Molecular Epidemiology Characteristics Of Caliciviruses In Patients With Diarrhea In Shenzhen, 2009

Background and ObjectiveNoV was first isolated from the patient feces in an outbreak of acute diarrhea in 1968, which occured in Norwalk City, the United States. And it was named as Norwalk virus according to the discovering place. Because the virus cannot be carried on cell culture, cannot build animal models, also did not have sensitive diagnostic technique, so in a long time (about 20 years), it can only be detected by using electron microscopy in small-scale feces samples. Initially, this kind of viruses were named and classified according to the forms under electron microscopy and discovering place. Along with the increase of virus types, this method was found to cause confusion easily and became obvious increasingly. At present, the virus classifications and naming mainly based on the morphological, antigen characteristics, the host range and clinical symptoms caused by virus, especially the viral genome sequence, structure and the results of genetics evolutionary analysis. Followed suggestions given by calicivirus study group in 1998, and the approval of the international committee on taxonomy of viruses(ICTV), Calicivirus is divided into four genera:(1) Lagovirus:rabbit hemorrhagic virus; (2) Norovirus(NoV); (3) Sapovirus (SaV); (4) Vesivirus:swine vesicular exanthema virus. Among them Lagovirus and Vesivirus infected animals; And NoV and SaV mainly infect human, both were collectively named as human Calicivirus (HuCV).In the early 1990s, Norwalk virus were successfully cloned and sequencing analysis, which greatly promoted the calicivirus' reserch, and along with the polymerase chain reaction(PCR) technology, The research on NoV became more conveniently, and the harm and significance caused by acute gastroenteritis were fully understanded. NoV was considered as one of the important pathogens causing outbreaks and sporadic acute gastroenteritis (AGE). It can spread all over the world, can infect all age groups, extremely cause outbreaks, become the hardly controlled gastroenteritis disease, which can affect significantly the public health. NoV have some common features:26-35nm in diameter, no coated, a rough surface, spherical, and 20 symmetry surface body; It can be isolated from the species of acute gastroenteritis patients, but could not be cultured in the cells or tissue, and could not build animal models. Genome is a non-package single strands RNA viruses, RNA span is 7.7 KB, including 3 open reading frames(ORFs):5100bp ORF1 gene full-length, located in the genome of 5-5104nt, encoding the polymerase unstructured protein which contains conservative sequence, this unstructured protein contains p48, NTPase, p22, VPG,3c-like protein and polymerase et al. six small proteins; ORF2 gene 1632bp, located in the genome stretches between 5085-6707tn, coding the capsid proteins VP1, whose molecular weight is about 56KD; ORF3 gene 807bp, located in the genome stretches between 6707-7513nt, coding a tiny structure protein VP2 about 22.5 KD.NoV has highly genetic diversity, which is divided into five genetic groups. Among them GⅠ, GⅡand GIV infect humans, GⅢand GV infect cattles and rats, respectively. GⅠand GⅡcan be further divided into 8 and 17 genotypes, GⅢis divided into two genotypes, in addition, the new genetic type is defined as GⅡ.b genotype.SaV was first detected in 1976, which belonged to HuCV, also cause outbreak and sporadic acute gastroenteritis, spread extensively worldwide, and can infect all the age groups. Because SaV infections were less serious than the NoV infections, so SaV was not caused more attention and research than NoV. SaV is a single strand RNA virus with no parcel, its genome length is about 7.3～7.5 KB, including 3 open reading frames(ORFs). ORF1 encodes structure protein (capsid proteins) and unstructured protein (RNA dependent RNA polymerase, RdRp); ORF2 encodes a basic small protein; ORF3 and ORF1 overlaps, which only exists in GⅠ, GⅣand GⅤgenotypes, encoding an unknown functional protein.According to the genetic diversity of RdRp and capsid proteins, SaV can be further divided into five genetic groups. Among of them, GI, GⅡ, GIV and GV genotypes mainly infect humans, and GⅢgenotype mainly affects pigs.Viral gastroenteritis is one of the high-risk diseases in the countries around the world, especially in developing countries. Because the number of children died to the gastroenteritis is higher than the developed countries. With the development of research, HuCV, especially NoV infection in acute non-bacterial diarrhea is considered more and more important. NoV infections are common in China, and the advantage strain is not always the same in different places, but mainly GⅡ.4 genotype. To identify and analyze the variation and changes about NoV can enrich NoV research material, which is significant to further understand NoV evolution.Although the study on SaV is not very deeply at home and abroad, but in recent years, data showed that the disease outbreaks caused by SaV is tend to increase. Therefore, we do research about the prevalence and type of HuCV infection in Shenzhen 2009, in order to understand the local genotype of NoV and SaV, and discuss the advantage strains.Analyzing HuCV popular features in shenzhen area can enrich diarrhea virus infection metrial in the South of China, and provide basic molecular biology and epidemiological background material for diarrhea infection.Methods1. There are several sentinel surveillance hospitals:Shenzhen Longgang Center hospital, Peking University shenzhen hospital, Longhua People's hospital and Xixiang people's hospital. Fecal specimens were collected from non-bacterial dirrhea patients by monitoring program in 2009. Each fecal sample was 5～10g or 5～10ml. The definition of viral diarrhea is three times/d, watery, egg pattern or thin paste sample, the conventional microscopy is WBC< 15, RBC=0, and to eliminate the common infection such as bacteria and parasites.2. RNA was extracted from the fecal suspension, all specimens were detected for NoV and SaV by amplifying the polymerase and capsid junction using primers of GISKF/GISKR,COG2F/GIISKR and SLV5317/SLV5749, The PCR products were purified, sequenced,comparing sequences of the all PCR products in GenBank using on line server BLAST and the sequences aligned viral sequences by using Clustal W,employing MEGA 4.1 program package to construct the phylogenetic trees.3. Using statistics software to analyse the data from the patient, to get the epidemiological data about HuCV infection in Shenzhen.Result1. HuCV infection can occur throughout the year in shenzhen, NoV infection predominantly occured in May to September, SaV infection mainly occured in Spring and Autumn; NoV and SaV infection in different months have differences, with statistical significance. The infection rates of NoV and SaV between different sexes and age groups are no significant differences.2.852 fecal specimens were collected from non-bacterial diarrhea patients in Shenzhen, and the positive rate of NoV GI, NoV GⅡand SaV genotype was 7.86% (67/852),0.35%(3/852) and 1.88%(16/852), respectively.32 random sequenced strains belonged to NoV GⅡ, in which 22 strains were GⅡ.4,6 strains were GⅡ.b,2 strains were GIL 12, and the other two strains was GⅡ.2 and GIL 14. Three NoV GⅠstrains belonged to GⅠ.1,GⅠ.3 and GⅠ.4.3. Among the 16 SaV strains, eight strains belonged to the G1 genotype (3 GⅠ.1 strains and 5 GⅠ.2 strains),7 belonged to GIV genotype, and the last one belonged to GⅡ.1 strain.4. JB030920397 and JB030920633 can't be returned to the existing NoV GII.3 subtypes a-c, so they are the newly discovered GⅡ.3 subtypes. SaV GIV was first reported genotype in China.ConclusionHuCV infection can occur throughout the year, but NoV infection mainly occurred in Summer, however SaV infection in spring and autumn; NoV and SaV infection between different months were statistically significant, but at different genders and ages, there were no statistical significance;There exist NoV GⅠ, GⅡand SaV infections in Shenzhen. NoV GⅡ.4 is still the advantage strains. We also found two new NoV GⅡ.3 subtypes(JB030920397 and JB030920633).SaV GI is the advantage strains of SaV infection. And we have detected SaV GIV for the first time in China, which is different from the genotypes detected by Chen Dongmei et al. These show that there are different SaV genotypes in China.