The Characteristics Of Human Metapneumovirus Infection In Scid Mice

PARTⅠ:Analysis of viral infection in infants and young children with severe pneumonia in Chongqing AreaObjective To investigate the prevalence of viral infections and putative association of viral infection and illness severity in young children with severe lower respiratory tract infection (LRTI) in Chongqing.Method One hundred and nineteen respiratory specimens were collected from hospitalized patients with severe pneumonia from December 2006 to March 2008. After being processed, the samples were detected for respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), human metapneumovirus (hMPV), human bocavirus (HBoV), parainfluenza virus 1,2,3 (PIV 1,2,3), influenza virus A and B (ⅣA andⅣB) either by PCR or RT-PCR. Clinical data were analyzed along with virological data by using appropriate statistical methods.Result Viral pathogens were identified in 86 (72.3%) specimens, among which RSV was detected in 49 (41.2%) patients, and hMPV 19(16.0%). More than one virus was detected in 23 individual (26.7%) samples, of which 19 were dual positive for RSV and another virus, and hMPV 42.11%(8/19). Bacterial cultures were performed in 69 patients. Both of bacterial and viral pathogens were identified in 53 (76.8%) patients. In 41 (59.4%) samples were co-infected with bacteria and virus.Conclusion Viral pathogens are the main etiology of severe pneumonia in young children in Chongqing area during the study period. RSV is the most frequent viral pathogens, followed by ADV and hMPV. Coinfection between respiratory common viruswes is relatively common, though co-infection of viruses does not appear to aggravate the patients' condition.PART II:Establishment of plaque assay titration for GFP-labeled recombinant human metapneumovirusObjective Human metepneumovirus (hMPV) is now known to be a commonly seen viral pathogen causing both upper and lower respiratory tract infections in young children. We sucessfully rescued a GFP-labeled recombinant hMPV from full length cDNA clone previously. This study, thus, was to establish plaque assay for the titration of GFP-labeled human metapneumovirus.Methods Vero-E6 cells were selected as host cells for titration. GFP-labeled hMPV was serially diluted and added to each well to infect the cells. The plates were covered with low melting agarose overlay and incubated for diferent days in incubator. The number of plaques was counted under fluorescence microscope for plaques with green flourecence and by immunostaining.Results Under the low melting agarose overlay, Vero-E6 cells grew well until the CPE caused by hMPV was seen. Clear green flourescence could be observed the first day post infection, much clearer on the third day post infection but showed somewhat fusion between plaques later on. The recombinant GFP-labeled hMPV could replicate up to 1×106 PFU in the Vero-E6 cells.Conclusion Plaque assay for titration of recombinant GFP-hMPV has been sucessfully established. This methodology will offer a solid base for further studies on pathogenesis and vaccine development of this virus.PARTⅢ:Characteristics of GFP-hMPV infection in BALB/C and SCID miceObjective To compare the characteristics of hMPV infection in BALB/C and SCID mice.Methods BALB/C and SCID mice were infected intranasally with GFP-hMPV, and sacrificed on day 3,5,7,9 and 14 post inoculation. Heart, liver, spleen, lungs, kidneys and brain of the animals were used for viral isolation, viral titration by a plaque assays, pulmonary histopathology and detection of GFP-hMPV mRNA expression by RT-PCR and Real-time PCR.Results Live viruses were Successfully isolated from the lungs of infected mice. Viral titers peaked on the 5th day post inoculation, (5.25±1.69)×104 PFU/g and (5.83±1.21)×105 PFU/g for BALB/C and SCID. Virus remained to be detectable on the 14th day post inoculation in SCID mice, but not in BALB/C mice, whereas genomic RNA of GFP-hMPV was able to be detected by RT-PCR targeting F gene in infected BALB/C mice. Live viruses were not able to be isolated from heart, liver, spleen, kidney and brain, neither was genomic RNA of hMPV able to be detected on the 5th day post inoculation by RT-PCR and Real-time PCR. Histopathology of lungs was characterized by interstitial pneumonia on 5 days post inoculation. Lung pathology score of BALB/C mice group was slightly lower than SCID, and the difference was not statistically significant.Conclusion (1) GFP-rhMPV intranasal infection can only replicate in the lungs, but not in other organizations. (2) As compared to BALB/C mice, lung virus titer was higher in SCID mice and replicate for a long time. There was no significantly difference of pathological lesion between BALB/C and SCID mice. PARTⅣ:Relication and pathogenicity of human metapneumovirus F mutants in SCID miceObjective Mutated human mepneumoviruses with N-linked carbohydrates deleted have been demonstrated replicate less well and cause significantly milder histopathology in immunocompetent host and thus might be candidate of live attenuated vaccine. This study was to verify these mutants show similar growth feature and also attenuated.Methods SCID mice were infected intranasally with wild type GFP-rhMPV, mutant viruses M1,M2 and M4 which habored point mutation(s) at 57th,172th, and 353th amino acid individually or together. The animals were sacrificed on day 3,5,7,9 and 14 post inoculation. Heart, liver, spleen, lungs, kidneys and brain were used for viral isolation, viral titration by a plaque assays, pulmonary histopathology and detection of GFP-rhMPV mRNA expression by RT-PCR and Real-time PCR.Results Live viruses were Successfully isolated from the lungs of infected mice. M1 viral titers peaked on the 5th day post inoculation(5.92±0.92)×105 PFU/g, remaining to be detectable on the 14th day post inoculation((0.67±0.52)×102 PFU/g. M2 was not isolated on the 3th day post inoculation. M2 titers on the 5th and 9th day post inoculation were (0.33±0.26)×101 PFU/g and (0.58±0.58) PFU/g. M4 viral titers peaked on the 3th day post inoculation (0.50±0.45)×101PFU/g. M4 can not be isolated on the 9th day post inoculation, but genomic RNA of GFP-rhMPV was able to be detected on the 9th day post inoculation by RT-PCR targeting F gene. Live viruses can not be isolated from heart, liver, spleen, kidney and brain, and genomic RNA of GFP-rhMPV was not able to be detected either on the 5th day post inoculation by RT-PCR and Real-time PCR. Histopathology of lungs was characterized by interstitial pneumonia in particular during 5 days post inoculation. As compared to lung pathology score of wild type, the difference with M1 group was not statistically significant(P> 0.05), but difference with M2 and M4 group was significant (P<0.05). As compared to M1, differences with M2 and M4 group was statistically significant (P<0.05).Conclusion (1) GFP-rhMPV can only replicate in the lungs,but not in other organizations. (2) As compared with wild-type, M1 replication capacity and replication did not change; However, replication capacity of M2 and M4 is significantly impaired. (3) As compared to wild-type and M1, lung injury in M2 and M4 groups was significantly less severe, and the difference was statistically significant.