Study On The Pathopoiesis And Molecular Characteristics Of Coxsackievirus B3 Originated From Sichuan Snub-nosed Monkey

The snub-nosed monkey, or golden monkey, is one of the most endangered primate species due to its dramatically shrinking distribution during the past 400 years. Populations of the snub-nosed monkey are found mainly in three isolated regions in China: Qinglin, Sichuan/Gansu, and Shennongjia. There are three species of snub-nosed monkey found in China, including the Sichuan snub-nosed monkey (Rhinopithecus roxellanae), the Yunnan snub-nosed monkey (Rhinopithecus bieti) and the Guizhou snub-nosed monkey (Rhinopithecus brelichi). There is also one species of snub-nosed monkey (Rhinopithecus avunculus) found in Vietnam. The snub-nosed monkey species are all under first-class state protection in China, and the International Union for Natural Conservation (IUCN) has formally recognized them as vulnerable or endangered species. Because of these designations, snub-nosed monkeys have drawn a lot of attention from various conservation efforts. In recent years, the population of the Sichuan snub-nosed monkey has increased by more than 10,000 because of improvement in the conditions of some nature conservation areas and also by artificial propagation. However, many diseases that threaten the snub-nosed monkey have developed because of multitude of factors. There are reports that Sichuan snub-nosed monkeys have died from septicemia due to infection with pneumoniae Diplococcus or Escherichia coli and Klebsiella, from acute pyelonephritis attributed to infected with Escherichia coli and Proteus mirabilis, from unspecified parenchymatous hepatitis, and from unspecified myocarditis. In addition, Staphylococcus aureus can cause spontaneous abortion or stillbirth in the snub-nosed monkey. Since 2001, five Sichuan snub-nosed monkeys had been living in the zoological garden in Changchun in the Jilin province of China. Four of them died between 2003 and 2004. The causes of death were unknown, but they all showed the homoplastic clinical symptoms on lack of activity and shortness of breath before death, if they had cardiac failure, no more evidence were acquired. The final monkey of the original five, a five-year-old female, died on September 18, 2005. She had been in good health but exhibited reduced activity and showed evidence of shortness breath after movement during the previous day. No dizziness or syncope was observed. The hind limbs were slightly edematous. She had been treated using several drugs, but with no effect. No other information about her was available. In order to ascertain the cause of death of the suub-nosed monkey, and the pathogenicity and molecular characteristics of its etiological agent,systematical experiments were done in this study.we dissected the died Sichuan snub-nosed monkey by the common practice and recorded its pathological changes, and then made manufacture pathological section. Pathology of the organs of the suub-nosed monkey appeared septicaemia. Cardiac pericardium, epicardium, and endocardium appeared bleeding point and ecchymosis; cardiac muscle appears white necrotic focus; lungs were swollen and congestive. Histopathologic examination showed that cardiac muscle fiber were abound with inflammatory cell infiltrate; part of them broke, degenerated, and the heart appears typical myocarditis symptom; blood capillary of alveolar wall highly stretched and congested; Other tissues appeared various degree legions. The viruses (Picornaviridae-like morphous) were found in the cardiac muscle tissue homogenate from the suub-nosed monkey by transmission electron microscope after negative staining. The animal regression test showed that the viruses isolates could make mouse died and it showed the similar pathologic symptoms with the suub-nosed monkey. The reverse transcriptase polymerase chain reaction (RT-PCR) performed on an extract of cardiac muscle tissue revealed a DNA sequence specific for Coxsackievirus B. According to the clinical symptom, pathologic histology character and results of pathogen detection, we could conclude that the myocarditis caused by the viruses was the immediate cause of the suub-nosed monkey's death.The tissues homogenates with PBS were centrifuged and the supernatants were treated with 100 U/ml penicillin, 0.1 g/l streptomycin, and 1 mg/l Fungizone. After centrifugation, the supernatant was added to duplicate monolayers of African green monkey kidney cells (Vero), porcine kidney cells (PK-15), and baby hamster kidney cells (BHK-21) in cell culture flasks in an attempt to isolate viruses. Five blind passages were performed at 5-day intervals, the viruses was successfully isolated from cardiac muscle tissue homogenates of the died monkey following inoculation of Vero. The viruses'property consistented with CVB by morphology, physicochemistry test, animal regression test and RT-PCR. Analysis of VP1 partial gene sequence of virus and detecting of mice specific serum IgG by virus were performing, the results showed that the strain isolated was a coxsackievirus B3. It was the first CVB3 case discovered in Sichuan snbu-nosed monkey in world and The virus was designated provisionally Sichuan snub-nosed monkey-origin coxsackievirus B3 (SSM-CVB3). Selected paraffin sections were examined for the presence of SSM-CVB3 antigens by immunofluorescence using an anti-CVB3 rabbit antibody. The fluorescence was detected using confocal laser scanning microscopy. CVB3 was found in the endochylema of myocardial cells and in the glomerular capillary basement membrane and mesangial matrix. This result can confirm furtherly that the snub-nosed monkey were infected with SSM-CVB3.Molecular cloning and sequencing of the genome of SSM-CVB3 were performed by special primers and 5'RACE kit and 3'RACE kit. From the poly (C) tract of the 5' non-translated region (NTR) to the 3' NTR, 7397 bp sequences of the whole genome were obtained by reverse transcription polymerase reaction (RT-PCR). The genome of SSM-CVB3 has been submitted to GenBank (access number: GU109481). The deduced amino acid sequences of the structural and nonstructural proteins of the SSM-CVB3 were analyzed for the sequence similarity among CVB3 strains. Comparison between SSM-CVB3 and other 12 strains indicates that overall the number of amino acids appears an amino acid deletion in VP2 region of capsid proteins, and corresponding amino acid sequences has changed from LGRTG to WSDW, and WSDW sequence can be think as a hereditary feature of SSM-CVB3. For another, the amino acid sequence AAKEFAGEPI in VP2 and RNKHFPVS in 2A can also be think as genetic mark of CVB3 that from China. There are high homology between SSM-CVB3 and CVB3F, CVB3B in 5'-NTR, and between SSM-CVB3 and CVB3G in 3'-NTR and coding region, thus, suggesting that SSM-CVB3 evolved with minor difference from domestic strains, and may be recombinate from CVB3G and CVB3F, CVB3B, but with major difference from the standard strains CVB3N (Nancy-CVB3). Molecular studies suggest that the agent of Sichuan snub-nosed monkey is a highly variant novel CVB3.Inoculate the SSM-CVB3 in healthy macaques by peritoneal injection, observe the pathogenicity to macaque. The macaque show hige body temperature, temple lack of activity, blood urine and albuminuria from 2 days post-inoculation. No other clinical symptoms were observed. Total white blood cells and neutrophil in blood decreased in some days post-inoculation. The myocardial enzyme, liver duty enzyme, kidney duty enzyme, amylopsin and blood sugar in serum were detected, the results showed that CK, CK-MB, ALT and AMY had different extent changing characteristic, but CRE and BUN no obviously change. Macaques were mercy killed and the representative tissues were divided into two portions: one was fixed in 10% neutral buffered formalin solution and processed for histology and immunofluorescence staining, and the other was stored at -80℃. Microscopically, the main lesion were mild extensive infiltration of lymphocytes in the heart, cells productive reaction in the Kiernan's space of liver, relatively severe infiltration of lymphocytes in the interstitial substance of kidney, and nerves cell degeneration, necrosis, and neuronophagia in the spinal cord and brain. Congestion, hyperaemia and oedema were found in most tissues, which indicated macaque infected with the SSM-CVB3 Strain can cause most organ damage. For most tissues, CVB3 antigens were detected by RT-PCR. Especially for the kidney, spleen, lung and liver of the monkey, CVB3 antigens in the cytoplasm of infected cells could be detected by immunofluorescence with a specific anti-CVB3 rabbit hyperimmune serum.In this study, the dead snub-nosed monkey infected with CVB3 were final diagnosis, the molecular characteristics of SSM-CVB3 were analysis systematically, and pathopoiesis were studied to macaque infected with SSM-CVB3 according to clinical pathology and pathologic histology. These results not only can provide the reference for the diagnosis and prevention and cure of snub-nosed monkey disease in clinic, know the genetic background and germline evolution relation of CVB3 in China, but also settle the grounding for further construct animal model of macaque infected with SSM-CVB3, it is very significant for the comparative medicine.