Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-gamma), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-gamma. In an in vitro study, ConA but not IFN-gamma enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4(+) (40%), CD8(+) (54%) and IgM(+) (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-gamma 12h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15dpi in non-treated and IFN-gamma-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-gamma-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo.     

Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs

Replication of porcine circovirus type 2 (PCV2) in pigs, as measured by spliced capsid mRNA (Cap mRNA) and viral DNA, was investigated following experimental infection. Peripheral blood mononuclear cells (PBMCs), and tissue from bronchial lymph nodes (BLN), inguinal lymph nodes (ILN), tonsils, lungs, liver, kidneys, spleen and thymus from infected pigs on different days post-infection (DPI) were assessed. PCV2 replication differed dramatically between tissues from the same infected pig. The virus actively replicated in most tested tissues at 14DPI in association with increased PCV2 associated lesions and PCV2 antigen levels, although no clinical signs correlated with PCV2 associated disease were observed in infected pigs during the course of the study. The PCV2 Cap mRNA was detected only at 13DPI in PBMCs from infected pigs, suggesting replication of the virus in circulating blood is transient and not a major site for PCV2 replication in vivo. Evaluation of the Cap mRNA and viral DNA synthesis in T and B lymphocyte and monocyte populations from PBMCs and BLN at various intervals post-inoculation revealed replication of PCV2 in all cell subpopulations; however, viral replication in B lymphocytes was greater than observed in mononuclear cells isolated from BLN at 14DPI indicating that B lymphocytes may be an important cell population for PCV2 replication. These findings further our understanding of the cell types permissive for PCV2 replication and the pathogenesis of PCV2 infection in vivo.     

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.     

Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2)

Porcine circovirus (PCV)-2, a newly described single-stranded circular DNA virus pathogen of swine is the cause of postweaning multisystemic wasting syndrome (PMWS). In gnotobiotic piglets, PCV-2 infection alone produces asymptomatic infection without evidence of overt PMWS. Gnotobiotic piglets infected with PCV-2 were injected with keyhole limpet hemocyanin in incomplete Freund's adjuvant (KLH/ICFA), and the effects on virus production and development of PMWS were determined. In the first experiment, piglets were injected subcutaneously on the left hip and shoulder, and viral burden was assessed in regional lymph nodes draining the injection sites and in contralateral lymph nodes 13-14 days after infection. Immune activation increased the number of virus antigen-positive cells in draining lymph nodes and increased the amount of infectious virus recovered by 1-4 log10. In a second experiment, the effects of injections of KLH/ICFA with or without concurrent stimulation of peritoneal macrophages by intraperitoneal injections of thioglycollate broth on induction of PMWS was assessed. All immunized piglets developed moderate to severe PMWS, whereas none of the piglets infected with PCV-2 alone developed PMWS. In PMWS-affected piglets, extensive replication of PCV-2 was documented by both immunocytochemistry and quantitative viral titrations. Thus, immune activation is a key component of the pathogenesis of PCV-2-associated PMWS in swine.     

Characteristics of porcine circovirus-2 replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection.

In this study, the characteristics of porcine circovirus-2 (PCV2) replication (infectious virus titrations, distribution, and immunophenotyping of infected cells) in lymphoid organs were examined and related to the development of clinical signs and histological lesions in 26 piglets that had been inoculated with PCV2 either in utero or at 1 day of age. Piglets inoculated in utero at 92 or 104 gestational days (n = 12) were collected by Caesarean section at term and either sacrificed immediately or kept in isolators and allowed to live postnatally until 35 days postinoculation (PI). Caesarean-derived piglets inoculated at 1 day of age (n = 14) were sacrificed at 10, 21, 35, 42, and 49 days PI. Spleen and lymph nodes were collected for virologic and histopathological examinations. Clinical signs were not observed in any of the piglets. High virus titers (10(4.5-5.7) TCID50/g [TCID refers to tissue culture infectious dose]) were detected in 6 of the 26 piglets. Three of these 6 piglets were euthanized at 10 days PI, and infected cells of the monocyte-macrophage lineage (SWC3+, CD14+, and sialoadhesin [Sa]+ cells) and infected cells bearing lymphocyte markers (CD4+, CD8+, and immunoglobulin M+ cells) were identified by double-immunofluorescence labeling on serial cryostat sections. The other 3 piglets were euthanized at 21 and 35 days PI, and the majority of infected cells were SWC3+, CD14+, and Sa-. The absence of Sa in these infected cells, together with their localization in lymphocyte-dependent regions, suggests that they were infiltrating monocytic cells. Sialoadhesin is highly expressed in differentiated macrophages and not in peripheral blood mononuclear cells. In all 6 piglets with high virus titers, lymphocyte depletion and infiltration of monocytic cells were observed. In the remaining 20 piglets with virus titers less than 10(4.5) TCID50/g, the majority of infected cells were SWC3+, CD14+, and Sa+. In conclusion, it can be stated that high PCV2 titers in lymphoid organs may lead to the development of histological lesions similar to those observed in pigs with postweaning multisystemic wasting syndrome without causing disease. Furthermore, in lymphoid organs with high virus titers, infection occurs mainly in infiltrating monocytic cells and to a limited extent in cells bearing lymphocyte markers.     

Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b

In late 2005, a postweaning, high mortality syndrome spread rapidly through finishing barns in swine dense areas of the United States. Diagnostic investigations consistently detected porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed that the infectious agent was a PCV2 type termed "PCV2b". Prior to late 2004, only the PCV2a type, but not PCV2b, had been reported in North America. In this communication, we produce severe postweaning multisystemic wasting syndrome (PMWS) in gnotobiotic pigs using infectious PCV2a and PCV2b generated from DNA clones constructed from field isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12h of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in these studies were comparable to those reported in experiments with gnotobiotic pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus or torque teno virus. The animals in these studies were confirmed to be free of detectable porcine parvovirus, porcine reproductive and respiratory syndrome virus, bovine viral diarrhea virus, swine hepatitis E virus, and aerobic and anaerobic bacteria. Seven out of 24 PCV2 inoculated pigs had a detectable congenital torque teno virus infection with no correlation to clinical disease. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any detectable infectious co-factor.     

猪圆环病毒2型的分离与鉴定

利用PCR方法，从疑似断奶猪多系统衰竭综合征(PMWS)的自然病例的淋巴结和脾脏中扩增出了预期长度的猪圆环病毒2型(Porcine Circovirus type 2，PCV2)的DNA片段，并用限制性内切酶对PCR产物进行了酶切鉴定。将经PCR扩增和酶切鉴定为PCV2阳性的组织样品匀浆液接种到无PCV污染的PK15细胞中传代，经间接免疫荧光检查，在接毒细胞内观察到了特异性免疫荧光；用接毒细胞制备超薄切片，在电镜下观察到了直径大约为17nm的圆形病毒样粒子和大量不同形态的胞浆内包涵体。由此表明分离出的病毒为猪圆环病毒2型。     

Viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus 2 infection

This publication reviews some pathogenetic features of the transplacental infection with porcine viruses in sows. Viremia either with virus freely circulating or associated to peripheral blood mononuclear cells (PBMC) is an essential part of such pathogenesis. Virus replication occurs either in fetal tissues only or both in fetal and maternal tissues and the outcome may be different.Since porcine circovirus 2 (PCV2) has been associated with reproductive failure in sows, the question was asked what type of viremia PCV2 causes and what the effect of PCV2 is on the pregnant uterus. Seronegative gilts were oronasally inoculated and plasma and PBMC were monitored for infectious virus and for quantity of viral DNA copies. Infectious virus was found in plasma only at 21 days post-inoculation (DPI). Virus associated to PBMC was detected between 14 and 49 DPI. Viral DNA was found in plasma between 14 and 49 DPI and associated to PBMC between 7 and 63 DPI (end of experiment). Direct intra-fetal inoculation at 57, 75 and 92 days of gestation and collection of fetuses 21 days later showed that the virus replicates highly in fetal tissues, particularly in the heart. Fetal death occurred in the 57 days sows while virus and antibodies were observed in the 75- and 92-day inoculated sows. Inoculation at 57 and 75 days of gestation and collection of the piglets at the end of pregnancy showed that intrauterine spread had occurred to fetuses adjacent to the inoculated ones and that fetal death occurred also in the presence of antibodies. The pregnancy was not interrupted.This study shows that PCV2 causes viremia which is largely cell-associated and that virus replication in fetuses causes fetal death with mummification. Whether such transplacental infection occurs in the immune sow population is questionable.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.     

小鼠感染猪圆环病毒2型的超微结构病变观察

为探讨猪圆环病毒2型（PCV2）对小鼠超微结构的病理损伤及其病毒在细胞内的复制,20只6周龄的昆明小鼠随机平均分成2组（即A组和B组）,A组小鼠经腹腔注射PCV2细胞培养物0.1mL/只（含病毒1 000TCID50）,B组以同样的方式和剂量注射无菌细胞培养液作为对照。于PCV2感染后14d,处死所有小鼠,取其组织做电镜观察和PCV2PCR检测。结果显示,在电镜下,所有PCV2感染鼠的超微结构病变基本一致,主要表现为淋巴器官、心脏、肝脏、肺脏、肾脏、脑、肠等脏器实质细胞凋亡或坏死,细胞内线粒体水肿、内质网扩张,间质毛细血管淤血、炎症细胞浸润,并在脾脏、胸腺、淋巴结的淋巴细、巨噬细胞,以及肝细胞,肾足细胞,脑神经细胞的胞浆或胞核内发现病毒包涵体。同时通过PCR检测,所有PCV2感染鼠的组织均可检出PCV2DNA。B组（对照组）小鼠除在淋巴结可见极少数淋巴细胞凋亡外,其他组织均无任何超微结构病变;同时,所有组织也未检出PCV2DNA。由此说明PCV2可在昆明小鼠多脏器实质细胞内复制,并诱导细胞凋亡。     

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.     

Evidence of porcine circovirus infection in pigs with wasting disease syndrome from 1985 to 1999 in Hokkaido, Japan

An epizootiological survey with histopathological methods was conducted for porcine circovirus in 220 diseased pigs (1-200 days old) in 49 farms from 1985 to 1999. Histopathological lesions containing PCV antigen were detected mainly in the lymphoid tissues from 42 of 189 diseased pigs (22.2%) in 4 of 45 farms (8.9%) from 1990 to 1999. The rate of positive pigs gradually increased from 1997 onward and PCV infection was found in 50% of diseased pigs in 1999. Histopathologically, the lesions in the lymphoid tissues (including lymph nodes, Peyer's patches, tonsil and spleen) were highly correlated with the presence of numerous spherical basophilic intracytoplasmic inclusion bodies with PCV antigen, and consisted of lymphocellular depletion and infiltration of macrophages. Although most affected cells showed cytoplasmic reactivity for PCV, intranuclear antigen was also seen in the lymphocytes, macrophages and ileal epithelial cells. Ultrastructurally, macrophages and giant cells contained electron-dense, round to ovoid lysosomal bodies, in which there were concentric circle or paracrystalline arrays of small nonenveloped icosahedral viral particles, approximately 15-17 nm in diameter. Other consistent infectious agents were present in 90.5% of cases, and porcine reproductive and respiratory syndrome virus infection was in 52.4% of the cases with PCV. The histopathological findings suggested that PCV induced systemic immunosuppression in the infected pigs and made them more susceptible to infection of the organisms. Because of the presence of PCV antigens in the intestinal epithelium, feces may play a significant role in dissemination of PCV.     

猪圆环病毒2型遗传标记毒株的构建及生物学特性鉴定

以临床分离的猪圆环病毒2型（PCV2）为材料，利用PCR法扩增病毒基因组，将2个基因组顺式连接插入到质粒载体中构建感染性分子克隆。通过引物设计替换碱基，在病毒基因组内插入Sal Ⅰ限制性内切酶位点作为遗传标记，经重组质粒转染细胞获得带有遗传标记的新毒株。采用免疫过氧化物酶单层细胞试验、免疫电镜、核酸序列分析表明，在病毒感染细胞中检出病毒特异抗原，其抗原性、病毒形态学及基因序列与亲本毒株一致。鉴于新病毒基因组内插入一个SalⅠ酶切住点，用PCR与限制性片段长度多态性分析法可与野生型病毒相鉴别。新毒株经细胞连续传60代，体外培养增殖性能稳定，毒价可达10^6.6CID50/mL。取病毒培养物经静脉和滴鼻途径接种35日龄抗体阴性仔猪4头，接种后表现出体温升高、进行性消瘦、被毛粗糙及体表淋巴结肿胀症状，迫杀后多种脏器中均能检测到病毒抗原与核酸。研究表明，利用感染性分子克隆手段构建带有遗传标记的PCV2新毒株，为进一步开展该病毒的致病性、疫苗免疫、分子诊断等研究奠定了基础。     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。     

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.     

Lack of an effect of a commercial vaccine adjuvant on the development of postweaning multisystemic wasting syndrome (PMWS) in porcine circovirus type 2 (PCV2) experimentally infected conventional pigs

The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the clinical and pathological outcome of PCV2 experimentally infected 8 to 9-week-old conventional pigs. Forty-four pigs were divided into four groups: non-infected control pigs, pigs that received a vaccine adjuvant, pigs inoculated with PCV2, and pigs inoculated with PCV2 together with the vaccine adjuvant. Infection was monitored until 69 days post-inoculation (PI). Some PCV2 inoculated pigs had hyperthermia, but no other clinical signs were recorded. No characteristic PMWS gross or microscopic lesions were observed in any of the pigs. PCV2 DNA was detected in lymphoid tissues by in situ hybridisation in 6 PCV2 inoculated pigs on day 69 PI. All PCV2 inoculated pigs seroconverted between days 21 and 49 PI, shortly after viremia detection. Moreover, viremia was detected between days 7 and 69 PI using PCR. A peak of the virus load was detected by real-time quantitative PCR between days 14 and 21 PI. There were no significant differences in the proportion of PCV2 positive serum and in the viral load between PCV2 and PCV2 + adjuvant inoculated pigs. Although PMWS was not reproduced in neither PCV2 nor PCV2 + adjuvant inoculated pigs, viremia detection and seroconversion indicated that all PCV2 inoculated pigs developed a chronic long-term asymptomatic infection. An increase of PCV2 replication was not observed in pigs inoculated with the adjuvant. These results indicate that the principle of immunostimulation may not be applicable under the experimental conditions used, suggesting that not all adjuvants used in commercial vaccines are capable of triggering mechanisms for PMWS development.     

Porcine circovirus type 2 in muscle and bone marrow is infectious and transmissible to naïve pigs by oral consumption

Pork products are a possible source of introduction of PCV2 isolates into a pig population. However, limited work has been done on the transmission through meat of porcine circovirus type 2 (PCV2), a virus associated with several disease syndromes in pigs. The objectives of this study were to determine if pork products from PCV2-infected pigs contain PCV2 DNA/antigen and to determine if the PCV2 present in the tissues is infectious by performing in vitro and in vivo studies. Skeletal muscle, bone marrow, and lymphoid tissues from pigs experimentally inoculated with PCV2 were collected 14 days post-inoculation (DPI). The tissues were tested for presence of PCV2 DNA by quantitative real-time PCR, for PCV2 antigen by immunohistochemistry (IHC), and for presence of infectious PCV2 by virus isolation and inoculation of PCV2 naïve pigs. Lymphoid tissues contained the highest amount of PCV2 (positive by PCR, IHC, and virus isolation), bone marrow contained a lower amount of PCV2 (positive by PCR and IHC but negative by virus isolation), and skeletal muscle contained the lowest amount of PCV2 (positive by PCR but negative by IHC and virus isolation). Naïve pigs fed for three consecutive days with either skeletal muscle, bone marrow, or lymphoid tissues all became PCV2 viremic as determined by quantitative real-time PCR on serum starting at 7 DPI. The pigs also seroconverted to PCV2 as determined by PCV2 IgM and IgG ELISA. In addition, PCV2 antigen was detected by IHC stains in lymphoid tissues and intestines collected from the majority of these pigs. Results from this study indicate that uncooked PCV2 DNA positive lymphoid tissues, bone marrow, and skeletal muscle from PCV2 viremic pigs contain sufficient amount of infectious PCV2 to infect naïve pigs by the oral route.     

Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages

Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS.     

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PCV212 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。     

Proliferative and necrotising pneumonia and severe vascular lesions in pigs naturally infected with porcine circovirus type 2

Severe disease induced by porcine circovirus type 2 (PCV2) was observed in three pigs originating from a large herd affected by respiratory and digestive signs as well as wasting. Proliferative and necrotising pneumonia (PNP) was diagnosed in two animals, while severe acute interstitial pneumonia characterised by the presence of abundant hyaline membrane in the alveoli and fibrin in the bronchioles was found in one pig. In all cases, large amounts of PCV2 antigen were found in each tissue sample collected from the lungs and mediastinal lymph nodes. Neither porcine reproductive and respiratory syndrome virus (PRRSV) nor swine influenza virus (SIV) was detected, and no bacteria could be cultured in any of the cases. Vascular lesions, e.g. degeneration of endothelial cells, perivascular and intramural oedema, fibrinoid necrosis, vasculitis, perivasculitis, and vascular thrombi were observed in all cases, associated with the presence of PCV2 antigen. The viral antigen was present in the intravascular mononuclear cells, endothelial cells, myocytes and infiltrating inflammatory cells in lymph and blood vessels. In one case, obliterating thrombi in the lymph and blood vessels were directly connected to areas of tissue necrosis and were associated with abundant PCV2 antigen. The results further suggest the causative role of PCV2 infection in PNP, and the importance of the vascular system in the pathogenesis of PCV2-associated diseases of swine.