Epidemiological And Genetic Characteristics Of Respiratory Viruses In The Children With Acute Respiratory Tract Infection

BackgroundAcute respiratory tract infection (ARTI) is one of the most common infectious diseases in the world with the first incidence rate in acute infectious diseases. About4millions patients diedfrom the lower ARTI every year. In recent years, SARS,avian influenza A (H5N1) viruses,influenza A (H1N1) pdm2009, the emergencing avian influenza A (H7N9) virus, cause humans to pay more attention to respiratory viruses. ARTI is the main factor for threatening children’ health, leading to2million children death each year and has become the second etiology of death in the children under5years old. Facing the current complex situationsfor prevention and control of respiratory infectious diseases, we should strengthen the monitoring report about infectious disease information and active to surveillance the respiratory viral pathogens in the populations, especially in the children. And thentry the best to forecast and warnabout the outbreak of respiratory infectious diseasesin order to reduce the influence on social and economic development.ObjectiveIn the current study, we performed sentinel surveillance for respiratory viruses among pediatric patients with ARTI presenting at a large referral hospital during2009-2012in Chongqing. China. The respiratory viruses mainly included respiratory syncytial virus (RSV). human rhinovirus (HRV), influenza virus (IFV), metapneumovirus (MPV), parainfluenza virus(PIV), human bocavirus (HBoV), human coronavirus (HCoV), human adenovirus (HAdV), and human enterovirus (HEV). This study was aimed to describe the spectrum and epidemiological characteristics of respiratory viruses in the children with ARTI and explore the association of respiratory tract viruses in the children with pneumonia and with asthma attack. Moreover we want to analysis the relationship between the positive rate of respiratory viruses and meteorological factors and to understand the symbiotic relationship between respiratory viruses and bacteria.This study demonstratedgenetic characteristics of complete genome or genes of HAdV、HRV、HEV and HBoV, especially emerging respiratory virusor subtypes, including HAdV-7, HAdV-55, HRV-C, HEV-68, HEV-118and HBoV-1. Analysis of genetic characteristics of respiratory viruses in order to explore the origin, evolution, epidemic strains and drug resistance type and other information of the viruses, and then provide a scientific basis and reference for treatment, vaccine research and epidemic control for virus infection.Materials and MethodsIn the current study, we performed sentinel surveillance for respiratory viruses among pediatric patients with ARTI presenting at Children’Hospital, Chongqing Medical University during2009-2012in Chongqing, China. Nasopharyngeal aspirates were collected at admission to hospital for the children. The clinical information were got form the medical records.The study performed the detection for respiratory viruses with different methods.HAdV hexon and fiber genes were sequenced for HAdV positive samples, VP4/2for HRV,5’NTR and VP1for HEV and complete genome for HAdV-55and HBoV.Genetic sequences analysis includedidentities, phylogenetic tree, recombination, molecular clock, selective pressure and spatial dynamical analysis. The dataset were built by Epidata3.0. Statistical analyses were conducted by SAS9.3software.Results1. Epidemiological characteristics of respiratory viruses(1) A total of2451children with ARTI were recruited in the study. The positive rate for RSV were36.7%, the highest among the nine respiratory viruses and then PIV23.3%、IFV19.6%、HRV/HEV18.4%、HBoV14.1%、HAdV7.9%、MPV3.4%as well as HCoV3.1%. The detection rate for at least one respiratory virus was80.3%and45.2%for single infection. (2) Compared to the group aged<6months, the positive rate for IFV were1.5～2folders in the other groups. The positive rate for HAdV increased with children’s age under5years old. Boys were easier to infect IFV-B and HRV/HEV than girls, ORs were2.19(95%CI:1.16-4.14) and1.28(95%CI:1.02-1.59), respectively. The clinical manifestation of the children with ARTI were fever, cough, expectoration, diarrhea, tachypnea and moist rales. RSV-A caused more dyspnea (28.2%, P<0.001) and RSV-B caused more expectoration (88.1%, P<0.001).The white blood cell count decreased in the children infected IFV-A. Neutrophil percentage decreased and lymphocyte percentage and platelet count increased in the childreninfected with RSV or PIV-3. While opposite for the children infected with HAdV.(3) Logistic regression model results showed respiratory viruses infection did not increase the risk for pneumonia compared to non-respiratory viruses infection. RSV and HAdV were related withsevere pneumonia (OR=1.58,95%CI:1.08-2.30; OR=5.10,95%CI:3.17-8.21). RSV-B, IFV-A and HRV/HEV were associated with moderate/severe asthma attacks, ORs were2.99(95%CI:1.28-6.97),2.28(95%CI:1.01-5.15) and2.94(95%CI:1.35-6.41).(4) The correlation coefficients were lower than0.30among respiratory viruses or between respiratory viruses and bacteria.The degrees of correlation were moderate between RSV and atmosphere, average month temperature and average sunshine duration, while were low between positive rates of other respiratory viruses and meteorologicalfactors, demonstrating meteorologicalfactors were less affected the epidemiology of respiratory viruses except for RSV.2. HAdV subtypes and genetic characteristics(1) Among all the tested children, HAdV subtypes included HAdV-7(48.7%), HAdV-3(37.3%), HAdV-55(3.1%), HAdV-5(2.62%), HAdV-1(2.1%), and HAdV-2(0.5%).(2) HAdV had a higher positively rate during July2010to June2011. HAdV-7became the predominant subtype from September2010, and caused more dyspnea, moist rales and a longer hospitalization. Compared to HAdV-3, HAdV-7was associated with severe diseases (45.7%vs.18.1%, OR=3.52,95%CI:1.60-7.74, P=0.002).(3)7d2was the main subtype of HAdV-7. Identities of hexon gene of HAdV-7were99.8%～100%. Phylogenetic tress showed the strains in the study distributed in the7d2and7d lineages with geographical characteristic. The strains from7d lineage were easier to cause severe pneumonia (OR=2.50,95%CI:1.05-5.94). Identities for fiber gene of HAdV-7were99.7%～100%and two mutations were found: C111T and A253G. The latter changed amino acid Thr85GLA.(4) The sequences of hexon gene of HAdV3formed multiple lineages with geographical differenece and migration events. Four mutations in the fiber gene of HAdV-3were found:T520C (CQ5685)、A567C、C722A (CQ5) and G742A (CQ483). C722A and G742A changed the amino acid:Pro261His and Glu278Lys. Phylogenetic tree demonstrated three lineages and the strains located in A lineage.(5) Identities of HAdV-55in the study were99.8%～99.9%with other strains from other regions,96.1%-98.7%with HAdV-B2and highest with HAdV-14.(6) CQ-814(China/2010) was most closely related to HAdV-11a (P14) from Beijing, followed by CQ-1657(China/2011) and CQ-2903(China/2012)(Figure1A). Surprisingly, the closest genetic relationship existed between the2012Chongqing strain (CQ-2903) and the2006Shaanxi strain (QS-DLL). The HAdV-11a strains from mainland China seems to have diverged from their TMRCA with other strains from Taiwan Province/Singapore/South Dakota around15years ago considering the hexon gene,10years ago considering the fiber gene and35years ago considering the whole genome, respectively.3. HRV and HEV subtype and genetic characteristics(1) Among1564samples,223(14.3%) were positive for HRV. which included HRV-A120(53.8%), HRV-B11(4.9%) and HRV-C92(41.3%);41(2.6%) were positive for HEV, mainly including HEV-687(17.1%), HEV-71and CV-A65(12.2%), respectively. (2) C2were the predominant subtype of HRV-C in Chongqing region, and the following were C43, C1and C17. In addition, another5sequences of VP4/2were different from the51known subtypes (more than10%differences). We named them C52, C53and C54.The overall evolutionary rate of VP4/2for HRV-C was6.9×10-3s/s/y (95%HPD:2.5×10-3～12.5×10-3s/s/y). The number of effective infection increased sharply before40years observed from Bayesian skyline plot.(3) Molecular clock analysis revealed the overall evolutionary rate of VP1for HEV-68was9.6×1O-3s/s/y (95%HPD:6.9×10-3～12.5×10-3s/s/y). On the phylogenetic tree based on VP1gene nucleotide sequences, all modern HEV-68strains were grouped into three separate lineages (Figure2). Six Chinese strains from the study fit into Lineage3, being divided into two branches with a high bootstrap value with divergence in1973. The lineage evolved with a high speed (3.2×10-2s/s/y,95%HPD:2.1×10-2～4.7×10-2s/s/y) before recovered to normal in2008. Positive selectively position was not found on VP1gene. The number of effective infection kept steady, with fluctuating in2～4years before. The migration events inferred across the parsimony genetic phylogenies for the160VP1sequences of HEV-68from19locations among five continents, and their concatenation showed the Japanese province of Yamagata and the American state of Texas acted as the geographic source.4. HBoV genetic characteristics(1) Identities forHBoV complete genome were99.4%～100%, and more than97%in the four genes. The sequences of HBoV were conserved.(2) The phylogenetic trees based on the four genes and complete genome were similar and formed branches with a bootstrap>750support. HBoV-1sequences did not reveal temporal and geographic distribution.(3) The evolutionary rate of HBoV-1was1.89×1O-4s/s/y (95%HPD:0.01～4.54×1O-4s/s/y), lower than that of HBoV. Selective pressure analyzed that Position40of VPl was the positive selection site, while none were found in NP, NS, and VP2gene. Conclusions1. Epidemiological characteristic of respiratory viruses(1) The positive rate of respiratory viruses was80.3%, demonstrating respiratory viruses was the predominant pathogen in the children with ARTI.(2) RSV and HAdV were associated with severe pneumonia, while RSV-B,IFV-A and HRV/HEV were related with moderate/severe asthma attack.2. HAdV(1) Multiple HAdV subtype circulated in Chongqing region, mainly including HAdV-7, HAdV-3and HAdV-55. HAdV-7caused more severe pneumonia than HAdV-3and others.(2) The sequences of hexon and fiber genes of HAdV-7and HAdV-3were steady and conserved and geographical distribution. HAdV-7existed with7d and7d2, and7d2may be related with severe diseases. HAdV-55complete genome sequences revealed temporal and geographical distribution.3. HRV and HEV(1) HRV-A and HRV-C were the main subtypes of HRV in the children with ARTI and C2was the predominant subtype of HRV-C.Three new subtype of HRV-C were found and named.(2) This is the first report to detect HEV-68in the children with ARTI in China. The strains in the study formed a branch. HEV-68revealed migration events in the world.(3) This is the first time to get the complete genome of HEV-118. Phylogenetic tree demonstrated the strain in China differed from that in Peru and Israel.4. HBoV(1) The sequences of HBoV-1were conserved and less variant.(2) The positive selectively site on VP1gene revealed co-existence of purifying and positive selection during the evolution of HBoV-1.