Porcine circovirus type 2 decreases the infection and replication of attenuated classical swine fever virus in porcine alveolar macrophages

Recently, it has been noted that porcine circovirus type 2 (PCV2) infection adversely affects the protective efficacy of Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), in pigs. In order to investigate the possible mechanisms of the PCV2-derived interference, an in vitro model was established to study the interaction of LPC virus (LPCV) and PCV2 in porcine alveolar macrophages (AMs). The results showed that PCV2 reduced the LPCV infection in AMs and the levels of PCV2-derived interference were dose-dependent. The PCV2-derived interference also reduced the replication level of LPCV in AMs. The full-length PCV2 DNA and its fragment DNA C9 CpG-ODN were involved in the reduction of LPCV infection in AMs, whereas UV-inactivated PCV2 was not. In addition, a moderate negative correlation between the LPCV antigen-containing rate and IFN-γ production was observed, and had a dose-dependent trend with the level of PCV2-inoculation. The results of the present study may partially explain how PCV2 infection interferes with the efficacy of LPC vaccine.     

猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响

采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应.     

The influence of maternal immunity on the efficacy of a classical swine fever vaccine against classical swine fever virus,genogroup 2.2, infection

In Thailand, where vaccination is routinely employed, there has been an increased incidence of chronic classical swine fever (CSF) outbreaks during the past decade. The major causative virus has been identified to be the moderate virulence, classical swine fever virus (CSFV) of the genogroup 2.2. An investigation was made into the efficacy of a CSF vaccine against this genogroup 2.2 challenge. Five-week-old pigs, grouped by their level of passive antibody titer were immunized with lapinized Chinese-strain CSF vaccine and challenged with CSFV genogroup 2.2, 13 days after vaccination. The group containing passive titers of lower than 64 at the time of immunization, had significantly higher number of CSFV-specific IFN-gamma secreting cells and was completely protected against the challenge. Interestingly, both cellular and antibody responses were inhibited in the pigs with the higher passive titer. Furthermore, following challenge, CSFV could be isolated from 50% of the pigs in this group. It was demonstrated that the CSF vaccine could induce complete protection in pigs, provided that the maternal derived titer at the time of vaccination was lower than 64. The result implied that an increase in CSFV outbreaks might be due to the inappropriate timing of vaccination as well as the nature of the CSFV genogroup 2.2.     

Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs

Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population.     

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.     

猪瘟病毒-猪圆环病毒2型疫苗联合免疫效果的评价

为建立一种既有效又简便的猪瘟和猪圆环病毒病疫苗联合免疫方法,通过细胞感染试验和动物免疫试验,评价了猪圆环病毒2型(PCV2)灭活疫苗和猪瘟病毒(CSFV)冻干活疫苗联合免疫的可行性.通过细胞感染试验证明了PCV2灭活疫苗稀释CSFV冻干活疫苗不影响CSFV疫苗的病毒活性;将PCV2灭活疫苗稀释的CSFV冻干活疫苗免疫试...     

Porcine circovirus type 2 (PCV2) viral components immunomodulate recall antigen responses

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus infecting domestic pigs worldwide. Interaction of this virus with the immune system apparently modulates the immune response of the host. In the present study, the implication of different components of PCV2 in the modulation of the immune response of the host were investigated by using PCV2 viral-like particles (VLPs) and 16 novel oligodeoxyribonucleotides containing CpG motifs (CpG-ODNs) based on the PCV2 genomic sequence. The role of these viral components was studied by evaluating the cytokine profiles (IFN-alpha, IFN-gamma, IL-10, IL-2 and IL-12) on porcine peripheral mononuclear cell (PBMC) and bone marrow-derived dendritic cell (BMDC) cultures. Also, the effect of PCV2 and its elements were examined in recall antigen (pseudorabies virus, PRV) responses. While PCV2 was a potent inducer of IL-10 by PBMCs, such effect was not observed using CpG-ODNs or VLPs. However, IFN-gamma and IL-2 production by recall antigen was repressed in presence of PCV2 and most of the studied CpG-ODNs. VLPs did not have such repressive effect. In BMDC cultures, PCV2 and most of CpG-ODNs were able to inhibit IFN-alpha secretion induced by PRV. Interestingly, CpG-ODNs with inhibitory effect were located within the PCV2 Rep gene. Additionally, PCV2 virus was a very strong IL-12 inducer in BMDC cultures. Whereas, IFN-alpha modulation on BMDC after PCV2 VLP treatment was neglectable, PCV2 VLPs were potent IL-12 inducers. Our data shows that PCV2 viral elements can distinctly regulate cytokine production depending on the cell population studied. Thus, the final immune response upon PCV2 infection seems to depend on the fine balance between the regulatory elements present in viral DNA and structural protein within the host immune system.     

猪瘟病毒与猪圆环病毒2型混合感染的检测

对广西南宁市、贵港市、崇左市共5个规模化猪场送检病猪的组织器官样品(脾脏和淋巴结),应用已建立的检测猪瘟病毒(CSFV)的RT-PCR技术和检测圆环病毒2型(PCV-2)的PCR技术,快速准确地扩增出了CSFV和PCV-2特异的目的基因片段,从而证实为猪瘟病毒和猪圆环病毒2型混合感染。     

Phylogenetic analysis of recent classical swine fever virus (CSFV) isolates from Assam, India

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5′ UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF.     

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.     

PRRSV、PCV2与CSFV多重SYBRGreenⅠ实时荧光PCR检测方法的建立与初步应用

旨在建立一种能快速、灵敏、同时检测出猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)与猪瘟病毒(CSFV)3种病毒的SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了3对引物分别用于扩增PRRSV ORF7基因、PCV2ORF2基因与CSFV 5′端保守序列的部分片段。将测序正确的3段基因片段分别克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和溶解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,PCV2、PRRSV与CSFV荧光定量PCR的标准曲线的Tm值分别为84.6、86.7、89.3℃,溶解曲线特异,灵敏度分别可达181、202、177拷贝/μL,是普通PCR检测方法的100倍。本试验建立的PRRSV、PCV2与CSFV的荧光定量PCR检测方法实现了3种病毒的同时检测,能够对PRRSV、PCV2、CSFV混合感染的临床病料进行快速诊断。     

一例猪瘟与猪圆环病毒2型混合感染的诊治

在生猪养殖过程中,多种病原混合感染的情况较为常见,并这种混合感染也导致传统的临床诊断容易造成误诊或漏诊,继而延误诊断治疗从而造成经济损失.2020年8月初,孟连县某养猪场保育猪群零散发病,临床主要表现为发热、咳喘、打堆、被毛凌乱无光泽、局部皮肤发绀、消瘦、饮食欲下降、生长滞缓,个别猪只伴便秘和腹泻交替.病理剖检变化显示...     

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.     

基于Fosmid文库平台快速构建PRV、PCV-2和CSFV多联疫苗及其应用研究

随着集约化、高密度养殖的迅速发展,生猪养殖遭受各种传染病暴发的困扰,严重影响了养猪业的发展。其中猪伪狂犬病、猪瘟和猪圆环病毒病是危害养猪业健康发展的主要疾病,三者在临床中常常混合感染,给养猪业造成巨大的经济损失。我国广泛采用疫苗接种的方法来控制上述传染病,为了降低免疫成本、减少免疫次数,研制“一针防多病”的多联疫苗显得格外重要。其中利用活病毒载体构建同时表达多种抗原蛋白的多联疫苗是抵抗传染病混合感染的有效策略。     

Comparison of Porcine circovirus type 2 (PCV2) infection in light and heavy pigs of market age on farms with routine PCV2 vaccination

Commercial vaccines against Porcine circovirus type 2 (PCV2) are widely used on swine farms. Marked body weight variation at marketing age is a problem on conventional pig farms using all-in/all-out barn management. The aim of this study was to investigate whether PCV2 infection could be a factor influencing body weight variation. Seven conventional farms that routinely used PCV2 vaccination were selected, and 60 serum samples from light and heavy pigs at each site were tested for PCV2 antibody titers and viremia. At 3 farms the mean antibody titer, proportion of viremic pigs, and virus load differed significantly between the light and heavy groups. These preliminary results suggest that PCV2 infection may be a factor contributing to weight variation in vaccinated market-age hogs.     

猪瘟疫苗中牛病毒性腹泻病毒2型的检测

为对上海某猪场送检的一份猪瘟疫苗进行牛病毒性腹泻病毒(BVDV)检测,本研究将猪瘟疫苗样品接种于MDBK细胞,盲传15代后仍无致细胞病变效应,但间接免疫荧光试验表明接种该疫苗后的MDBK细胞能够被单克隆抗体BZ-53(BVDV-2)识别。采用BVDV-1和BVDV-2的5’-UTR的通用检测引物和针对BVDV E2的引物,对样品RNA进行RT-PCR检测,结果显示,样品能够扩增出约288 bp的BVDV特异性片段;此外,5’-UTR和E2基因片段的测序分析结果表明分离株属于BVDV-2,并且其E2基因与牛源XJ-04株(BVDV-2)的E2基因同源性最高(92.3%),而与猪源ZM-95株(BVDV-1)的E2基因同源性较低(64.5%)。由此证明,该猪瘟疫苗中的确污染有一株BVDV-2株。     

Porcine circovirus type 2 (PCV2) induces cell proliferation,fusion, and chemokine expression in swine monocytic cells in vitro

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs.     

Porcine circovirus type 2 (PCV2) and Campylobacter infection induce diarrhea in piglets: Microbial dysbiosis and intestinal disorder

Diarrhea is considered to be associated with microbial dysbiosis caused by infection of pathogens but poorly understood. We herein characterized the colonic microbiota of diarrheal early-weaning piglets infected with porcine circovirus type 2 (PCV2) and Campylobacter. Campylobacter infection significantly decreased species richness and Shannon diversity index of colonic microbiota together with a significant increase in the proportion of Campylobacter and Enterobacteriaceae, whereas no significant difference on the above indexes was observed in piglets infected with PCV2 compared with healthy piglets. PCV2 and Campylobacter infection could disturb the homeostasis of colonic microbiota through deterioration of ecological network within microbial community, and specially Campylobacter performed as a module hub in ecological networks. The microbial dysbiosis caused metabolic dysfunction and led to a remarkable reduction in production of short chain fatty acids, following by a higher pH level in colon cavity. Campylobacter infection disturbed the function of colonic tract barrier observed in terms of significant lower relative expression of claudin-1, occluding, and zonula occludens protein-1 genes, and PCV2 infection induced intestinal inflammation together with a higher permeability of colon. Generally, these results suggested that PCV2 and Campylobacter infection could induce microbial dysbiosis and metabolic dysfunction, and cause intestinal disorder, all of which finally were associated to contribute to the diarrhea of early-weaning piglets.     

Porcine circovirus type 2 (PCV2) vaccination reduces PCV2 in a PCV2 and Salmonella enterica serovar Choleraesuis coinfection model

We previously reported that prior porcine circovirus type 2 (PCV2) infection potentiates the severity of clinical signs, lung lesions, and fecal shedding and tissue dissemination of Salmonella enterica serovar Choleraesuis in infected pigs. Here, we evaluated whether PCV2 vaccination is effective in reducing fecal shedding and tissue dissemination of S. Choleraesuis and improving clinical signs associated with PCV2 and S. Choleraesuis infection in 15 Cesarean-derived, colostrum-deprived pigs randomly assigned to 3 groups (n = 5/group). The vaccinated and co-infected (VAC-COINF) group received 2 ml of a commercial PCV2 vaccine at age 3 weeks. The VAC-COINF and co-infected (COINF) groups were inoculated intranasally with PCV2 and S. Choleraesuis at 5 and 7 weeks of age, respectively. The CONTROL group pigs received a similar volume of PBS for sham-vaccination and sham-inoculation. PCV2 vaccination clearly reduced PCV2 DNA load in the serum and postmortem tissue samples and decreased PCV2 antigen levels in tissue samples of the VAC-COINF group. After S. Choleraesuis infection, the incidence of several clinical signs increased in the VAC-COINF group compared to that in the COINF group. The microscopic lung lesions and weight gain, fecal shedding and tissue dissemination of S. Choleraesuis except in the spleen were not significantly different in the VAC-COINF and COINF groups. Thus, PCV2 vaccination reduced PCV2 in the S. Choleraesuis and PCV2 coinfection model and the effects on S. Choleraesuis were minimal.     

猪瘟病毒感染仔猪接种猪瘟疫苗后抗原的跟踪检测

为了解猪瘟病毒感染仔猪免疫猪瘟疫苗后带毒情况,并比较实验室几种猪瘟抗原检测方法的适用性,采用(CSFV)RT-nPCR、猪瘟兔化弱毒疫苗荧光定量RT-PCR(HCLV-FQ-PCR)和CSFV实时荧光定量RT-PCR(CSFV-FQ-PCR)3种检测方法对田间感染CSFV仔猪疫苗免疫前后带毒情况进行定期跟踪检测.结果显示:本实验室建立的CSFV-FQ-PCR灵敏度高于CSFV-RT-nPCR;猪瘟疫苗免疫48 d后,采用HCLV-FQ-PCR方法检测不到血液中的HCLV;猪瘟病毒感染猪免疫疫苗后仍存在持续带毒现象,因此对猪瘟病毒感染猪必须彻底淘汰.