Surveillance Of Respiratory Viral Pathogens In Selected Regions Of China

Acute respiratory tract infections (ARTI) are among the most common infectious diseases of human worldwide, causing influenza like illnesses. ARTI is the secondary cause of mortality in children and nearly 90% of pneumonias among this population are caused by viral pathogens. Respiratory viral infections are easy to transmit in population because of their special route for transmission. A series of newly discovered or recombination pathogens had been confirmed to be related with respiratory infections since 2001. Influenza-like illness (ILI) definitions have been used worldwide for influenza surveillance. However, ILI may be difficult to distinguish influenza infection based on the symptoms alone from symptomatic infections caused by other respiratory viruses. Therefore, ILI is usually used for surveillance of respiratory infections. According to ILI surveillance, the spectrum of viral pathogens in respiratory infections could be described, and the evolutionary pattern of virus may be further highlight and even clues for novel pathogens discovery would be offered.In this thesis, the most common respiratory viral pathogens, influenza A, B C(Flu-A, -B, -C), parainfluenza viruses 1-4 (PIV-1~IV), adenovirus(ADV), coronavirus-229E, -OC43 (CoV-229E, CoV-OC43), respiratory syncytial viruses-A, -B (RSV-A, -B), human metapneumovirus (MPV) and human bocavirus(BoV), were included in the surveillance for respiratory infectious diseases. The regions were Beijing, Tianjin, Chongqing, Shandong, Shanxi and Henan. In addition, clinical characteristics of viral infections, evolutionary dynamics of influenza and coronavirus were further studied based on the surveillance.The main results:1. Surveillance of respiratory viral pathogens in some regions of China During a two-year period of surveillance in six sentinel hospitals from Apr 2009 to Mar 2010, 3865 ILI cases (person-time) with monthly of 160 were included in this study. Surveillance data showed that ILI cases increased from September, lasting for 7 months, to March next year. The least detected positive rate for viral pathogen was 37.85% (1463/3865) and the most common virus was influenza with positive rate of 21.57%(834/3865). Following influenza were PIV(6.65%,257/3865), ADV(6.57%, 245/3865), RSV(5.46%, 211/3865), BoV(5.02%, 194/3865), MPV(2.25%, 87/3865) and CoV(1.81%,70/3865). Co-infections with more than two viruses, which accounted for 23.30%(341/1463) of all positive samples, were also observed (positive rate 8.82%,341/3865). Among co-infections, 75.07% (256/341) were two-virus, 19.94%(68/341) were triple-virus and 4.99%(17/341) were more than four viruses. Co-infections of Flu and ADV, ADV and PIV, BoV and RSV were the most common. MPV (80.46%, 70/87) and BoV (65.46%, 127/194) were more frequently detected with co-infections.Epidemiological results showed that the viral positive rates were higher in children(62.08%, 650/1047) than in the adults(29.05%, 700/2410)(P<0.00). Among children, the group of less than 5-years old was more frequently detected with viruses infection (69.62%, 550/790) and there was a remarkable decrease trend with age in this group(P<0.00). In addition, co-infection rates were much higher in children (26.36%, 276/1041) than that of the adults (2.45%, 59/2410)(P<0.01).Though there were yearly occurrences of respiratory viral infections, the seasonality was virus-dependent. For example, PIV, ADV and MPV reached their climax in September, RSV in October, BoV in the middle of November, Flu in December and CoV in the end of January.For ILI cases, the symptoms were cough(72%), expectoration(39%), sore throat(35%), runny nose(30%), headache(22%), hypodynamia(18%), muscular soreness(14%), diarrhea(10%), dyspnea (6%), chest pain (5%) and bellyache(3%). However, in viral positive cases, cough, expectoration, and runny nose were more common compared with negative cases among which runny nose, sore throat and cough were more frequently seen.Associated factors, which may affect the detection of viruses including age, disease histories, preclinical treatment of diseases and duration from onset to visit doctor, were studied and positive correlations were observed in univariate analysis. Opposite to age in which increase with decrease the detection rates(OR=0.97, 95% CI 0.97-0.98), disease histories, preclinical of diseases and duration from onset to visit doctor may lower the detection rates. However, only age and duration from onset to visit doctor displayed significantly affecting the detection rates when adjusted by other factors. And, the factors mentioned above may differ by infection type(single and coninfection), population (adults and children) and virus. 2. Seroprevalence and genetic characteristics of swine influenza viruses in Chinese swine population: a pooled data analysis.A literature review and data pooling analysis were made on the antibodies prevalence against five influenza viruses subtypes in pigs in China for the last 10 years. The grand average of seropositive rates of subtypes H1, H3, H5, H7 and H9 were calculated as 32.55% (3402/10451), 28.60%(2900/10139), 1.20%(77/6392), 0%(0/1815) and 2.15% (86/3944), respectively. Moreever, there was a higher level of antibodies against H1 than against H3(P<0.01). There were large geographical variations in seroprevalence of subtypes -H1, with South and East China as the top regions with high seroprevalence in pigs. In addition, the region with high densities of pigs raising had higher seroprevalence than that of the regions with median and low densities (P=0.02, P=0.04).BLAST analysis of genetic sequences revealed that genome segments with moderate homology to the 2009 pandemic influenza A (H1N1)(pH1N1) virus were present among swine influenza viruses isolated in China, especially in South and East China.3. Influenza virologic surveillance during Apr 2009- Mar 2011 in some regions of ChinaIn order to cope with ourbreaks of pH1N1, virologic surveillance for influenza was carried out during 2009-2011 in Beijing, Tianjin, Chonqging, Shandong, Henan and Shanxi. Based on respiratory viral surveillance, two more sentinel hospitals were added in this svrveillance and 6143 ILI cases (person-time) with monthly 255 cases (person-time) were included. ILI cases increased from August to March next year during 2009-2010 influenza season. However, in 2010-2011 season, ILI cases increased from November to January next year.One thousand six hundred and forty five cases were diagnosed (26.78%,1645/6143) positive for influenza, in which pH1N1, seasonal H3N2(sH3N2), seasonal H1N1(sH1N1), untyped and influenza B accounted for 31.98%(526/1645), 41.76%(687/1645), 1.88%(31/1645), 3.22%(53/1645) and 21.15%(348/1645), respectively.Different epidemic pattern was observed between 2009-2010 and 2010-2011 influenza season in this surveillance. Firstly, in 2009-2010 influenza season, the highest detected rate was 52.84%, obviously greater than that of the 2010-2011 season (24.12%). Secondly, in the 2009-2010 season, influenza viruses composed of sH3N2, pH1N1, influenza B and sH1N1. However, in 2010-2011 season, influenzas were mainly sH3N2, pH1N1, influenza B, and sH1N1 was less detected. Thirdly, in the 2009-2010 influenza season, the order of subtype scheduled as sH1N1, sH3N2, pH1N1 and influenza B and this pattern was changed with sH3N2, pH1N1 and influenza B in 2010-2011 season. Finally, no obvious changes of sH3N2 intensity in both seasons were observed though the epidemic strength of pH1N1 and sH1N1 lowered very much. In addition, no oseltamivir resistance mutations were observed according to drug resistance surveillance.Epidemic data showed that the infections of the males were more frequently seen with influenza A than with influenza B. The age distribution was different among (sub)types of influenza, in which pH1N1 with the highest age specific rate in group of 10-14 years old, sH3N2 in 30-39 age group, sH1N1 in elders than 70 years and B in 5-9 age group.For influenza cases, the symptoms were cough(76%), sore throat(47%), expectoration(38%), runny nose(35%), headache(26%), hypodynamia(18%), muscular soreness(12%), diarrhea(8%), chest pain (5%), dyspnea (4%) and bellyache (2%). Moreover, a different list of symptoms was observed among children and adults. For example, symptoms in adults were mainly cough, sore throat, runny nose and headache, however, in children the mainly symptoms were cough, expectoration, diarrhea and runny nose.Factors analysis showed that age, preclinical treatments were associated with rates of influenza detection what was (sub)type dependent. Compared with the influenza negative cases, the risk for positive detection of pH1N1 and sH3N2 increased without preclinical treatments with odds ratio of 2.5 and 1.7, respectively. However, to influenza B, preclinical treatment decreased the detection rates. In adults, the detection rate was positively associated with age for seasonal influenza (sH1N1 and sH3N2), yet, in children, age increased the detection of novel influenza (pH1N1).4. Evolutionary pattern of seasonal H3N2 during pandemic of H1N1 There was no positive selection pressure (dN/dS <1) for seasonal H3N2 during the pandemic of influenza A/H1N1, 2009. Higher dN/dS ratios for PB1-F2, HA, NA, M2 and NS genes existed, which imply higher evolutionary rate in these gene segments. Phylogenetic analysis showed that NA gene had the highest evolutionary rate with substitution rate of 4.30×10-3/nt/year. The most likely ancestor of influenza in this study may come from seasonal influenza strains in 2007. Among the eight segments, PB1 gene originated from strains in Mar 2007 and NA from strains in Nov 2007. Recombination existed in PB1 gene and the region between 40-821bp might from other influenza strains.5. Molecular study of coronavirus in some regions of China Among coronavirus infections, CoV-229E and CoV-OC43 were the main strains. Phylogenetic analysis showed that CoV-229E was in the main branch of Chinese strain reported in 2009 but closer to strains reported by Australia, USA and Germany. However, CoV-OC43 was closely to strains reported by Belgium in 2003 and France in 2006. No positive selection pressure was observed in CoV-229E and CoV-OC43 when evaluated by dN/dS. Two polymorphism sites with nonsynonymous nucleotide substitutions located at 1681(A561S) and 2692(Y898H) in CoV-229E and three such sites in CoV-OC43 located at 3073(D1024H), 3085(E1028Q) and 3202(S1064A). Deduced amino acid sequence showed that there was a 16aa and a 17aa signal peptide in CoV-229E and CoV-OC43, respectively. Homology protein modeling of the receptor binding domain(RBD) was success for CoV-229E and CoV-OC43. Successfully docking of CoV-229E with angiotensin-convertion enzyme-2(ACE-2) was modeled, however, no docking of CoV-OC43 with ACE-2 was observed and this difference partial explained the difference of the infection caused by CoV-229E and CoV-OC43.Conclusions:1.The common respiratory viral pathogens spectrum were influenza virus, parainfluenza virus, adenovirus, respiratory syncytial viruses, human bocavirus, human metapneumovirus, and coronavirus. Co-infections with more than two viruses were frequently seen in respiratory viral infections and two-virus infection was the major of co-infections. Flu and ADV, ADV and PIV, BoV and RSV were the most common mix infections, and MPV and BoV had the high proportion of combining infection with other viruses. An obvious age specific distribution pattern existed in respiratory infections. The seasonalities of virus infection as following: PIV, ADV and MPV reached their climax in September, RSV in October, BoV in the middle of November, Flu in December and CoV in the end of January. Cough, expectoration, and runny nose were the most common symptoms. Age and duration from onset to visit doctor may affect the detection of respiratory viral infections.2. H1 and H3 were the main serotypes in pig in China during 1999-2007 and a large geographical variation in seroprevalence of subtypes -H1 existed in South and East China, which related with densities of pig-raising. Genetic sequences blast revealed that swine influenza virus isolated in China were moderate homologous to the pH1N1, especially those isolated in South and East China.3. Virological surveillance showed the major (sub)type of influenza were seasonal H3N2, pH1N1 and influenza B during Apr,2009 to Mar, 2011. The pandemic of H1N1 influenza in 2009 affected the epidemic influenza, in which the intensity, the duration of epidemic, and the peak of (sub)type were changed. The clinical symptoms of influenza were (sub)type dependent in population. Age and preclinical treatment may determine the detection of influenza.4. Stochastic mutations without selection pressure may be the evolutionary manner of seasonal influenza H3N2 during pandemic of H1N1 in 2009. Among the segments, NA had the highest evolutionary rate. The most likely ancestor of influenza in this study may come from seasonal influenza strains in 2007 and PB1 gene might recombine whit other influenza strains.5. The major strains of coronavirus were CoV-229E and CoV-OC43 and stochastic mutations without selection pressure was the source of mutations. Two polymorphism sites with nonsynonymous nucleotide substitutions located at 1681(A561S) and 2692(Y898H) in CoV-229E and three such sites in CoV-OC43 located at 3073(D1024H), 3085(E1028Q) and 3202(S1064A). Successfully docking of CoV-229E receptor binding domain with angiotensin-convertion enzyme-2(ACE-2) may partial explain the difference of the infection caused by CoV-229E and CoV-OC43.