| BackgroundBats are Chiroptera in the mammals, the number of bat is only less than that of the rodents in mammals, there are about 180 groups of 17 families, about 960 kinds bats all over the world, of which China has 7 families 29 groups 107 kinds. Bats have two suborder: one is Megachiroptera, also named as Fox bats, there are one family 5 guoups 7 kinds in our country; The other is Microchiroptera, known as the bat, there are 6 families 24 groups 100 kinds in our country . Megachiroptera lived in the tropical and subtropical regions of the Eastern Hemisphere, they have more original physical structure and big body, only a fruit bat family. Microchiroptera distributed in the tropical and temperate regions of , the Eastern and Western Hemisphere,they are small including Rhinolophidae、Hipposideridae、Phyllostomatidae、Desmodontidae、Vespertilionidae etal more than 10 families.Bats, valuable for human beings, some kinds of which contacted closely human beings, are an important species that can coexist harmoniously with human beings. However, it is found that bats are hosts for various human viral pathogens. It is already known that 25 viraceaes can affect vertebrates, in which 10 viraceaes are related to bats, mainly are RNA virus. In the 16 RNA viraceaes affecting vetebrates, at least 9 can affect bats. So far, more that 80 kinds of virus have been isolated from bats. Some of them are source of infection for the serious diseases caught by both man and animals, so they pose a great threat to human health. In the past several years, the occurrence of new viral diseases and the recurrence of old viral diseases are related to bats, such as the Hendra virus disease in Australia in 1994, the Nipah Virus disease in Malaysia in 1998, and Timan virus disease.Arbovirus are important pathogens for human infections, there are five major arbovirus diseases: Japanese encephalitis (JE), dengue fever, hemorrhagic fever with renal syndrome, Xinjiang hemorrhagic fever and forest encephalitis in China. Among these diseases, Japanese encephalitis and dengue fever whose pathogens belong to Flaviviridae are mosquito-transmitted. Among Flaviviridae, West Nile fever is caused by West Nile virus which also spread by mosquitoes. Since 1999, outbreak and/or epidemic of West Nile fever hapended in many countries with high mortality rate in the Western Hemisphere where no case reported before, which has aroused global concern. Although China has no West Nile fever case reported until now, considering that the geographic latitude similar to that of American endemic area, coupled with increasing globalization, it is possible for epidemic of West Nile fever in China. Studies abroad have reported that bats can infect Japanese encephalitis virus, dengue virus and West Nile virus. In China, researchers have detected Japanese encephalitis virus and dengue vims antigens in bats, however no detection of virus nucleic acid was reported. In addition, there is no report about bats infected Japanese encephalitis virus and dengue virus in Hunan province.Therefore, to understand the situation of bats carrying human viral pathogens in different regions has the theoretical and practical significance in delving into the evolutionary relations of relevant virus in the human beings and animals and in scienctifically preventing and responding to the possible occurrence of diseases caught by both human beings and animals. So,We intend to investigate whether Bats can carry Japanese encephalitis virus, dengue virus and West Nile virus in part region of Hunan Province.ObjectiveTo carry out investigate and molecular epidemiological study of bats carrying Dengue virus, encephalitis B virus and West Nile virus in part region of Hunan Province. , and to inquire into the possibility of bats as natural storage hosts for these diseases and as sources of infection for relevant infectious diseases. Methods.1. The selection of regions for investigation:①Convenient for carrying out the investigation of bats and the collection of samples②Relevant diseases were once popular here.2. The collection of bats and making of samples2.1 The collection of batsCarrying out spot studies in regions for investigation and understanding the general information of the existence of bats、Habitat typesand the category of bats. we capture a small amount of bats by the corresponding method in accordance with the bat habitat characteristics for research. the mount of captured bat won’t influence activities and ecological changes of bat.2.2 The making of samplesThe brain tissues of the bats collected were taken out by sterile anatomy after the bats were determined into different kinds. The tissues were put into a freezed 1.5ml tube with RNAlater, transported at 4℃.The brain tissue samples were sterile ground into pulp with 5ml grinder which soaked with 0.1%DEPC water and sterilized by autoclave with DEPC water, then centrifugated 15min at 3000 r/min.The supernatant was testing sample, stored at minus 70℃.3. Virus detection and identification methods3.1 Dengue virus①We detect the bats brain tissue by Taqman real-time RT-PCR, our primers and probe have been confirmed and recognized as specificity for the dengue which cited from reference.②We inoculate supernatant of positive or Suspicious positive samples into C6/36 cells to Separate viruses, then daily observation and three blind- generation.③We inoculate culture solution of cell or supernatant of bat brain into the encephalocoele of BALB/c suckling mice and observe the symptom, if there are Suspicious positive symptom we will continue to blind passage three generation, we will identity it with Taqman real-time RT-PCR.when symptom emerging.3.2 West Nile virus①We detect the bats’ tissue by Taqman real-time RT-PCR, our primers and probe have been confirmed and recognized as specificity for the dengue which cited from reference.②We inoculate supernatant of positive or Suspicious positive samples into BHK-21 cells to Separate viruses, then daily observation and three blind- generation.③We inoculate culture solution of cell or supernatant of bat brain into the encephalocoele of BALB/c suckling mice and observe the symptom, if there are Suspicious positive symptom we will continuet to blind passage three generation, if symptom emerging we will identity it with Taqman real-time RT-PCR.3.3 Japanese encephalitis virus①As described in previous papers, associated with alignment of all known Japanese encephalitis virus, we design specific primer against E gene of Japanese encephalitis virus. Then we detect the tissue of bats with Taqman real-time RT-PCR.②We inoculate supernatant of positive or Suspicious positive samples into encephalocoele of BALB/c suckling mice, then daily observation . If there are Suspicious positive symptom we will continue to blind passage three generation, we will identity it with Taqman real-time RT-PCR when symptom emerging.③Cutting the gelatin、weighing、reclamatiing through depuration with kit, then linking to the pGEM-T vector、converting to the colibacillus DH5α、applying to LB flat plate、cultivating in AMP 5mlLB nutrient medium and gene sequencing, so we will get the sequence of DNA.④Compare sequences of the PCR products with known sequences of the genes of Japanese encephalitis virus in GeneBank using on line server BLAST: Align viral sequences by using Clustal W.4. Quality control①Tips, EP tubes, gloves and eta are disposable; During reverse transcription, all tips, EP tubes and grinding devices were treated by DEPC, in order to prevent contamination of RNase;②RNA extraction and RT-PCR were all proceeded on the Class II biosafety cabinet in a biological clean room; During procedure, wear respirator and head-cov -ering to prevent RNase from handler.③Positive、negative and blank controls were included in each run of the Taqman real-time RT-PCR assay、RT-PCR assay、animal experiment and cell experiment.Results1. General information of BatsWe collected bats from Hunan districts twice from 2007 April to 2007 September. 2 families, 8 species, 344 bats altogether were collected, in which 2 families were Rhinolophidae, and Vespertilionidae, and 8 species were 156 Rhinolophus affinses, 2 Rhinolophus macrotises, 20 Rhinolophus sinicuses, 1 Myotis rickettis, 2 Rhinolophus pearsonis, 2 Rhinolophus blythis, 152 Miniopterus schreibersis, 9 Rhinolophus ferrumequinums. The 344 bats collected for the research are all insectivorous bats, assayed by experts. The habitats of bats are mainly crevice and eaves of houses, deserted houses and caves for tourism. They contact closely human beings. It was found that local people had no habit of eating bats, but some hospitals taking bats and bats stools as traditional Chinese medicine to treat diseases. Meanwhile some local people even took bats as folk prescription medicines for polio, convulsion and mental diseases and caught bats without any protection.2. Bats carring Dengue virus①We obtained 344 brain tissues from 344 bats collected and tested 4 encephalitis virus positive samples with the method of Taqman Real-time RT-PCR. Ct values respectively are: 28.62, 31.36, 34.46, 34.78; Positive contrast Ct value is 24.80. Positive samples: Rhinolophus sinicus 1 case( carrying rate 50%), Rhinolophus affinis 3 cases( carrying rate 1.92%), other bats were not detected positive. The carring rate of Dengue virus of these bats was 1.16%,②We inoculate these four brain supernatant into C6/36 cells and found no cytopathic effect(CPE),after daily observation .there was still no CPE after three blind- generation.③We inoculated these four brain supernatant into the encephalocoele of BALB/c suckling mice, there were a little symptom after three days. But these suckling mice were all engulfed overnight because we found not in time. then thawed the supernatant again and Continued to inoculate but there found no symptom .3. Bats carrying West Nile virus①The experement did not test and isolate West Nile virus from 344 bats brain tissue samples with the methods of Taqman Real-time RT-PCR, animal vaccination and cell isolation.②There was still no positive result after inoculated the supernatant into BHK-21 cells and BALB/c suckling mice .4. Bats carring Japanese encephalitis virus①We found 7 Japanese encephalitis virus positive samples from 344 bats brain tissue samples with the method of RT-PCR. The earring rate of bats encephalitis B virus is 2.04%, 2 Rhinolophus affinis, the carrying rate of encephalitis B virus for Rhinolophus affinis 1.28%, 5 Miniopterus schreibersi, the carrying rate of Japanese encephalitis virus for Miniopterus schreibersi was 3.29%. Other species were not detected with Japanese encephalitis virus.②we vaccinated serum of 7 positive samples tested by RT-PCR on BALB/c baby rats for 1 -3 days. Most of them were attacked after 3 days, and the symptoms were resisting latex, convulsion, sideways, roaching back. All died after 8 days. The encephalitis B virus was isolated successfully.③The result display that the homology with known sequences of the genes of Japanese encephalitis virus ranges between 99%-100% after depuration and sequencing. Conclusion1. Although this study did not test and isolate West Nile virus from collected 344 bats brain tissues and hinted that revelant bats might not carry West Nile virus in this region, it was still not reasonable to exclude the possibility of bats carrying West Nile virus and as its host, considering the number of samples in this study was not big enough and that previous studies reported that there existed a certain relationship between bats and West Nile virus.2. positive samples of Dengue virus was detected from 344 bats brain tissues with the method of Taqman real-time RT-PCR.( The carrying rate was 1.16%); The baby rats were attacked after these 4 samples were vaccinated, however, because they were not timely found, the attacked baby rats were devoured by mother rates overnight. It was not successful to re-vaccinate. But it could still be decided that several positive samples had Dengue virus.3. 7 positive samples of Japanese encephalitis virus was detected from 344 bats brain tissues with the method of RT-PCR (The carrying rate was 2.04%). These 7 samples could cause regular attack and death to baby rats once vaccinated. Besides the result display that the homology with known sequences of the genes of Japanese encephalitis virus ranges between 99%～100% after depuration and sequencing. So we concluded that there did exist bats carrying Japanese encephalitis virus in the nature in our country. |
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