A Study On The Feature Of Norovirus Infection In Adults And The Comparison Between Rotavirus Infection And Norovirus Infection In Children In Ji’nan

BackgroundAcute gastroenteritis is one of the most important diseases which make a threat to public health. Researches during the past twenty years suggested that virus had replaced the bacteria to become the most important pathogen that could cause acute gastroenteritis, of which rotavirus and norovirus were the most common.Norovirus, also known as the Novak-like virus, is the first virus to be recognized to cause acute gastroenteritis. It can infect people at any age group. The high attack rate of norovirus is due to their high environmental stability, low infectious dose, the short-term host immunity and mutation and recombination of their genome. Norovirus infection can cause outbreak easily based on the sporadic cases. So it also was named "stomach flu". Although norovirus infection was reported as early as in1995in China, the data associated to the molecular epidemiology and clinical manifestation of norovirus infection is limited because of the lack of rapid and sensitive detection methods and the test of norovirus excluded from the routine test in hospital, which hindered the progress in prevention and treatment of norovirus infection.Rotavirus is believed to be the most important pathogen that causes acute gastroenteritis in children, especially under the age of5years old children. Children of this age group experienced at least one episode of rotavirus infection. Based on the previous reports from CDC of USA, rotavirus infection caused25million clinic visits,2million hospital admissions and about600000deaths annually worldwide in children. However, there was no specific drug in the light of rotavirus infection. So, development and application of high valent vaccine is the hope for controlling rotavirus infection. Norovirus is the second common pathogen to cause acute gastroenteritis in children under the age of5years old. Although at most200000 children died of norovirus infection annually and the clinical manifestation is lighter than that caused by rotavirus infection, there was no effective vaccine to be developed. Therefore norovirus infection can not be ignored. If the infections were identified in the clinical diagnosis and treatment, the spread and prevalence of rotavirus and norovirus infection would be avoided.This study is divided into two parts:the purpose of the first part is to study the molecular epidemiology and clinical feature of norovirus infection in adults in Ji’nan; the purpose of the second part is to study the molecular epidemiology feature of rotavirus and norovirus infection in children in Ji’nan and the comparison of the clinical feature between rotavirus and norovirus infection. The aim of the present study is to provide useful epidemiological and clinical data which may help for the development of vaccine and treatment. Part1The clinical and molecular epidemiology features of norovirus infection in adults with acute gastroenteritis in Ji’nanObjective:1. To study the incidence of norovirus, epidemiologic feature and clinical manifestation in adults with acute gastroenteritis in Ji’nan.2. To identify the presence of the recombinant virus strains in adults with norovirus infection in Ji’nan.Methods:1. From June2010to May2011,480fecal specimens and clinical data were collected from adult patients with acute gastroenteritis at the department of infectious disease, Shandong University Qilu Hospital. Samples were converted to suspensions and centrifuged. The supernatants were collected to store at-70℃until they were ready for viral RNA extraction.2. Viral RNA was extracted from the stored fecal supernatants. The extracted RNA was dissolved in RNase-free water and stored at-70℃until used.3. Extracted viral RNA was amplificated by RT-PCR. In order to identify the presence of recombinant virus strains, each of viral RNA was amplificated with two primers for different sections of genome.4. Amplification productions were purified and sequenced. All sequences were compared with norovirus sequences from NCBI GenBank database using BLAST program. Multiple sequence alignments were analyzed using Clustal X. Phylogenetic trees were generated using the neighbor-joining method with MEGA.5. The RNA from potential recombinant strains was amplificated with another primer covering the RNA polymerase and the capsid encoding region. Then, the amplification products were purified and sequenced. Simplot software was used to compare sequences to identify potential recombinants.Results:1. Of480fecal specimens,42samples were positive for norovirus, and the positive rate is8.75%. 2.7genotypes were observed:GI-1,GⅠ-4,GⅡ-3,GⅡ-4,GⅡ-6and GⅡ-13. Of which GⅡ-4(29/42,69.0%) was the most common genotype.3. The clinical manifestations were diarrhea (97.6%), abdominal pain (40.5%), fever (28.6%) and vomiting (16.7%).The frequency of diarrhea and vomiting was6.08(±1.42)episodes/day and2.61(±1.37)episodes/day, respectively.4. Norovirus infections were observed throughout the year. Higher activity of NoV infection was seen from September to December and lower positive rates were seen in June and July.5. Of42norovirus strains,3strains were recombinant virus strains:two strains divided into GⅡ-4cluster based on RNA polymerase sequence were classified into GⅡ-3cluster based on capsid sequence; One strain grouped into GⅡ-6cluster based on RNA polymerase sequence was classified into GⅡ-4cluster based on capsid sequence.Conclusions:Norovirus infections were an important cause of acute gastroenteritis in adults in Ji’nan. Infected cases were found throughout the year with peak from September to December. The most common clinical manifestation was diarrhea, abdominal pain, fever and vomiting. The viral gene was diversity and GⅡ-4strain was the dominant strain, which was consistent with the popular trend worldwide. Two recombinant types were seen:GⅡ-4polymerase/GⅡ-3capsid and GⅡ-6polymerase/GⅡ-4capsid. GⅡ-6polymerase/GⅡ-4capsid recombinant strain was observed for the first time, which should be paid more attention to and observed continuously avoiding large-scale outbreak. Part2The clinical and molecular epidemiology features of rotavirus and norovirus infections and the comparison between rotavirus and norovirus infection in children under the age of5years in Ji’nanObjective:1. To study the incidence of rotavirus, epidemiologic feature and clinical manifestation in children under the age of5years with acute gastroenteritis in Ji’nan.2. To study the incidence ofnorovirus, epidemiologic feature and clinical manifestation in children under the age of5years with acute gastroenteritis in Ji’nan.3. To study the comparison between rotavirus and norovirus infection in children under the age of5years in Ji’nan.Methods:1. A total of767fecal specimens were collected from children under5years of age (502outpatients and265inpatients) with acute diarrhea between February2011and January2012at Shandong University Qilu Hospital and Qilu Children’s Hospital in Ji’nan. Simultaneously, the clinical information from these children was collected from their parents who were asked to sign an informed consent. Samples were converted to suspensions and centrifuged. The supernatants were collected to store at-70℃until they were ready for viral RNA extraction.2. Viral RNA was extracted from the stored fecal supernatants. The extracted RNA was dissolved in RNase-free water and stored at-70℃until used.3. Stool specimens were tested for rotavirus by enzyme-linked immunosorbent assays (ELISA). Rotavirus positive specimens were further characterized for G and P type by RT-PCR with type-specific primers. Negative specimens for rotavirus tested by Elisa were selected at random (252samples,50%) and amplificated to confirm the presence of rotavirus.4. The PCR products were resolved on agarose gels, stained with ethidium bromide and visualized under UV light to determine the G and P type. 5. The viral RNA was amplificated using specific primers for norovirus. Amplification products were purified and sequenced. All sequences were compared with norovirus sequences from NCBI GenBank database using BLAST program. Multiple sequence alignments were analyzed using Clustal X. Phylogenetic trees were generated using the neighbor-joining method with MEGA.6. The clinical data collected from patients was analyzed using Chi-square and U test. The results were shown as proportion and the mean value with standard deviation (SD). For all analysis, statistical significance was defined as P values less than0.05.Results:1. Of767stool samples, the overall detection rates of rotavirus and norovirus were34.3%(263/767) and10.4%(80/767), respectively. The mixed infections were identified in8patients (8/767,1.0%).2.5serotypes and3genotypes were detected in263rotavirus strains. The most common serotype and genotype was G3(40.7%) and P[8](46.8%), respectively.3.80norovirus positive samples were genotyped for GⅡ, and no GⅠ strains were detected based on the capsid sequences. Three genotypes (GⅡ.3, GⅡ.4and GⅡ.6) were detected and GⅡ.4was the most prevalent genotype (61.2%). Phylogenetic analysis of the GⅡ.4sequences showed that18GⅡ.4strains belonged to GⅡ.42004-2006cluster and31GⅡ.4strains were divided into GⅡ.42006b cluster.4. The mean age of patients positive for rotavirus and norovirus were24.2(±14.0) months and19.9(±15.8) months, respectively, which was statistically significant.5. Rotavirus infections peaked in the months from November to January, while norovirus infections were observed to peak from September to November.6. The major clinical symptoms related to rotavirus and norovirus infections were acute diarrhea, vomiting and fever. The frequency of diarrhea and vomiting for rotavirus infection was5.08(±1.85) episodes/day and2.61(±1.35) episodes/day, respectively. The mean degree of fever in rotavirus infected patients was38.0(±0.65)℃. The frequency of diarrhea and vomiting for norovirus infection was4.99(±1.55) episodes/day and2.20(±1.47) episodes/day, respectively. The mean degree of fever in norovirus infected patients was37.8(±0.62)℃. There was no significant difference among the frequency of diarrhea and vomiting and the degree of fever by U test analysis.Conclusions:rotavirus and norovirus were the important pathogen in childhood diarrhea in Ji’nan. The most common genotype was G3P[8]and GⅡ-4.Rotavirus incidence was significantly higher than that of norovirus and the patients infected with rotavirus showed more severe clinical symptoms. Infections mainly occurred under2years of age. The mean age of patients infected with norovirus was younger than that of rotavirus. The common clinical symptoms were diarrhea, vomiting and fever. Infections were observed throughout the year and peaked in autumn and winter. The peak of norovirus infection appeared earlier than that of rotavirus. This study provided useful data for epidemiologic and clinical features of rotavirus and norovirus infections, which would provide scientific support for the development of effective vaccines to reduce the morbidity and mortality.