Isolation Of Human Metapneumovirus(HMPV) Strains And Construction Of Dicistronic Minigenomes

Human metapneumovirus can cause respiratory infection in children,adults with compromised immunity and the elderly.Studies have shown that the HMPV virus has been circulated in the population for at least 65 years,and almost every child have been infected by the age of 5.Symptoms of infection include cough,fever,runny nose and even more severe pneumonia and bronchitis.HMPV is a single-stranded,negative-sense,RNA virus.The genome length of the virus is about 13kb,and there are 8 gene regions encoding 9 proteins,namely N,P,M,F,M2,SH,G and L protein.The F and N gene of HMPV are relatively conserved genes in the four subtypes,so they are often used as the target genes for virus detection.At present,the detection methods of the virus mainly include traditional RT-PCR,immunofluorescence detection and quantitative PCR detection.In this experiment,by comparing the full-length F gene sequences of four different subtypes of viruses,a pair of PCR primers were designed at the conservative part,and the theoretical length of sequences was 526bp.Traditional RT-PCR method was used to detect the clinical pharyngeal swab samples or cell cultures.At the same time,we synthesized a 526bp sequence and inserted it into the clone vector as a positive control template.By agarose gel electrophoresis analysis,it can be determined that which sample is negative or positive for HMPV.This method has relatively high amplification efficiency and is simple and fast.Further sequencing and alignment can make sure whether the sample contain HMPV.Since virus isolation is the gold standard for virus detection,however,there is no mature and unified virus culture method in the world.Therefore,we refer to relevant studies at abroad and optimize and explore virus culture method by using pharyngeal swab samples or cell culture samples from Qingdao and Guangzhou regions.We respectively optimize TPCK concentration and cell line,finally chose LLCMK2 cell and medium with 1 ?g/ml TPCK pancreatic enzyme to culture virus,and Guangdong samples are HMPV nucleic acid positive from P1 to P4 in cells,however,pharyngeal swab samples in Qingdao region failed to adapt to the cell.We speculated that the main reasons were as follows:firstly,the virus load in the samples was low and the virus was unstable;secondly,the virus culture method and system still needed to be further optimized.Studies have shown that the G gene of HMPV varies greatly among the four subtypes and often used as the basis for typing.Therefore,in this study,PCR primers in the G gene conserved regions were designed,full-length G gene(theoretical length 942bp)was amplified.By RT-PCR,we got four G gene respectively from samples(including clinical swabs and cell cultures),and sequencing,80 G gene sequences in China mainland were alignment,established the evolutionary tree,the results showed that 3 samples of from Qingdao were A2 subtypes,one sample was B1 subtype,one sample from Guangzhou was A2 subtype,and the 65%reference sequences were A2 subtypes,this show that A2 subtype is still major HMPV virus subtype in China mainland.In this study,10 pairs of degenerate primers were designed and RT-PCR amplification method was used to amplify and sequence the entire genome sequence of HMPV virus in cell culture samples.The sequencing results were spliced and about 13kb sequences were obtained.Currently,about 100 bp at 3'end of the genome and about 200 bp at the 5'end were not finished.In order to study the regulation effect of different intergenic sequences of this virus,we constructed 4 dicistronic minigenome plasmids,which contained two reporter genes at upstream and downstream.NP junction(containing gene end of N and gene start area of P and characteristic sequence),FM2 genes junction,M2SH junction and GL junction,during and after transcription gene start signal and gene end signal were further analyzed in transcriptional regulation mechanism.BSRT7/5 cells were transfected with plasmids(N,P,L and M2-1 expression plasmids)and minigenome plasmids to detect and compare the expression levels of reporter genes,and to analyze transcriptional efficiency of different intergenic sequences of the virus.It was found that different GE sequences may have different transcriptional termination activities,and different GS sequences have different activities of binding polymerase to initiate transcription.However,the specific transcriptional regulation mechanism of viral genome still needed to be further studied,and the minigenome system constructed in this study can provide a convenient research tool for further study.