| Objective:To investigate the viral pathogen composition and epidemiological characteristics of Severe acute respiratory infection(SARI)in hospitalized cases from October 2017 to June 2019 in Luohe City,Henan Province.In order to provide etiological basis for the clinical diagnosis,treatment as well as respiratory disease prevention and control of SARI cases.The epidemiological and clinical characteristics of HMPV positive cases were described in China.To learn more about the gene characteristics and gene variation of HMPV virus in Luohe,Henan Province.Methods:Based on the SARI case surveillance program in China,throat swabs were collected from hospitalized SARI cases in Luohe Central Hospital of Henan Province from October 2017 to June 2019.And their epidemiological and clinical information were gathered.The specimens were detected for 19 common respiratory viruses by Mutiplex Real-time Reverse Transcription Polymerase Chain Reaction(Real-time RT-PCR)to describe the composition of viral pathogens.Epidemiological analysis was conducted on the screened HMPV positive cases.The HMPV G genes and whole genome of strains were amplified by reverse transcription polymerase chain reaction(RT-PCR),and the gene sequences were obtained by Sanger generation sequencing.The gene variation and genetic relationship of the G gene or genome strains abtained in this study,together with the global G gene or genome-wide representative strains,were analyzed by MEGA.Bioedit and BEAST software were used for homology and evolutionary analysis respectively.Results:1.A total of 1021 cases of SARI were included in this study,and 64.38%of them were positive for at least one virus.The positive rate of influenza virus was 19.78%,and that of respiratory syncytial virus(HRSV)was 19.39%,followed by rhinovirus(9.58%)and parainfluenza virus(9.32%).HRSV was the main infection in children under 5 years old,while influenza virus took an advantage in people over 5 years old.The co-infection rate of virus was 15.84%.2.The positive rate of HMPV was 7.05%.Children under 2 years of age had the highest HMPV positive rate,and children under 5 years of age account for 84.72%of HMPV positive cases.The prevalence of HMPV was mainly from October to May of the following year,and the prevalence peaked from March to May.The common clinical manifestations of HMPV infectious patients were upper respiratory tract infection,mainly causing bronchopneumonia.3.A total of 43 strains of G gene were amplified in this study,and the genotype distribution was as follows:There was 1 case of A2b genotype and 31 cases of A2c genotype in subtype A.In subtype B,there were 5 cases of B1 genotype and 6 cases of B2 genotype.But A1 and A2a genotypes were not detected.4.The sequences of 31 strains of HMPV A2c genotype obtained in this study were compared with the prototype strains of A2c genotype(Genebank No.JX082176).Among the 31 A2c genotypes,22 of the 31 A2c genotypes showed repeated insertion of 111 nucleotide fragments in G gene,which formed an independent branch with the Japanese A2b111nt-dup sequence in the evolutionary tree.Another six strains showed repeated insertion of 180 nucleotide fragments in G gene,which clustered in the phylogenetic tree with A2b111nt-dup prevalent in Japan,Spain,Croatia,etc.In this study,all the strains included 111 and 180 nucleotide duplication were renamed as HMPV A2c111 nt-dup and A2c181 nt-dup duplication variants based on their genetic relationships and the latest references.The two variants added 37 or 60 amino acids to the G protein,and 12-13 or 25 potential O-glycosylation sites were predicted in the insertion region,respectively.There was no duplication insertion mutation in subtype B.5.In this study,a mutant containing 111 nucleotide fragments duplication insertion,namely A2c111 nt-dup variant(SA20180159),was selected for full genome amplification.Sequence analy sis confirmed that the full length of its genome was 13.45 kb.In the whole genome,there were 111 nucleotide dulication inserted in G gene,but no insertion or deletion mutation occurred in other genes.The homology of the nucleotide sequences of SA20180159 variant and the three Japanese strains with 111 nucleotide duplication fragments were high up to 99.26%-99.33%.6.The results of this study on the degree of nucleotide variation of different gene fragments in the whole genome showed that the mean homology of nucleotide sequence of G gene was 61.2%,which was lower than that of SH gene(77.9%)and other genes(86.20%-90.07%),indicating that G gene had the largest variation in the whole genome sequence.7.The A2c111 nt-dup variant obtained in this study(SA20180159 variant),was analyzed in relation to the HMPV representative strains prevalent in different countries and regions in the world.The phylogenetic tree constructed by the whole genome or the coding region of each protein gene(except M2-1),which showed the same topological structure,divided into six independent lineages,namely A1,A2a,A2b,A2c,B1 and B2 genotypes.Conclusions:1.From October 2017 to June 2019,more viral pathogens were detected in respiratory tract samples from hospitalized SARI cases in Luohe City,Henan Province,with a total positive rate of 64.38%.Influenza and respiratory syncytial virus are the main pathogens,followed by rhinoviruses and parainfluenza viruses.Co-infection was common with HRSV,influenza virus and rhinovirus.2.HMPV mainly infected children under 5 years old,especially children under 2 years old,in SARI inpatients in Luohe city,Henan Province.,the positive rate of HMPV decreased with the increase of age.The epidemiological season was from late autumn to early summer of the following year,and reached its peak from March to May.3.HMPV was circulating with four genotypes:A2B,A2c,B1 and B2,and A2c was the dominant genotype(82.35%)in Luohe City,Henan Province4.In this study,the prevalence of A2c180 nt-dup and A2c111 nt-dup were detected for the first time in China,and these two variants gradually became dominant strains in Luohe City,Henan Province,China from 2017 to 2019.The BEAST analysis estimated that the origin time of these two variants was about 2009.Further surveillance and studies of HMPV in other areas of China were needed to understand the prevalence and evolution pattern of these two variants.Simlarly,the BA9 and ON1 genotypes of HRS V also have similar duplication insertions(60 and 72 nucleotide duplication in G gene,respectively).Since their first discorvered,the prevalence of BA9 and ON1 genotypes had gradually transmitted into more than 20 countries in a short period of time.Is this similar duplication mutation occurring in HMPV also a key pattern in HMPV evolution?Will the duplication variants,which were possibly associated with viral adaptation,leading to a dominant spread of the mutant in humans?These are key questions that need to be addressed by further scientific research.5.The genotyping of HMPV based on the whole genome were consistent with that based on the coding region of each protein(except M2-1 protein).The G gene has the highest degree of nucleotide variation in the entire genome,with duplication of 111 or 180 nucleotide fragments.Based on the whole genome sequence analysis,it was further confirmed that G gene could be used as target gene for HMPV genotyping,which could be better for tracing the viral transmission path and analyzing the variation or evolution of HMPV gene. |
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