Investigation On The Prevalence Of Murine Norovirus Among Experimental Mice In Beijing And Analysis Of Infection Models

Objective:The global prevalence of Human norovirus（Hu No V）in acute gastroenteritis was about 16%and was the most common cause of acute gastroenteritis outbreaks in all age groups worldwide.Hu No V’s disease poses a huge economic burden.Murine norovirus（MNV）shares many features with Hu No V,including fecal-oral transmission,capsid structure,intestinal replication and prolonged shedding after acute infection,exhibiting similar performancer to asymptomatic Hu No V infection.MNV is the most prevalent virus in the mouse population,and infection with this virus affects the results of experimental models such as mouse viral microorganisms and mouse immune system.In recent years,the infection rate of MNV virus in animal laboratories in China has been between 16.9%～60%.The 2014 survey showed that the infection rate of MNV in Beijing experimental mice was 11.67%.The prevalence of MNV in Beijing experimental mice is not clear yet.In order to control laboratory MNV virus infection and improve the quality of experimental mice,this study investigated the prevalence of MNV in experimental mice in Beijing,established an infection model of immunodeficient mice,and further clarified the transmission rule of MNV among mice in different concentration groups,the change rule of viral load in nude mice feces during continuous infection and the distribution of MNV virus in nude mouse organs.Method:1.A total of 1396 cages of mice were selected from various companies in Beijing.Stool samples were collected from each cage.Taq Man probe fluorescence quantitative PCR method was used to detect MNV infection of each strain of mice with MNV-1primer,and whether the mice were infected with MNV was determined according to Cycle threshold（Ct value）.Chi-square test was used to analyze the differences between the positive rate of fecal samples of five mice fecal samples and the differences between the two strains of mice（BALB/c,C57BL/6,BALB/c-nude are inbred mice,ICR and KM are closed group mice）;The Ct values of the five kinds of mice’s positive samples were statistically described;the Non-parametric test was used to analyze the difference of fecal viral load（P<0.001、P<0.05）.2.Fourteen SPF nude mice were randomly divided into two groups:high-dose group（n=7）and low-dose group（n=7）.Two mice in the high-dose group were randomly selected and treated with 200μL high concentration MNV(10^4.08copies/μL)by gavage.Two mice in the low-dose group were randomly selected and given 200μL MNV(10^2.08copies/μL)by gavage.The two groups were referred to as high and low dose artificial infection groups,respectively.The remaining nude mice in the two groups were gavaged with 200μL PBS solution,which was defined as high and low dose natural infection group.Stool samples were collected from each mouse for 48consecutive days and MNV was detected by Taqman probe method.Then,the heart,liver,spleen,lung,kidney,small intestine,cecum and colon of the mice were dissected to detect MNV viral load in each organ.SPSS software was used to analyze the data obtained from the experiment,and local weighted regression scatter smoothing（LOWESS）method was used to fit the curve of fecal viral load in each group of mice over time from day 48 after exposure.Result:1.The positive detection rates of feces in BALB/c,C57BL/6,ICR,KM,BALB/c-nude mice were 17.65%,39.33%,10.57%,18.32%,and 27.4%,respectively.According to the chi-square test results,The positive rate of feces in C57BL/6 mice was higher than BALB/c,ICR and KM mice（P<0.05）,and the positive rate of BALB/c-nude mice was higher than ICR and BALB/c mice（P<0.05）.There was no significant difference in fecal positive rate between inbred mice（26.01%）and closed group mice（13.92%）（χ²=24.460,P<0.05）.The viral load of BALB/c-nude and C57BL/6 mice was generally greater than that of KM mice.2.There was a correlation between the infection rate of high and low dose groups and the positive rate of environmental samples in the dose group,that is,the higher the infection rate,the higher the positive rate of environmental samples（P<0.05）;The fluctuation curve of fecal viral load in the two dose groups was similar.The artificially exposed mice in the low dose group reached the peak 3 days earlier than the artificially exposed mice in the high dose group,and the naturally infected mice in the high dose group reached the peak 2 days later than the naturally infected mice in IVthe low dose group,but the difference was not statistically significant（P>0.05）;The peak time of viral load in the artificial infection group was later than that in the natural infection group,but the difference was not statistically significant（P>0.05）;The median viral load of artificial infection group was lower than that of natural infection group,and the median viral load of high dose naturally infected mice was lower than that of low dose group,but there was no significant difference in viral load（P>0.05）;The top three positive detection rates of each organ in nude mice were cecum>colon>heart.There was no significant difference in the viral load of cecum,colon,and heart between the high and low dose groups（P>0.05）,but the viral load of cecum and colon in the two groups was higher than that in the heart（P<0.05）.In the low-dose group,the viral load in the cecum was higher than that in the colon（P<0.05）.Conclusion:1.The positive rates of MNV in five kinds of mice in Beijing ranged from 10.37% to 39.33%.The results showed that C57BL/6 and BALB/c-nude mice had the highest positive rate of MNV.The Ct values of mice feces were 3.5%in the range below 34,indicating that only a small part of mice feces carried high concentrations of MNV virus.2.Fecal viral load of most mice in the high-low dose group reached its peak during the 26 to 32 day after infection.The low-dose group and the natural infection group reached the peak earlier than the high-dose group and the artificial infection group.Therefore,compared with the high-dose group,the low-dose group was presumed to exhibit faster and more efficient replication dynamics in the body.The spleen and liver of nude mice were dissected and the infection rate of MNV was low,indicating that these two organ parts may not support persistent replication of the MNV virus strain.