs. The start codon of ORF6 was mutated and an infectious clone was used to create an ORF6-deficient mutant virus. Viral DNA replication curves and immunofluorescence analysis indicated that ORF6 is unnecessary for viral replication and ORF6 deletion reduces viral DNA replication in PK-15 cells. The activities of caspases 3 and 8 in ORF6-deficient virus-infected cells were significantly different from those in wild-type virus-infected cells. The ORF6 protein can increase the expression of IFN-β, TNF-α, IL-1b, IL-10, and IL-12p40. These results demonstrated that the newly discovered ORF6 protein may be involved in caspases regulation and the expression of multiple cytokines in PCV2-infected cells. The functions of this gene in viral pathogenesis remain to be further elucidated.     

猪圆环病毒Ⅱ型ORF2基因在酵母中的表达

为探索猪圆环病毒Ⅱ型(PCVⅡ) ORF2基因的高效表达,将ORF2基因整合到酵母表达载体pPICZαC中,构建重组质粒pPICZαC-ORF2,通过SacⅠ酶切线性化,经电穿孔法转到毕赤酵母菌SMD1168。经Zeocin^TM抗性筛选得到转化子,通过SDS-PAGE分析和Western blotting鉴定,结果表明衣壳蛋白在酵母菌中成功分泌表达。     

北京市猪场猪圆环病毒3型感染状况调查及其ORF2基因遗传进化分析

为了解猪圆环病毒3型(PCV3)在北京市不同区县猪场中的流行情况,研究建立PCR检测方法,对来自北京市8个区县56个养殖场的1177份临床样品进行检测,并对获得的部分ORF2全基因序列进行遗传进化分析。结果显示,PCV3总体阳性率为1.0%(12/1177),猪场阳性率为8.9%(5/56)。对PCV3阳性样本进行ORF2基因测序及同源性比对,共测得12株PCV3 ORF2全长基因序列,其中包括6株不同的ORF2全长基因序列。结果显示,该6株序列之间的核苷酸相似性为97.7%～99.5%,推导氨基酸序列的相似性为97.2%～100%;与参考毒株之间的核苷酸同源性为96.0%～99.5%,推导氨基酸同源性为92.1%～100.0%;进化树显示北京毒株属于PCV3b基因型。试验表明,PCV3在北京多个猪场呈现一定的流行趋势,流行毒株以PCV3b基因型为主。     

杂交野猪猪繁殖与呼吸综合征病毒分离及其ORF6、ORF7基因的序列分析

试验旨在研究杂交野猪猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) 辽宁分离株的遗传变异情况及分子生物学特征.用Marc-145细胞从辽宁某杂交野猪场疑似猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome, PRRS)病猪血液中分离到1株病毒,该分离毒株经Marc-145细胞6次传代后出现稳定的细胞病变,采用RT-PCR方法对分离病毒进行ORF6和ORF7基因的扩增、克隆和测序,并与已知序列毒株的相应片段进行同源性比对.结果表明,分离毒株的ORF6、ORF7基因与国内外美洲型毒株的核苷酸同源性分别为96.0%～100.0%、94.5%～99.4%;氨基酸同源性分别为89.6%～100.0%、87.3%～98.7%;与欧洲型代表毒株LV的ORF6、ORF7基因差异较大,核苷酸同源性分别为70.4%、70.1%,氨基酸同源性分别为48.8%、49.7%.推测辽宁杂交野猪体内分离毒株在基因型上属于美洲型毒株.     

ORF9基因缺失突变PCV2的构建与部分生物学特性

设计1对针对猪圆环病毒2型（PCV2）全基因组的特异性引物,从疑似断乳仔猪多系统衰竭综合征（PMWS）的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后构建重组质粒pMD18-T Simple-PCV2（命名为P-S-PCV2）。用ORF9特异的限制性内切酶Bpu10Ⅰ对P-S-PCV2进行酶切、补平连接反应,构建了ORF9基因缺失突变的重组质粒（命名为P-S-PCV2-J）。用SacⅡ对基因缺失突变的重组质粒进行酶切,获得的线性化基因突变PCV2基因组在体外进行自身环化,形成了相应缺失基因DNA（命名为PCV2-J）。用PCV2-J缺失突变株进行细胞转染、动物致病性和免疫原性、T淋巴细胞亚群的动态变化等部分生物学特性的研究。结果显示：PCV2-J转染IBRS-2细胞后,电镜观察可见病毒颗粒,PCR-RFLP检测有突变株生长;PCV2-J接种仔猪后无临床典型大体病变,PCR-RFLP检测淋巴结中有PCV2-J突变病毒的感染;PCV2-J免疫仔猪后的抗体水平在第2周开始上升,与对照组相比差异显著,CD3＋下降,与对照组无差异,CD4＋下降,第1周与对照组差异显著,CD8＋与对照组差异不显著。结果表明,ORF9基因缺失突变的PCV2仍具有复制感染能力,但免疫原性减弱。     

猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5和ORF6在真核细胞中表达

用PCR方法从重组质粒pOKCH-1a扩增出猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5、ORF6,将它们分别定向克隆到含有鸡β-actin启动子的高效真核表达载体pCAGGS的多克隆位点,经酶切、PCR及测序分析,筛选鉴定出含有ORF3、ORF5、ORF6基因的重组质粒,分别命名为pCAGGS-ORF3、pCAGGS- ORF5、DCAGGS-ORF6。将重组质粒纯化后转染293T细胞,用RT-PCR扩增出了特征性的基因片段,并采用特异性单抗进行间接免疫荧光检测,结果在胞浆内有亮绿色荧光,而在细胞膜上没有见到荧光,这说明表达的蛋白在胞浆内而不在细胞膜上。通过Westerm blot检测确定表达的GP3、GP5和M蛋白的分子量分别为42 Ku、25 Ku和19 Ku。本试验结果为进一步研究PRRSV膜蛋白伪病毒粒子及DNA疫苗奠定了基础。     

PRRSV ZQ-GD-2010株的分离及ORF5和ORF7基因的序列分析

为了给研制基因工程疫苗选取毒株奠定基础,研究采集广东肇庆某猪场疑似患高热症猪的病料,通过RT-PCR检测、病毒分离传代,证实分离到1株猪繁殖与呼吸综合征病毒,命名为ZQ-GD-2010株,并对其ORF5和ORF7基因进行序列测定,绘制遗传进化树。结果表明:该毒株为美洲型,其ORF5和ORF7基因核苷酸与近年来我国分离到的美洲型毒株的相似性为96%～100%,而与欧洲型毒株LV株的相似性仅为58.9%～62.8%;其与2007—2009年国内分离株的遗传演化关系较近。     

The effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets

The effects of transplacental porcine circovirus type 2 (PCV2) infection on porcine epidemic diarrhoea virus (PEDV)-induced enteritis were examined in neonatal piglets. Six pregnant sows were randomly allocated to an infected (n=3) or control group (n=3). Three pregnant sows were inoculated intranasally with 6 mL of tissue culture fluid containing 1.2 x 10(5) tissue culture infective doses 50% (TCID(50))/mL of PCV2 strain SNUVR000470 three weeks before the expected farrowing date. Three control pregnant sows were similarly exposed to uninfected cell culture supernatants. Thirty piglets from PCV2-infected sows were randomly assigned to two groups (A and B) of 15 piglets each. Another 30 piglets from noninfected sows were randomly assigned to two groups (C and D) of 15 piglets each. The piglets in groups A and C were dosed orally at three days of age with 2mL of virus stock (1 x 10(6.5) TCID(50)/mL) of the PEDV strain, SNUVR971496, at the third passage. The mean villous height and crypt depth (VH:CD) ratio in PEDV-infected piglets from PCV2-infected sows (group A) were significantly different from those of the PEDV-infected piglets from PCV2 negative sows (group C) at 36, 48, and 72 h post-inoculation (hpi) (P<0.05). In PEDV-infected piglets from PCV2-infected sows (group A), significantly more PEDV nucleic acid was detected in the jejunal tissues (P<0.05) at 24 hpi than in the same tissues of the PEDV-infected piglets from PCV2 negative sows (group C). Thereafter, at 36, 48, 60, and 70 hpi significantly more PEDV nucleic acid (P<0.05) was detected in the jejunal tissues of the PEDV-infected piglets from PCV2 negative sows (group C) than those of the PEDV-infected piglets from the PCV2-infected sows (group A). It is concluded that the clinical course of PEDV disease was markedly affected by transplacental infection of PCV2.     

2013年-2014年江西地区PRRSV分离株NSP2及ORF5基因的遗传变异分析

为研究江西地区猪繁殖与呼吸综合征病毒(PRRSV) ORF5基因的变异情况及NSP2基因的结构特征,采用RT-PCR方法扩增了12份江西地区猪场疑似患PRRS的猪肺脏样品中的ORF5全序列和NSP2部分序列,应用DNAStar和Mega 6.0等软件对所得序列进行同源性比对及遗传变异分析。12株PRRSV ORF5核苷酸同源性为83.7%~99.8%,氨基酸同源性为82.1%~99.5%;与参考毒株JXA1、VR-2332和LV的核苷酸同源性分别为84.9%~99.7%、85.2%~91.0%和62.4%~64.8%。对阳性病料进行了NSP2基因部分序列的扩增,测序结果显示12株PRRSV均属于美洲型毒株,12株PRRSV的NSP2部分序列均存在30个氨基酸的不连续缺失,与高致病性PRRSV有相同的缺失特征。12株PRRSV的ORF5遗传进化树分析显示,10株与高致病性PRRSV处在同一进化分支,进一步说明高致病性PRRSV已成为江西地区的优势流行毒株。     

高致病性猪繁殖与呼吸综合征病毒强弱毒ORF1a、ORF1b、ORF2-ORF7片段互换嵌合病毒的构建及其生物学特性的分析

为研究高致病性猪繁殖和呼吸综合征病毒(HP-PRRSV)强弱毒之间毒力差异的分子基础,本实验分别以HP-PRRSV强毒HuN-F5株及其传代致弱的疫苗病毒株HuN4-F112为亲本病毒,利用反向遗传操作技术分别将ORF1a、ORF1b或ORF2-7编码序列在强弱毒之间互换。将6种含有不同嵌合基因的全长病毒基因组的重组质粒体外转录后转染BHK-21细胞,然后在Marc-145细胞中传代,拯救的重组病毒经RT-PCR、测序和免疫荧光鉴定,并分别命名为rHuN4-F5-ORF1a、rHuN4-F5-ORF1b、rHuN4-F5-ORF2-7(以强毒为骨架)和rHuN4-F112-ORF1a、rHuN4-F112-ORF1b、rHuN4-F112-ORF2-7(以弱毒为骨架)。进一步测定这些病毒在Marc-145上的生长曲线,结果显示:以强毒为骨架的嵌合病毒rHuN4-F5-ORF1a生长滴度显著高于亲本强毒rHuN4-F5,而以弱毒为骨架的嵌合病毒rHuN4-F112-ORF1a在细胞上的生长滴度低于其亲本弱毒rHuN4-F112,其他片段替换对病毒在细胞上的生长没有明显影响。本实验结果提示ORF1a对于PRRSV在体外细胞培养上的生长调节起重要作用。