| Background and objectiveBats are being increasingly recognized as reservoir hosts for zoonotic viruses which can cross species barriers to infect humans and other domestic and wild mammals.A large number of viruses have been isolated from or detected in bats, including at least 18 families 70 species of viruses. But most of these viruses have not been shown to be transmitted from bats to other animals or to cause human disease. Transmission from bats of viruses causing highly pathogenic disease has been demonstrated for rabies virus and related lyssaviruses, Nipah and Hendra viruses, and inferred for SARS-CoV-like virus of bats. Other viruses such as certain alphaviruses, flaviviruses, and bunyaviruses, may infect bats via arthropods, but it is not clear whether bats are important reservoir hosts for these viruses. Clearly, a great deal of additional research is needed to document the roles of bats of different species in the natural history of the many viruses for which these remarkable animalscan serve as hosts.In China, more than 120 species of bats are found. However, the data about bats was scare, especially about these questions:what virus do bats host? What is the relationship between virus and bats? Do they threaten human health? By now, several findings about bats carrying virus are achieved, some virus were isolated from bats, such as West Nile virus, Japanese virus, Kysanaur forest virus, SARS-CoV-like virus, rabies virus, astrovirus, adenovirus and Nipah virus. Viral gastroenteritis is an infection caused by a variety of viruses that result in vomiting or diarrhea. The main symptoms of viral gastroenteritis are watery diarrhea and vomiting. The affected person may also have headache, fever, and abdominal cramps. The condition is usually of acute onset, normally lasting 1-6 days, and is self-limiting. In general, the patient has short disease course and low death rate. Viral gastroenteritis is very common diseases in infants and young children, which is the leading cause of children death. Viruses causing gastroenteritis include rotavirus, norovirus, adenovirus and astrovirus. At least 50% of cases of gastroenteritis due to foodborne illness are caused by norovirus. Another 20% of cases, and the majority of severe cases in children, are due to rotavirus. Other significant viral agents include adenovirus and astrovirus.Viral gastroenteritis is mainly transmitted by the fecal-oral route. Viruses are transmitted directly from person to person and indirectly via contaminated fomites, water and food. In retrospect, outbreak of Nipah and Hendra viruses were also owing to the foodborne infection. They were believed to be result of bat virus contaminating water, following by a bat-to-pig (horse) spillover event. This was followed by massive animal transmission and eventually led to animal-to-human transmission, raising the outbreak in humans.Astrovirus, belonging to the family Astroviridae, was first discovered in 1975 using electron microscopes following an outbreak of diarrhoea in humans. They have been identified from human as well as a variety of animal species including sheep, pigs, cattle, mink, dogs, cats, chickens, turkeys and several marine mammals. Recently, two studies have shown that a high prevalence rate and high genetic diversity of astroviruses in insectivorous bats, and some bat astroviruses may be phylogenetically related to human astroviurses. However, the information on the ecology and evolution of bat astroviruses is still scarce. More extensive surveillance for astroviruses in bats of different species and in different geographic areas is needed.Recent studies have shown that a high prevalence rate and high genetic diversity of astroviruses in insectivorous bats, and some bat astroviruses may be phylogenetically related to human astroviurses. However, the information on the ecology and evolution of bat astroviruses is still scarce, and more extensive surveillance for astroviruses in bats of different species and in different geographic areas is neededThe original norovirus was called the "Norwalk virus," after an outbreak of intestinal flu at an elementary school in Norwalk, Ohio, in 1968. It is an RNA virus (taxonomic family Caliciviridae) that causes approximately 90% of epidemic non-bacterial outbreaks of gastroenteritis around the world. Norovirus gastroenteritis starts suddenly, transmit rapidly and spread widely. However, noroviruses (NoV) are genetically diverse. The virus outbreak in the same community during the same period may eventually diverse. Nowadays, there was not available vaccine to prevent getting infected with the viral gastroenteritis.Norovirus have a broad host range and cause a wide spectrum of diseases and lesions in their respective hosts. Complete sequencing of the capsid gene has allowed the classification of NoVs into five genogroups (GG).Human NoV strains are found in GGⅠ,ⅡandⅣ. Bovine NoVs fall in GGIII, the murine NoVs in GGV, the porcine NoVs in GGII.Epidemiological studies have repeatedly shown that NoVs are widespread and that infection is common in the human population as well as in the bovine, porcine and murine species. However, the epidemiology is not well understood and few studies have been carried out on animal Nov infections. A serological prevalence of 22.1% was found in laboratory mice in North America, making murine NoV the most prevalent virus infecting these animals. In the UK, NoVs were detected in 11% of the cases of bovine diarrhoea tested. However, the detection rate of porcine GGII NoV was low:0.35% in Japan and 2.0% in the Netherlands.The detection of NoVs in animal faeces (calves and pigs), with or without clinical signs of gastroenteritis, is frequent. Molecular analyses have shown that animal and human strains are closely related, especially porcine NoVs, which are included in the same gene group (GGII) as some human strains. A resent study showed that a high prevalence of antibodies against human NoVs was found in pigs in Venezuela, which strengthened the hypothesis that animals may act as ahuman NoV reservoir. Moreover, simultaneous presence of human and animal NoVs has been detected in shellfish. These observations raise concern about the risk of co-infection of humans with human and animal NoVs, resulting in possible recombination and emergence of new strains. The emergence of such recombinant has put humans and animals in close contact so increasing the risk for cross species transmission.Do bat host noroviruses? It has never been reported according to author's knowledge. As we know, NoVs have been identified from human as well as a variety of animal species, and they are highly genetic diversity. As the second largest order within mammal and widely distributed in the world, bats may carry these virus, eventually they may be the reservoir hosts for norovirusHence, to further study prevalence of astroviruses in bats of different species and different geographic areas, and to explore the relationship of bat astrovirus and human astrovirus, this study select the Guangdong and Hainan province to be study field. We we describe the prevalence and the phylogenetic characterization of astroviruses isolated from bats in three areas in Hunan and Hainan Provinces of Southern China, where were not involved in previous reports. Meanwhile, these samples were screened for norovirus, in the hope to explore whether bats carry these viruses.Methods1. Collection and treatment of bat samplesThe sampling of bats was carried out at four natural habitats in three regions in Hunan (Shaoyang and Yueyang) and Hainan (Haikou) provinces of Southern China between June 2007 and June 2008. Bat species identification was determined by Professor Wu Yi (bat expert, academy for life science, Guangzhou University). There were two ways of samples collection, including rectal swab samples and rectum specimens. The sample was taken at locations of bat natural habitats and placed immediately into a tube containing 300μ1 of RNAlater (Qiagen, Germany), then transported to the laboratory in a foam box with ice packs and then stored at -80℃until used.2. Virus detection and identification2.1 Astrovirus①Detection of astrovirus by SYBR Green real-time PCR According to previous papers, specific primers were designed targeted to the conserved region of norovirus RNA, which were used to screen the samples.②Detection of astrovirus by semi-nested PCRThe primers of Semi-Nested PCR were designed targeting the highly conserved region of the RdRp gene as previously described, containing four forward primers and one common reverse primer, which were used to screen the samples.③Sequences identificationPCR amplicons were purified with Agrose Gels DNA Purification Kit (TaKaRa), and then sequenced by TaKaRa Biotechnology (Dalian) CO., LTD.④Sequences analysisNucleotide sequences were compared to reported sequences in the GenBank using the basic local alignment search tool (BLAST) and aligned using the Clustal W. Sequence manipulation and identity calculation were performed using DNAstar software. Phylogenetic analysis was performed using Mega version 4.1. Phylogenetic tree was constructed by the Neighbor-joining method.2.2 Norovirus①Detection of NoV GGII by TaqMan real-time PCRAccording to previous papers, specific primers and TaqMan probe were designed targeted to the conserved region of norovirus RNA, which were used to screen the samples.②Detection of NoV by SYBR Green real-time PCRAs described in previous paper, two pairs of consensus primer were synthetized targeted to the conserved region of norovirus RNA, which were used to screen the samples, respectively.③Detection of NoV GGV by RT-PCRAccording to previous papers, a pair of consensus primer was designed targeted to the ORF2 region of norovirus, which were used to screen the samples.④Suspicious positive amplicons by RT-PCR were purified with Agrose Gels DNA Purification Kit, and then sequenced by TaKaRa Biotechnology CO., LTD. Suspicious positive amplicons by TaqMan real-time PCR were purified then sent to Biotechnology company for cloning and identifying,3. Quality control①Pipette tip, EP tube, frozen pipes, gloves and other supplies are disposable.②During RNA extraction and reverse transcription, pipette tip, EP tube and grinding devices were treated by DEPC, in order to prevent contamination of RNase.③All the experiments were processed on the biosafety cabinet in a biological clean room to prevent from pollution.④Positive, negative and blank controls were included in the process of experiments.Results1. General information of batsA total of 321 bats were collected at four natural habitats in three cities of Southern China. Nine bat species in two suborders were involved, including Rhinolophus blythi, Rhinolophus ferrumequinum, Rhinolophus macrotis, Miniopterus schreibersi, Scotophilus kuhlii, Myotis ricketti, Hipposideros pomona, Hipposideros larvatus, and Rousettus leschenaultia.2. Detection of astrovirus (Astv)2.1 DetectionA total of 29 of 321 (9.03%) samples were tested as positive. Three species of bats screened were observed to carry astroviruses, including two insectivorous bats, Miniopterus schreibersi and Scotophilus kuhlii, and one frugivorous bat, Rousettus leschenaultia.2.2 Phylogenetic characterization of bat astrovirusesPhylogenetical tree revealed that the bat astroviruses are not only highly divergent from previously described human and other animal astroviruses, but also remarkably diverse within them. Five bat astroviruses (in this study) were mostly related to mink astrovirus, ovine astrovirus and recently discovered human astroviruses VA. Eight astroviruses isolated from Miniopterus schreibersii in a single habitat concentrated in the same phylogenetic branch with highly amino acid identity (97%-100%)3. Detection of norovirus (NoV)TaqMan real-time PCR, SYBR Green real-time PCR and common RT-PCR were employed to screen samples for NoVGGⅠ-Ⅴ, respectively. However, the virus has not been found in these samples, though some samples were suspiciousConclusions1. A total of 29 of 321 (9.03%) samples were tested as positive for astrovirus. Positive bats come from megachiroptera and microchiroptera. These indicate that the viruses have adapted to the Chiroptera although the host susceptibility may be different in various species of bats.2. This study revealed that bat astroviruses were highly divese. Bat may transmit astroviruses to humans and other animals, or vice versa.3. The information on the ecology and evolution of bat astroviruses is still scarce, and more study about molecular characteristic of astroviruses in bats is needed.4. This study did not isolate norovirus from collected 321 bat samples. It implied that the relevant bats might not harbor norovirus in theses region. However, the gene of norovirus is such diverse that no single pair of primers could detect all five gene groups of norovirs. It was still not reasonable to exclude the possible of bats harboring norovirus, farther studies are need. |
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