Molecular And Epidemiological Study On Viral Diarrhea Among Children In Guangzhou, China,2012through2013

Background:Acute gastroenteritis is one of the leading causes of childhood morbidity and mortality worldwide, with more than2million deaths in children every year from low-income countries. Most episodes of diarrhea were attributable to bacteria, viruses and parasites. With application of antibiotics and sanitary condition improvement, bacterial diarrhea has been effectively controlled; the viruses were identified as the major agents causing acute gastroenteritis. Virus infection has the characteristics of fast spread, acute onset and immunity un-sustained, can cause higher incidence rate, pandemic, even death.Human Rotavirus (HRV), human caliciviruses (HuCV), adenovirus (AdV), astrovirus (AstV) are known to be important etiologies of diarrhea. Group A rotavirus is the most common cause of severe diarrhea in infants and young children worldwide, Group A rotavirus diarrhea is currently the focus of prevention and control. With the development of research, NoV infection in acute diarrhea is considered more important. Recent studies indicated that NoV was the second most common cause of viral gastroenteritis and the prevalence of NoV infection was second only to HRV. It can infect all age groups, extremely cause outbreaks. In addition, AdV and AstV can cause diarrhea and should be given more attention. Although most cases of gastroenteritis are associated with infection by known pathogens, up to40%remain undiagnosed, thus the role of other infectious agents (especially viruses) in diarrhea should be given more attention.Human bocavirus (HBoV), a recently described newvirus species belonging to the Parvoviridae family, was identified as a human pathogen in September2005. Phylogenetic analysis showed that this virus was most closely related to bovine parvovirus and minute virus of canines. The virus was thus named human bocavirus.The genome has three open reading frames (ORFs). The first ORF encodes for a non-structural viral protein (NS1) of unknown function for HBoVs. The second ORF encodes for two major structural proteins (VP1and VP2) that comprise the viral capsid. The third ORF encodes for a non-structural nuclear phosphoprotein (NP1).HBoV1is predominantly found to be a respiratory pathogen. In addition, HBoV2and HBoV1have also been found mainly in stool samples. HBoV3and HBoV4are occasionally be detected, but the significance of this virus may be regarded as unknown.Human parechoviruses (HPeVs)-are single-stranded, positivesense RNA viruses belonging to the large family of Picornaviridae. HPeV possess a single-stranded RNA genome of approximately7300bases in length, and encodes a single polyprotein flanked by5’and3’untranslated regions (UTRs). The polyprotein is post-translationally cleaved into three structural viral proteins (VP1-VP3) and seven nonstructural proteins (2A-2C and3A-3D).HPeV were discovered in USA from children with diarrhea in1956. Since the first report of HPeV infection, the new genotypes of virus have been continuously reported. To date,16types of HPeV (HPeV1-16) have been identified. Many studies have been described HPeV detection in association with acute gastroenteritis in children especially in infant. In China, epidemiological surveillance of HPeV infection in children with diarrhea has been reported since2009. Little is known about this viral agent causing acute gastroenteritis in China, except for three studies of HPeV detection in acute gastroenteritis in infants and children less than5years old. It should be noted that HPeV7to16are a new genotypes that have been recently discovered. Thus, epidemiology and prevalence of these genotypes have not been fully established.HPeV are frequent reproducting in gastrointestinal tract, usually causing acute gastrointestinal and respiratory infections in young children. The associated spectrum of symptoms is similar to that shown by enteroviruses, Infections are enteric and often associated with mild gastrointestinal and respiratory symptoms, although severe diseases including sepsis, meningitis, myocarditis and flaccid paralysis have been described. Several studies showed that co-infection of HPeV and other enteric virus was frequent, and HRV was the predominant co-detected pathogen. The prevalence and distribution of HPeV in children with acute gastroenteritis may provide new insights into their epidemiology.The birth rate of China is the second rank in the world. Infectious diarrhea is a common disease of children. We sought to determine the prevalence of common viral etiologic agents (HRV, NoV, SaV, AdV and AstV) to evaluate the role of common viruses in childhood gastroenteritis in Guangzhou, China to analyze of their etiological role for the prevention and control of viral diarrhea in China. At the same time, we determined the prevalence of HBoV and HPeV to provide useful epidemiological data for future disease control and prevention in Guangzhou, China.Objectives:1. Epidemiological study on common diarrheal viruses was conducted in Guangzhou, China,2012-2013, to enrich material about diarrhea virus infection in Southern China and provide basic molecular biology and epidemiological background material for diarrheal prevention.2. The present study was also aimed to study the prevalence of human parechovirus in Guangzhou, China,2012-2013and to clarify the role of HBoV and HPeV in diarrhea and provide useful epidemiological data for future disease control and prevention.Methods:1. Sample collection:A total of322stool samples were collected from children under5years of age with diarrhea in the outpatient department of the third Affiliated Hospital of Southern Medical University from July2011to June2013. The demographic and epidemiological data were also collected.2. Detection methods:Group A rotavirus was screened by using the Rotavirus Enzyme-linked immunosorbent assay (ELISA) Diagnostic Kit according to the manufacturer’s instructions. Viral DNA and RNA were extracted manually Stool DNA Kit (Omega Bio-tek, USA) and TRIzol (Invitrogen, Carlsbad, CA) respectively. NoV, SaV, AstV and AdV were detected using PCR or RT-PCR. Samples testing were amplified by nested PCR for HBoV. For parechovirus genotyping, samples testing positive by the5’UTR were amplified by nested PCR using primers from the VP3/VP1junction region. The sequences of PCR products positive were compared with those of reference strains available in the GenBank database using BLAST server. Multiple sequence alignment was conducted using ClustalW and phylogenetic tree from nucleotide and deduced amino acid sequences of the partial gene using MEGA version5.0.3. Statistical methods:All the data were used Epidata3.1for double entry. All of the statistical analyses were conducted by SPSS13.0statistical software. A P-value of <0.05was considered significant.Results:1. The female to male gender ratio was0.84(150/178). The age of children with acute gastroenteritis ranged from6days to59months, with a mean age of28.8 months, and the difference in the age distributions of the boys and girls was not statistically significant (t=0.903, P=0.367).2.(1) A total of322stool specimens were collected from children under the age of5years with acute diarrhea between Jul2012and Jun2013. At least one viral agent was found in39.1%of children with gastroenteritis. The infection rate of group A rotavirus, norovirus, sapovirus, adenovirus, and astrovirus were25.3%,8.7%,1.4%,7.6%, and3.9%, respectively.(2) The detection rate of NoV was9.63%(31/322). GII4(25strains) was the most common norovirus genotype (85.5%), followed by GI.3(3strains), GII.6(2strains) and GII.5(1strain). SaV genotypes GI.l, GI.2and GII.2strains were respectively two, two and one of sapoviruses recovered. All14AstV strains belonged to genotype1. Among the25samples positive for AdV in this study,20(80%,20/25) belonged to serotypes41; there were four (16%,4/25) AdV7and one (4%,1/25) AdV5.(3) The majority of the five common viruses infected children were younger than two years of age. The incidence of gastroenteritis caused by group A rotavirus peaked between September and January. NoVs were observed throughout the year without obvious seasonal trends, but have one peak periods in "July in2012through March in2013". SaV were more frequently detected during the Sep-Oct and Mar-Apr. The highest detection rate of astrovirus infection was in Jun-Sep. AdV were detected almost all year around except February and December, with the peak incidence in August.(4) More than one viral agent was found4.97%(16/322) of the study population. HRV was the most common co-infecting pathogen. HRV combined with AdV accounted for the largest portion of coinfection (5/16,31.3%). Clinical data of the HRV-infected alone and co-infected subjects were compared. None of the comparisons were statically significant. 3. Among322fecal specimens collected from children with diarrhea younger than5years in China,31(9.63%) were positive for HBoV. The incidence of gastroenteritis caused by HBoV peaked between October and January. The majority of the infected children were younger than two years of age. Nucleotide sequence analysis revealed that the strains could be identified as HBoVl (54.8%,17/31) and HBoV2(45.2%,14/31). Co-infection with other enteric viruses was found in14(45.2%) patients, while co-infection with HRV was found most frequently (71.4%,9/14).4.(1) A total of322fecal samples that were collected from children outpatients in Guangzhou, China. This study incorporated42(13.04%,42/322) were HPeV-positive with the majority of the infected children being younger than two years of age. HPeV were detected almost all year around with the peak incidence in July (28.2%,11/39) and August (28.6%,8/28). Phylogenetic analysis of the VP1/VP3segments of reference HPeV strains and the strains studied showed that the strains could be identified as HPeV1(29strains), HPeV3(2strains), HPeV4(2strains), HPeV5(1strain) HPeV6(5strains), HPeV8(2strains) and HPeV14(1strain). HPeV1was the predominant genotype, and more than half of them clustered with the prototype Harris strain (HPeV1A).(2) Among of42positive samples, co-infection with other enteric viruses was found in21(50.0%) pediatric patients. Co-infection with HRV was found most frequently (31.0%,13/42). The differences in the detection rate of these five enteric viruses between HPeV-infected and uninfected children were not statistically significant. Clinical data of the HPeV-infected and non-infected subjects who were negative for any of the five viruses were compared. In addition, we also compared clinical data of any of these common viruses-positive children with or without HPeV infection. None of the comparisons were statically significant. Conclusions:1. Enteric viruses were confirmed as the most common cause of acute gastroenteritis among infants and children in Guangzhou, China. Rotavirus was the most important pathogen for viral gastroenteritis, but AdV, NoV, SaV and AstV also play a important role in children diarrhea. The seasonal distribution of the five common viruses was different, so further studies on viral diarrhea are necessary.2. The diversity in genotype of NoV, SaV, AstV and AdV was found. GII.4was the most common norovirus genotype in childhood gastroenteritis in Guangzhou, China,2012-2013. The AdV41genotype was predominant over other AdV. All astrovirus strains belonged to genotype1.3. The high frequency of human bocavirus detection was found, and HBoV1and HBoV2were the common genotype in childhood gastroenteritis in Guangzhou, China,2012-2013. Co-infection is common for HBoV. Rotavirus was the most common partner.But infection with HBoV does not significantly influence the severity of gastroenteritis.4. HPeV infections were very common in diarrheal children and the prevalence of HPeV infection was second only to rotavirus. HPeV1was the predominant pathogenic genotype in Guangzhou, China,2012-2013, and most identified HPeV1strains were clustered in the HPeV1A. HPeV14, a rarely reported genotype, was reported for the first time in children with acute gastroenteritis in China. Co-infection is common for HPeV. Rotavirus was the most common partner. The clinical profiles of children with HPeV positive alone were not significantly different from the cases without pathogens detected. The result indicates that infection with HPeV does not significantly influence the severity of gastroenteritis. The high frequency of HPeV detection was found, and should be given more attention.