Observations On Fitness And Pathogenicity Between A And B Subtype Of Human Metapneumovirus

Part1Fitness of human metapneumovirus in vitro and in vivoObjective Human metapneumovirus (hMPV), a recently described respiratory virus in2001,causes acute respiratory illness such as bronchiolitis or pneumonia in all age groups, with more severe disease occurring in infants, elderly people, and immunocompromised hosts.Further more, hMPV is associated with asthma. Up to now,no reliable vaccines and therapy are discovered to hMPV infection.The information acquired on viral fitness on hMPV is important to the design of the vaccine, the evaluation of disease severity of virus infection, the drug selection and disease prognosis. However, the report on fitness of hMPV has not been found. This study aims to offer the first report about fitness on two standard genotypes of hMPV, NL/1/00(A subtype) and NL/1/99(subtype B), through in vivo and vitro competitive replication experiment.Methods:1. Two subtypes of hMPV NL/1/00(A subtype) and NL/1/99(subtype B) were mixed into three different proportion of hybrid virus strains(50:5020:8080:20). The two separate strains and three mixed strains were respectively inoculated into Vero-E6cells grown on6-well plate.Competitive replication was used to evaluate the relative fitness of the two subtypes. The passaging process was continued for12passages. RNA was extracted from the infected Vero-E6cells at each passage.To determine the relative proportions of NL/1/00and NL/1/99strains of each passage, a TaqMan probe-based Real-time PCR assay was conducted.2. Female BALB/c mices (specific pathogen-free) aged6-8weeks were randomly assigned to six groups:NL/1/00-infected, NL/1/99-infected,50:50-infected,20:80mixed strains infected,80:20mixed infected and DMEM control groups.Each mice of group was infected intranasally with50ul1*109copies/ul virus under chloral hydrate anesthesia. NL/1/00-infected group received NL/1/00,50:50mixed strains-infected group received a 50:50mixture of NL/1/00AND NL/1/99and control group received50ul DMEM.Every six mices of each experimental group were sacrificed atl,2,4,6,8,13,17,20days after infection, lungs were harvested and viral RNA was extracted from lung tissue.To determine the relative proportions of NL/1/00and NL/1/99strains in BALB/c mice lung, a TaqMan probe-based one-step Real-time RT-PCR assay was conducted.Result:hMPV replicate well in the BALB/c mice lungs and viral titers peaked on the4th day post inoculation,remaining to be detectable on the20th day post inoculation.In BALB/c mice model, within20days of infection were NL/1/99dominant in3mixed groups.However,NL//99dominated the first5passages in cell lines,followed6to8passages NL/1/00follow the lead.Finally,starting from the ninth passge NL/1/99disappeared. hMPV infection didn’t appear in blank hole. In separate NL/1/00or NL/1/99infection in animals model and cell line, no other genotype was detected.Conclusion:Animal experimental showed that the growth of NL/1/99adwantage of hMPV can be confirmed.From the cell lines system to keep alternating between the two genotypes of growth advantage resulting in alternating fashion with the phenomenon of population coincide. Part2Observations on pathogenicity betweenA and B subtype of hMPV in micesObjective:To investigate the difference of pathogenicity between the two genotypes of hMPV for the further research, we observe the changes:weight, viral load, pathology and airway responsiveness in mices after infected by subtypes A and B of human metapneumovirus (hMPV)Methods:At various time after hMPV infection in BALB/c mice, viral titers of lung tissue were measured by real-time RT-PCR, pathology was assessed by a hstopathological scoring system, airway responsiveness was assayed by animal lung function monitoring equipment. Pathogenicity was then measured by detailed evaluation through the results above.Results:There is no significant difference in weight of mices between control group and experimental group through dynamic monitoring; though the difference was exists in airway responsiveness and pathological changes of mices between control group and experimental group,the differences were not statistically in airway responsieness, pathological changes and virus drops among the three groups of experimental group.Conclusion:There is no difference in pathogenicity between the two subtypes of hMPV in infection of BALB/c mices, viral genotype do not appear to be associated with pathogenicity.