A Molecular Epidemiological Survey Of Bat Coronaviruses

Background and objectiveBats as a continuing source of emerging infections in humans attract more and more attention after SARS epidemic.1. Bat biology and ecologyThe bats represent the second largest order Chiroptera within the mammals. Order Chiroptera has about 960 species of bats, 180 genuses, 17 families. There are two suborders within Chiroptera, the Megachiroptera and Microchiroptera. Pteropodidae (flying foxes) is the only family within Megachiroptera, there are 7 species, 5 genuses, 1 family in China. Microchiroptera has 16 families all over the world, while 100 species, 24 genuses, 6 families in China.Except the Polar Regions and some oceanic islands, bats are found in all continents, including high latitude regions, deserts and isolated islands. However, most species of bats exist in tropical and subtropical regions. Bats with diverse biology and ecology are relatively longevous, and have powerful flying ability; even some species have habit of long-term migration.Humans have a lot of opportunities to contact with bats directly and indirectly. Bats can roost under the eaves and wall's cracks of houses; bats might fly into house while predating; bats can roost in the cave located in tourist sites; in Chinese medicine, the stools of horseshoe bats are believed to cure eyes diseases. In Guangdong province, fruit bats are viewed as a delicious and tonic dish; In Hunan province, some people use bats to treat mental diseases.2. Bats and coronavirusesThe coronaviruses have become the focus of research since the 2003 global epidemic of SARS caused by the SARS-CoV. After studies indicating that SARS was a possible zoonosis, researchers from HKSAR and mainland of China detected bat-SARS coronaviruses from horseshoe bats (Rhinolophus spp.) respectively, which were 88% nucleotide identities with SARS coronavirus, moreover highly serological prevalence among a group of Chinese horseshoe bats. Therefore, it is reasonable to consider that bats might be the natural reservoir of coronaviruses. After serial surveillances of bats, researchers found that bat coronaviruses are quite diverse, until now a lot of novel coronaviruses, belonging to group 1 or group 2, were discovered from bats. However, genome sequence comparison of the spike(S) genes from bat SARS-like coronavirus and civet SARS-like coronavirus revealed only 64% genetic homology, suggesting that the evolutionary pathway of SARS-CoV remains to be fully described. Additionally, genetic analysis indicated that there are still a big number of coronaviruses undiscovered.Given the high biodiversity of bats, along with significant population size, broad geographical distribution, and the ability to migrate and along with the detection of many emerging viruses, it is reasonable to consider that bats may contain the direct progenitor of SARS-CoV. Therefore, to explore the natural distribution of the virus in bat populations and also to understand the possible role of bats in the ecologies of viruses, we conducted a molecular epidemiological survey about coronaviruses and rabies viruses in some regions of Hainan, Hunan and Guangdong provinces. Methods1. Collection and treatment of bat samplesFrom May to April 2007, 468 bats from 12 species (belonging 4 families) were captured and sampled from their natural habitats in some regions of 3 provinces. Bat identification was determined by Prof. Wu Yi (Bats expert, academy for life science, Guangzhou University). Abandon houses, wall's cracks and eaves of houses, or natural damp caves are common sampling roosts of bats.①Treatment of sera. Obtain blood from hind limb vein and heart, still at 4℃for 5h, centrifuge 3000r/min for 10min, transform supernatant to another new and sterile EP tube, label each specimens properly, transport to lab at 4℃, test immediately or store them at -70℃for long-term storage.②Treatment of rectum specimens. Bats were anatomized sterilely. Remove recta, store immediately in RNAlater, transport to lab at 4℃, test immediately or store it at -70℃for long-term storage.2. Detection of bat coronaviruses antibodies in sera by SPA-ELISA①Based on the property that Staphylococcus protein A (SPA) can bind with Fc fragment of IgG, a novel SPA-ELISA array was established to detect coronavirus antibodies in bat sera;②Employ SPA-ELISA array to test coronavirus IgG in serum specimens collected from some regions of Hainan, Guangdong and Hunan provinces.3. Detection of bat coronaviruses in recta by RT-PCR①As described in previous papers, associated with alignment of all known coronaviruses, a pair of consensus primers designed targeted to the conserved region of coronavirus RNA-dependent RNA polymerase (RdRp) sequences was used to screen the RNA samples;②Extract RNA and detect coronaviruses by RT-PCR established.4. Sequence and genetic analysis①Sequence RT-PCR products;②Compare sequences of the PCR products with known sequences of the RdRp genes of coronaviruses in GeneBank using on line server BLAST;③Align viral sequences by using Clustal W;④Employ MEGA 4 program package to construct the phylogenetic trees by using the neighbor-joining (NJ) method with 1000 bootstrap replicates.5. Quality control①Tips, EP tubes, gloves and eta are disposable;②During reverse transcription, all tips, EP tubes and grinding devices were treated by DEPC, in order to prevent contamination of RNase;③RNA extraction and RT-PCR were all proceeded on the Class II biosafety cabinet in a biological clean room;④During procedure, wear respirator and head-covering to prevent RNase from handler;⑤Positive and negative controls were included in each run of the RT-PCR assay, and no false-positive result was observed in the negative-control reactions.Results1. Sample collectionFrom May to April 2007,468 bats from 12 species (belonging to 4 families) were captured and sampled from their natural habitats in some regions of 3 provinces (i.e. Hainan, Guangdong and Hunan provinces). There were 274 serum specimens, 468 brain specimens, 468 rectum specimens respectively.2. Detection of bat coronaviruses antibodies①SPA-ELISA established is sensitive and convenient, and can be employed in the field, concluded through color changing without other devices;②36 serum specimens were positive for coronavirus antibodies by SPA-ELISA, including 4 species (i.e. common long bent wing bat 20.45%, Chinese horseshoe bat 11.11 %, intermediate horseshoe bat 9.26%, fruit bat 3.64%).3. Detection of bat coronaviruses13 rectum specimens were positive for coronaviruses by RT-PCR, all positive specimens collected from two regions in Hunan province. Among positive specimens, 4 were screened from common long bent wing bats with positive rate 2.63% (4/152); 6 from intermediate horseshoe bats with positive rate 3.85%(6/156); 2 from Chinese horseshoe bat with positive rate 6.90%(2/29); and 1 from blyth's horseshoe bat with positive rate 16.67%(l/6).4. Sequence and genetic analysisGenetic analysis indicated that coronaviruses screened from common long bent wing bat were bat-CoV HKU7 and bat-CoV 1 A; coronaviruses from intermediate horseshoe bat and Chinese horseshoe bat were bat-CoV HKU2 and bat-SARS-CoV; bat-CoV HKU7, bat-CoV 1A and bat-CoV HKU2 belonged to group 1, while bat-SARS-CoV belonged to group 2.Conclusion①Build a SPA-ELISA assay for detection of coronavirus antibodies in bat sera; ②Bats in some regions of Hunan province can infect coronaviruses, with coronavirus antibodies positive rate 13.14% (36/274) and coronavirus positive rate 2.78% (13/468) ;③Coronaviruses in this survey were homological to those previously described, but screened from different species; it is first time to screen bat-CoV HKU2 from intermediate horseshoe bat, bat-CoV HKU7 from common long bent wing bat, and bat-SARS-CoV from intermediate horseshoe bat and blyth's horseshoe bat;④Different bat species had different coronaviruses, while same bat species sampled from different locations had coronaviruses which were highly nucleotide homological;⑤Bat coronaviruses in this survey were diverse, nevertheless, the direct progenitor of SARS-CoV was still not found.