Detection Of Murine Norvirus From Naturally And Experimentally Infected Laboratory Mice By Rt-Pcr Method And Isolation

Noroviruses (NVs), also known as norwalk-virus or small round structured virus, was first discovered in the United States in1972and until in1995only reported in China. NVs is an important pathogen causing acute aseptic gastroenteritis worldwide. The virus had caused more than90percent of nonbacterial gastroenteritis cases. NVs is an important food-borne virus, which has a serious hazard to human health, leading people of different ages to acute viral diarrhea. The main transmission route of NVs is faecal-oral route and the source of infection is NVs infected food or water or droppings. The characteritices of NVs are high incidence and low dose pathogenicity and resistant to the outside world. NVs is mainly prevalent in schools, families, hospitals, army, kindergartens and tourist areas. The clinical symptoms of acute gastroenteritis infection include:nausea, vomiting, diarrhea, abdominal pain, fever, anorexia etc.NVs belong to the family Caliciviridae. Caliciviruses are small non-enveloped viruses approximately27-35nm in diameter with a positive-sense, single-stranded RNA of around7.5kb genome and contains three open reading frames (ORF). In proper order, it is ORF1, ORF2and ORF3. NVs can be divided into five gene group (GG), GGⅠ, GGⅡ, GGⅢ, GGⅣ and GGⅤ. GGⅠ, GGⅡ and GGIV infect humans, GGⅢ infect cattle, GGⅤ infect murine. GGⅤ only found in murine in vivo, so it is called murine norovirus (MNV). Studies have shown that MNV shares biological, genetic, and molecular properties with NVs. The viral size, shape, buoyant density, and genomic structure of MNV are very similar to those of human NVs, which means that MNV is a useful surrogate for human NVs. In addition, MNV is easily cultivated in mouse macrophage cells, such as RAW264.7cell line, unlike NVs. Therefore, MNV becomes an important surrogate virus for studying the human norovirus and also the only ideal research model. MNV disseminated in the experimental animals widely and almost all of the experimental mice susceptible to this virus. Reported that MNV is shed in the feces for a longer period of time. Therefore, we collected experimental mice feces or cecal contents from shanghai region and used RT-PCR method to detect MNV. In addition, we successfully isolated MNV virus from RAW264.7cell.1. In order to elucidate the natural infection situation of murine norovirus (MNV) in laboratory mice, feces samples from eighty laboratory mice were tested for partial ORF2region (187bp, MNV nt5473-5659) of MNV genome using RT-PCR method. PCR positive feces samples were diluted with10volumes of PBS and the dilutions were orally administrated to four ICR mice. One week later,3ICR mice were cohabited with4mice which were orally administrated with positive feces dilutions. To study the experimental infection situation, we observed whether the mice had abnormal symptoms or not and feces were detected for MNV using RT-PCR method at fixed periods. One month later, all mice were killed and samples were collected from feces, cecum, duodenum, spleen, liver, brain, kidney, lung, heart, thymus, uterus, ovary and salivary gland of all mice. All the samples were detected for MNV using RT-PCR method to study the main infection sites of MNV. The results showed that the natural infection rate of MNV was13.75%(11/80). Mice from experimental infection looked like healthy and didn’t appear diarrhea symptom. The experimental infection rate was100%and the main infection site was digestive system of mice. This study is the first report about MNV which was detected from feces of laboratory mice in Shanghai.2. RAW264.7cells were seeed onto onto60-mm plates at a density of5×106cells/plate and allowed to adhere for24h at37℃in5%CO2so that an approximately80-90%cell monolayer formed. Then,500μL of stool suspension from PCR positive results was inoculated onto the RAW264.7monolayer from which the medium was removed. The plates were incubated for1hour at37℃in5%CO2, and then washed twice with serum-free medium. After the two washes,10ml of medium supplemented with5%fetal bovine serum was added into the plates. The plates were then incubated for48hours, until approximately90%viral-induced cytopathic effects (rounding of cells, loss of contact inhibition and cell death) were observed. The plates were then stored at-80℃. After24-hour storage, the plates were then allowed to thaw at room temperature (RT). Following an additional freeze-thaw cycle, the content of the plates was completely removed and centrifuged at3000rpm for5minutes to remove all cellular debris. Supernatant were then removed and aliquoted into1.5mL microfuge tubes. The supernatant were used for MNV RNA extraction, reverse transcribed into cDNA, and subjected to PCR. Through electrophoresis on a1%agarose gel and observed under ultraviolet light. The results show that: the target band is187bp. which is consistent with the expected resuts. This study had successfully isolated MNV virus from RAW264.7cell.