| BackgroundEmerging infectious diseases pose constant threats for people’s health.Over the past decade,with the development of biotechnology,novel viruses have been increasingly found,such as HCo V-NL63,HKU1,Ebola virus,Boca virus,humanmetapneumovirus.Discovery and identification of novel viruses,as well as determination of the correlation between new viruses and diseases is essential for thediagnosis,treatment and prevention of emerging infectious diseases.In the current study,we are designed to investigate the epidemiological and genetic characteristics of two novel virus that have been identitied in recent years: WU Polyomavirus and Saffold Cardiovirus.WU polyomavirus is a new virus found,through a high-throughput sequencing of nasopharyngeal aspirates from a 3-year-old child with pneumonia in 2007.Saffold Cardiovirus was discovered in the United States from a fecal sample from an 8-month-old child with fever of unknown origin in 1981.The virus was subsequently identified as a new type of human infection in the Cardiovirus by applying DNase sequence-independent single-primer amplification in 2007.Studies have reported that WU polyomavirus and Saffold Cardiovirus could be detected from nasopharyngeal aspiration,feces,serum and other specimens of the patients.Their role in causing infection has been increasingly studied,however there is still lack of research on the prevalence,pathogenicity and genetic characteristics of these two viruses in China based on large sample size.The previous studies are limited to only ne syndrome,or based on small sample size.The genetic characteristics of the viruses warrant further investigation.\ObjectiveIn this study,we made a hospital based susrveillnce to detect the major viral pathogens among with respiratory tract infection,acute diarrhea and hand-foot-mouth patients in children’s hospital affiliated to Chongqing Medical University,by doing so,we are designed to understand the epidemiological characteristics of these two newly identified viruses,the clincal features that might be related to their infection,the genetic features that are associated with the viral evolution.These knowledge might provide scientific basis for the diagnosis and therapy in clinical practice,also the informed guidance of the infection in public health practice.MethodsThe study was performed at the children’s hospital of Chongqing Medical University,Chongqing,from January 2012 to December 2015.The nasopharyngeal aspirate specimens of pediatric patients with acute respiratory tract infection,fecal samples from diarrhea pediatric patients and stool specimens from hand-foot-mouth disease pediatric patients were collected.WU polyomavirus(WUPy V)and Saffold Cardiovirus(SAFV)were detected from all three kinds of patients.Other viruses,such as the human adenovirus(HAd V),human Rhinovirus(HRV/HEV),influenza virus(Flu),Respiratory syncytial(RSV),metapneumovirus(MPV),parainfluenza(PIV),human bocavirus(HBo V),human coronavirus(HCo V),were detected from the patients with acute respiratory tract infection.Rotavirus(Rt V),norovirus(No V),adenovirus(Ad V),sapovirus(Sp V),astrovirus(At V)were detected from the patients with diarrhea,the Enterovirus(EV),Enterovirus 71(EV 71),Coxsackievirus A16(CVA16)were detected from patients with hand-foot-mouth disease.The detection was performed by PCR,RT-PCR,Real-Time PCR and Real-Time RT-PCR for different viruses as appropriate.From samples with positive detection of WU polyomavirus(WUPy V),the whole viral genome was amplified by PCR.For the Saffold Cardiovirus(SAFV)positive samples,VP1 fragments were amplified based on which phylogenetic analysis was performed.The dataset were built by Epidata 3.1,and SPSS20.0 software was used for statistical analysis.The test level was α= 0.05.The phylogenetic tree was constructed using the software Mega 7.0,using the maximum likelihood method,and the bootstrap value was set to 1000 for building and testing.Selective pressure analysis using FEL,IFEL,MEME,SLAC four methods of screening,the same site at least three methods were identified as positive selection sites,the use of Data Monkey website submitted to analyze the sequence(http://www.datamonkey.org /).Results1.Epidemiological and genetic characteristics of WU Polyomavirus in children(1)Amon totally 3612 recruited pediatric patients,170 were detected to be positive for WU polyomavirus by Real-time PCR,with the overall detection rate of 4.7%.Estimated 7.8%(127/1623)of patients with acute respiratory tract infection,2.4%(25/1036)of diarrhea patients,and 1.9%(18/953)of hand-foot-mouth disease patients,were detected to be positive for WU polyomavirus.The positive rates differed significantly among three groups of patients,with the highest positive rates found among patients with acute respiratory tract infection.(2)Altogether 35 patients were solely infected with WU polyomavirus.Co-infection rate f WU polyomavirus with other viruses was 79.4%(135/170),among them 97 were coinfected with other one virus,31 with other two viruses,7 with other three viruses or more.The coinfection rate among patients with respiratory infection was 79.5%(101/127).The most frequently seen co-infected virus was HRV / HEV(28 children).The co-infection rate of diarrhea patients was 76.0%(19/25)and the most prevalent co-infection was found to be with Rt V(11 children).The co-infection rate of hand-foot-mouth disease patients was 83.4%(15/18),with the EV71(9 children)deteremind as the predominant co-infection virus.(3)For patient with acute respiratory infection,the highest positive detection rate of WU Polyomaviruswas observed from May to June 2013.A peak positive detection rate in both acute diarrhea and hand-foot-mouth patients were observed in July 2013.This suggested the possibility of WU polyomavirus outbreaks during this period.(4)For patients with respiratory tract infection,significant difference was observed for the occurrence of cough,dry rales and wet rales between WU polyomavirus single infection and co-infection patients(P <0.05).WUPy V infection rate was 11.4% in patients with upper respiratory tract infection,and the difference was statistically significant compared with non-upper respiratory tract infection(P=0.018,OR=1.770,95%CI: 1.103-2.842).The infection rate of WUPy V in pneumonia patients was 7.8%.(5)The whole genome sequence of 57 WU polyomaviruses were obtained,with their homology ranging from 98.7% to 100%.The phylogenetic analysis based on our sequences and other public sequences supported the clustering of three branches I,II,III.All the sequences obtained in the current study were clustered in Ia,Ic and IIIc.Among the 35 strains from respiratory infections,20 were clusterd into Ia,8 into Ic,and 7 into IIIc.Twelve strains from diarrhea patients were all grouped into the Ia branch.Ten strains from hand-foot-mouth disease were clustered into Ia(8 strains)and IIIc(2 strains),respectively.(6)The selection of pressure analysis showed that position 82 of VP1 fragment was positive selection site,VP2,VP3,STAg and LTAg did not show positive selection sites.2.Epidemiological and genetic characteristics of Saffold Cardiovirus in children(1)Altogether190 children were detected positive for Saffold Cardiovirus by Real-time PCR,with an overall detection rate of 2.0%,including 44(1.3%)children with respiratory infection,28(0.9%)with diarrhea,118(3.5%)with hand-foot-mouth disease.The positive rates of three groups differed significantly,with the highest positive rate found among hand-foot-mouth disease patients.(2)Altogether 51 patients were solely infected with Saffold Cardiovirus.Co-infection rate of Saffold Cardiovirus with other viruses was 73.2%(139/190),among them 116 were coinfected with other one virus,18 with other two viruses,5 with other three viruses or more.The coinfection rate among patients with respiratory infec tion was 84.1%(37/44).The most frequently seen co-infected virus was RSV(12 children).The co-infection rate of diarrhea patients was 67.9%(19/28)and the most prevalent co-infection was found to be with Rt V(16 children).The co-infection rate of hand-foot-mouth disease patients was 70.3%(83/118),with the EV71(36 children)deteremind as the predominant co-infection virus.(3)For patients withrespiratory tract infection,The positive rate of SAFV in patients older than 36 months was higher than that in 1-6 months(0.95% vs 2.9%,P = 0.005,OR = 3.047,95% CI: 1.396-6.651).The positive rate of SAFV in patients with upper respiratory infection was 1.3%,the positive rate of SAFV was 1.1% in pneumonia patients,and the difference was statistically significant compared with non-pneumonia patients(P = 0.001,OR = 0.308,95% CI: 0.152-0.627).(4)In patients with HFMD,the positive rate of SAFV in patients with neurological symptoms was higher than that in patients without neurological symptoms(P = 0.040,OR = 1.475,95% CI: 1.016-2.140)and the positive rate of patients with severe hand-foot-mouth diseasewas higher than mild hand-foot-mouth patients(P = 0.021,OR = 1.535,95% CI: 1.063-2.219).(5)For patients with hand-foot-mouth disease,the coinfection of EV71 and SAFV has increased the disease severity significantlycompared with the EV71 single infection group(P=0.007).(6)The VP1 fragment sequences were amplified from 151 SAFV positive samples.The phylogenetic analysis revealed existence of four types,S AFV-1(17 strains),SAFV-2 type(70 strains),SAFV-3 type(59 strains)and SAFV-6 type(5 strains).The specific geographical distribution of these four types was disclosed.Conclusions1.Epidemiological and genetic characteristics of WU Polyomavirus in children(1)WUPy V was first detected in patients with HFMD,with a positive rate of 1.9%.The peaking detection rate for three groups of patients was seen from May to July in 2013,suggesting the possibility of WU polyomavirus outbreak during this time.(2)The whole genome sequence of 57 WU polyomaviruses was obtained,which forms a new sub-branch IIIc,and displayed specific geographical features.Ten whole genome sequences obtained from hand-foot-mouth patients belonged to Ia and IIIc type.Twelve whole genome sequences obtained from diarrhea patients all belonged to Ia type.(3)WUPy V gene mutation is not only the choice of purification,but also the effect of positive selection.2.Epidemiological and genetic characteristics of Saffold Cardiovirus in children(1)SAFV was detected in patients with HFMD for the first time,with a positive rate of 3.5%.and the positive rate was higher than in the other two syndromes.(2)SAFV may be associated with central nervous system disease and severehand-foot-mouth diseasein patients with HFMD;EV71 and SAFVco-infection may be aggravate the condition.(3)The phylogenetic analysis showed that the sequences in this study were SAFV-1,SAFV-2,SAFV-3 and SAFV-6,which had certain geographical distribution characteristics. |
