Research Progress of Porcine Cirovirus-like Viruses
and Porcine Circovirus Disease
Porcine circovirus disease (PCVD) is one of major newly-found diseases caused by porcine circovirus type 2 (PCV2) which can cause weaned pigs multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory syndrome (PRDC), proliferative and necrotizing pneumonia (PNP), as well as sow reproductive disorders and some other diseases. The average herd incidence rate is about 60% and the mortality rate is 10% -30%, and even as high as 40% in some areas. It has brought huge economic losses to the global pig industry. In addition, PCV2 can also induce immunosuppression, which causes immune failure.
Vaccination is the most effective way to control the disease. Currently, PCV2 vaccines in Chinese market are comprised of inactivated whole virus. Since the titer of PCV2 propagation is low, it is time-consuming to produce the vaccines. Due to the strong resistance of PCV2, it is difficult to inactive the virus completely. Additionally, oil adjuvant has strong immune stress in pigs, which could bring about biosecurity risks.
Zoonosis Prevention and Control Laboratory of the Institute of Veterinary Medicine, JAAS, first discovered porcine circovirus-like virus P1, which has the smallest genome of an animal virus by far. Similar to PCV2, P1 also has circular DNA genome with a length of 648 nucleotides. It demonstrates that they have both connections and distinctions in pathogenic morphology, genome, the changes of the anatomy, histology, virus tissue distribution, cytokine (IL-8, and IL-10 in particular) transcriptions. To be more specific:
- P1 genome has single strand and circular DNA. The sequence is highly homologous with PCV2 ORF2 except 16 nucleotides presumably derived from porcine retrovirus.
- The detection of PCR shows that P1 infection rate is about 30%. Meanwhile, an ORF1 protein-based indirect ELISA for P1 antibodies has been established, and the results showed that P1 positive rate is about 30% on swine farms in Jiangsu, Zhejiang, Shandong, and Shanghai, and the infection of P1 with symptomatic manifestations of PMWS was significantly higher than those without symptoms of PMWS swine herds.
- When P1 molecular clones were used to transfect PK15 cells, inclusions could be visible in the cell cytoplasm and nucleus, which showed circular shape with a diameter of about 0.1-0.3μm and had larger quantities compared with PCV2. There were two types of nucleus inclusions: one was a small and round, with a diameter of about 0.1μm high electron density thereof, the other one was about 0.4-0.8μm in diameter of a substantially hexagonal inclusion body. Meanwhile, P1 could lead the transfected cells to be autophagy and apoptosis. It could rescue spherical, non-enveloped viruses with a diameter of about 25nm, and it also could be limited to subculture in the PK15, ST and some other cells.
- When the double copies of molecular clones of P1 were injected in the inguinal lymph nodes of pigs, inoculated pigs began to appear in various degrees of viremia at 14 d.p.i, some of them had the symptoms of weight loss, pale skin and diarrhea, which was similar to PMWS. 35 days after inoculation, dissection pig showed visible lesions, including hyperemia, bladder mucosal bleeding, bleeding inguinal lymph nodes, lung atrophy and bleeding. Histology showed subarachnoid vascular congestion, interstitial pneumonia, myocardial contraction, tonsil tissue follicular cell hyperplasia, pancreatic cell necrosis. By means of PCR and immuno-histochemical methods, the nucleic acid and antigens of P1 were detected from lung, brain, heart, liver, bladder, pancreas, gonads and some other organs in varying degrees. The results showed that the immune cells in organs and tissues of P1 infection appeared cell apoptosis by TUNEL.
- The results also showed that P1 infection had different degrees of impact to the host antiviral protein, Toll-like receptors, cytokines, and processing of exogenous antigen presenting molecules .
In the past decade, our research team has been carrying out a large-scale epidemiological investigation of PCV2 in the PMWS infected swine as well as healthy swine in Jiangsu and its surrounding areas. We mastered the epidemiological characteristics and variation laws of PCV2, so we presented a theoretical basis for the prevention and control of PCV2.
For the study of PCV2, two subtype strains of PCV2a and PCV2b were separated and proliferated on PK15 cells and porcine alveolar macrophage cell line 3D4/21. The researches were also conducted on PCV2a and PCV2b Kinetics in infected mice and pigs, as well as autophagy mechanism in porcine alveolar macrophages. It has been found that PCV2 infected pigs can cause alveolar macrophage autophagy, and the phenomenon may be related to the virus replication and cell apoptosis.
The research group first demonstrated that PCV2 both in vitro serial passage and naturally infected pigs could form intramolecular rearrangement of genomic molecules, which belonged to the defect of PCV2 virus. Their genome was annular entirely from the nucleotide sequence of the parent strain. It did not contain an exogenous nucleic acid fragment, but in different cleavage sites.
Currently, the genome sizes of PCV2 molecular rearrangements were found at the large range from 358bp to 1125bp, and they could be classified by the presence or absence of a specific stem-loop structure, the ORF1 and ORF2, the composition and encoding amino acid number of ORF1 and ORF2. The codon-optimized ORF1 and ORF2 recombinant baculovirus series of PCV2 have been constructed, which was confirmed with good immune protection and cross-protective immunity.
The research group has also successfully developed a safe and effective genetic engineering PCV vaccine. The immune effect of the vaccine is even better than similar foreign products. It is characterized that the baculovirus expression system is easy to produce, the water adjuvant is easy to innoculate with low adverse reactions, and the vaccine produces antibodies early with high titer and long immune periods.
On this basis, our group is now working on the creation of porcine circovirus - swine mycoplasma pneumonia combined inactivated vaccine, porcine circovirus - porcine reproductive and respiratory syndrome bivalent inactivated vaccine.
In this project, more than 40 papers have been published including 7 SCI papers. 11 national patents were applied, 4 of them have been obtained.
- WEN LiBin1, HE KongWang, YANG HanChun, NI YanXiu, Zhang XueHan, Guo RongLi & Pan QunXin. Complete nucleotide sequence of a novel porcine cir-covirus-like agent and its infectivity in vitro. Science in China(Life Sciences). 2008,51(5):453-458.
- PAN Qunxing, HE Kongwang, HUANG Kehe. Development of recombinant porcine parvovirus-like particles as an antigen carrier formed by the hybrid vp2 protein carrying immunoreactive epitope of porcine circovirus type 2.Vaccine. 2008, 26(17):2119-2126
- Libin Wen, Kongwang He, Hanchun Yang, Zhengyu Yu, Aihua Mao, Shulin Zhong, Yanxiu Ni, Xuehan Zhang, Bin Li, Xiaomin Wang, Junming Zhou, Rongli Guo, Lixin Lv and Jieyuan Jiang. Prevalence of porcine circovirus-like agent P1 in Jiangsu, China. Virology Journal. 2011, 8:543.
- Bin Li*, Junjie Ma, Yanling Liu, Libin Wen, Zhengyu Yu, Yanxiu Ni, Xuehan Zhang, Junming Zhou, Rongli Guo, Xiaomin Wang, Zhixin Feng, Yiyi Hu, Lixin Lv and Kongwang He. Complete genome sequence of a highly prevalent porcine circovirus 2 isolated from piglet stool samples in China. Journal of Virology. 2012,86(8):4716.
- Libin Wen, Kongwang He*, Bin Li, Xiaomin Wang, Rong li Guo, Zhengyu Yu, Aihua Mao, Yanxiu Ni, Xuehan Zhang,Junming Zhou, Lixin Lv and Jieyuan Jiang. In Vitro and in vivo isolation of a novel Rearrangement of Porcine Circovirus Type 2. Journal of Virology. 2012,86(23):13120.
- Libin Wen, Kongwang He*, Qi Xiao, Zhengyu Yu, Aihua Mao, Yanxiu Ni, Xuehan Zhang, Bin Li,Xiaomin Wang, Rongli Guo, Junming Zhou, Lixin LV, Jieyuan Jiang. A Novel Porcine Circovirus-Like Agent P1 Is Associated with Wasting Syndromes in Pigs. PLOS ONE. 2012, 7(8): e41565.
- Libin Wen, Kongwang He*, Yanxiu Ni, Xuehan Zhang, Bin Li, Xiaomin Wang, Rong li Guo, Zhengyu Yu, Aihua Mao, Junming Zhou, Lixin Lv and Jieyuan Jiang.In Vitro Intragenomic Rearrangement of Porcine Circovirus Type 2. Journal of Virology.2012, 86（16）: 8911.
- LiBin Wen, KongWang He*, ZhengYu Yu, AiHua Mao, YanXiu, Ni, XueHan Zhang, RongLi Guo, Bin Li, XiaoMin Wang, JunMing Zhou and LiXin Lv. Complete genome sequence of a novel porcine circovirus-like agent. Journal of Virology. 2012, 86(1):639.
- LiBin Wen,Kongwang He*,Yanxiu Ni, Xuehan Zhang, Bin Li, Xiaomin Wang, Rong li Guo, Zhengyu Yu, Aihua Mao, Junming Zhou,Lixin Lv, and Jieyuan Jiang. Complete Genome Sequence of the Rearranged Porcine Circovirus Type 2. Journal of Virology. 2012, 86(10):5963.
- Xiaomin Wang, Lichang Sun, Yanhua Li, Tao Lin, Fei Gao, Huochun Yao,Kongwang He, Guangzhi Tong*,Zuzhang Wei*, Shishan Yuan. Development of a differentiable virus via a spontaneous deletion in the nsp2 region associated with cell adaptation of porcine reproductive and respiratory syndrome virus. Virus Research.2012.