Changchun Tu

Virome Profiling of Bats from Myanmar by Metagenomic Analysis of Tissue Samples Reveals More Novel Mammalian Viruses

Bats are reservoir animals harboring many important pathogenic viruses and with the capability of transmitting these to humans and other animals. To establish an effective surveillance to monitor transboundary spread of bat viruses between Myanmar and China, complete organs from the thorax and abdomen from 853 bats of six species from two Myanmar counties close to Yunnan province, China, were collected and tested for their virome through metagenomics by Solexa sequencing and bioinformatic analysis. In total, 3,742,314 reads of 114 bases were generated, and over 86% were assembled into 1,649,512 contigs with an average length of 114 bp, of which 26,698 (2%) contigs were recognizable viral sequences belonging to 24 viral families. Of the viral contigs 45% (12,086/26,698) were related to vertebrate viruses, 28% (7,443/26,698) to insect viruses, 27% (7,074/26,698) to phages and 95 contigs to plant viruses. The metagenomic results were confirmed by PCR of selected viruses in all bat samples followed by phylogenetic analysis, which has led to the discovery of some novel bat viruses of the genera Mamastrovirus, Bocavirus, Circovirus, Iflavirus and Orthohepadnavirus and to their prevalence rates in two bat species. In conclusion, the present study aims to present the bat virome in Myanmar, and the results obtained further expand the spectrum of viruses harbored by bats.

Identification of Diverse Alphacoronaviruses and Genomic Characterization of a Novel Severe Acute Respiratory Syndrome-Like Coronavirus from Bats in China

ABSTRACT Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. IMPORTANCE Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province.

Characterization of a novel G3P[3] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses

ABSTRACT Bats are considered important animal reservoirs for many viruses pathogenic to humans. An approach based on viral metagenomics was used to study gut specimens from 78 insectivorous bats in Yunnan Province, China. Seventy-four reads were found to be related to group A rotavirus (RVA). Further reverse transcription-PCR screening and viral isolation on cell cultures confirmed the presence of a novel RVA strain, named RVA/Bat-tc/MSLH14/2012/G3P[3], in 1 (6%) of 16 lesser horseshoe bats. Full genomic sequencing analyses showed that MSLH14 possessed the genotype constellation G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is akin to human and animal rotaviruses believed to be of feline/canine origin. Phylogenetic analysis indicated that VP7 was most closely related to bovine RVA strains from India, whereas VP4 was most closely related to an unusual human RVA strain, CMH222, with animal characteristics isolated in Thailand. The remaining gene segments were only distantly related to a range of animal RVA strains, most of which are believed to be related to feline/canine RVAs. Experimental infection showed that bat RVA strain MSLH14 was highly pathogenic to suckling mice, causing 100% mortality when they were inoculated orally with a titer as low as 5 × 102 50% tissue culture infective doses. As this virus is not closely related to any known RVA strain, it is tempting to speculate that it is a true bat RVA strain rather than a virus transmitted between species. However, further screening of bat populations, preferably juvenile animals, will be crucial in determining whether or not this virus is widely distributed in the bat population.

Hepatitis virus in long-fingered bats, Myanmar.

During an analysis of the virome of bats from Myanmar, a large number of reads were annotated to orthohepadnaviruses. We present the full genome sequence and a morphological analysis of an orthohepadnavirus circulating in bats. This virus is substantially different from currently known members of the genus Orthohepadnavirus and represents a new species.

Prevalence and genetic characterization of Toxoplasma gondii infection in bats in southern China

Toxoplasma gondii can infect a wide variety of warm-blooded animals, including bats. Limited information on T. gondii infection in bats is available in China. The objective of the present study was to determine prevalence and genetic diversity of T. gondii infection in bats in southern China. A total of 608 bats representing 12 species, including 120 Aselliscus stoliczkanus, 59 Myotis chinensis, 11 Miniopterus schreibersii, 53 Rhinolophus affinis, 32 Rhinolophus pusillus, 81 Hipposideros armiger, 28 Hipposideros fulvus, 32 Cynopterus brachyotis, 14 Cynopterus sphinx, 45 Eonycteris spelaea, 109 Hipposideros larvatus, and 24 Taphozous melanopogon, were collected from Yunnan and Guangxi provinces, southern China. They were examined for the presence of T. gondii DNA by amplification of the B1 gene using a nested PCR, and the positive samples were genotyped at 11 genetic loci (SAG1, 5- and 3-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Fifty-nine (9.7%) of these bats were detected positive by PCR but only five of these positive DNA samples were completely typed at all loci; of which 4 samples, 2 from A. stoliczkanus, and 2 from H. larvatus, belonged to ToxoDB Genotype #10, and the other one from H. larvatus was identified as ToxoDB Genotype #9 (http://toxodb.org/toxo/). To our knowledge, this is the first report of molecular detection and genetic characterization of T. gondii infection in bats in China. The results show that these bats are potential reservoirs for T. gondii transmission, which may pose a threat to human health.

Filovirus RNA in Fruit Bats, China.

To the Editor: Filovirus-associated diseases, particularly those caused by Ebola and Marburg viruses, represent major threats to human health worldwide because they have extremely high death rates and antiviral therapies or vaccines against them are not available (1). Members of the family Filoviridae are classified into 3 genera: Marburgvirus, Ebolavirus, and the recently approved Cuevavirus (2,3). Marburg virus (MARV) and Ebola virus (EBOV) were initially isolated in Africa, but other filoviruses have been identified on other continents. The initial Cuevavirus, Lloviu virus (LLOV), was identified in Europe (Spain) (3), and Ebola-Reston virus has been found in pigs in Asia (the Philippines) (4). n nBats are natural reservoirs for filoviruses (5). Viral isolation and serologic studies indicate that filovirus infections have occurred in various bat species in central Africa countries (6), the Philippines (7), China (8), and Bangladesh (9). However, identification of these viruses in bats has been difficult; although isolates of MARV have been obtained (6) and the genome of LLOV has been fully sequenced (3), very short sequences of EBOV have been obtained from bats, and only in Africa (5). Reports of molecular detection or isolation of filoviruses in bats in Asia are lacking. We conducted a study to investigate the presence of filoviruses in bats in China. n nIn June 2013, twenty-nine apparently healthy Rousettus leschenaultia fruit bats were captured in Yunnan Province, China. All bats were humanely killed, and their intestines, lungs, livers, and brains were collected and subjected to viral metagenomic analysis by a previously described method (10). As a result, we obtained and reassembled de novo 10 million reads into 590,010 contigs. Of these contigs, 3 (129–354 nt) were genetically close to filovirus, corresponding to the nucleoprotein gene of LLOV (74% nt identity), the viral protein 35 gene of Sudan Ebola virus (69% nt identity), and the L gene of Tai Forest Ebola virus (72% nt identity) (Technical Appendix Table 1). n nFor further screening, we used the longest contig as a template for design of specific seminested primers. Nested degenerate primer pairs were also designed and focused on the most conserved region of the L gene of all currently known filoviruses (Technical Appendix Table 2). After screening, 2 reverse transcription PCRs of tissues from 1 bat (Bt-DH04) showed positive amplification in specimens from its lung but not from intestine, liver, or brain tissue. Moreover, 5 blind passages in Vero-E6 cells failed to isolate the virus from the lung homogenate. In an attempt to obtain its genomic sequence, 24 primer pairs covering the full genome were further designed by alignment of these contig sequences with the full genomes of representative filoviruses within the 3 genera. All amplifications used ddH2O as a negative control; positive controls were not available because filoviruses were not available in China. Two fragments of 2,750-nt (F1) and 2,682-nt (F2) were successfully amplified from lung tissue of Bt-DH04; attempts to amplify the remaining regions failed. Alignment with sequences of 26 representative filoviruses of 7 species from 3 genera revealed that F1 covered the 3′ end of the nucleoprotein gene and almost the entire viral protein 35 gene, and that F2 covered the middle region of the L gene, corresponding to nt 1,313–4,085 and nt 12,613–15,302 of the full genome of EBOV (GenBank accession no. {type:entrez-nucleotide,attrs:{text:HQ613402,term_id:355344222,term_text:HQ613402}}HQ613402). The 2 fragment sequences were submitted to Genbank (accession no. {type:entrez-nucleotide,attrs:{text:KP233864,term_id:859262346,term_text:KP233864}}KP233864), and the strain has been tentatively named Bt-DH04. n nPhylogenetic analysis showed that the Bt-DH04 strain is placed, together with LLOV, at basal position and intermediate between EBOV and MARV (Figure). It is divergent from all known filoviruses, with F1 sharing the highest nucleotide identities (46%–49%) to members of the genus Ebolavirus, followed by 44% to LLOV and <40% to MARV (Figure, panel A). The L gene is the most conserved region of filoviruses, and F2 of Bt-DH04 strain shared relatively closer 66%–68% nt identities with members of the genus Ebolavirus, followed by 64% with LLOV and ≈60% with MARV (Figure, panel B). This sequence diversity is likely the main factor for unsuccessful amplification of the full genome of Bt-DH04. n n n nFigure n nPhylogenetic analysis of 2 fragments of filovirus Bt-DH04 and other filoviruses. Full genomes of representatives from the family Filoviridae were trimmed and aligned with F1 (partial nucleoprotein/viral protein 35 gene, panel A) and F2 (middle L gene, ... n n n nIncreasing PCR evidence has identified the existence of filoviruses in bats in Africa and Europe (3,5); however, although serologic studies have shown that filovirus antibodies are prevalent in bats in a few countries in Asia (e.g., the Philippines, Bangladesh and China [7–9]), filovirus or filovirus RNA have not been reported in bats in Asia. Our results show that the Bt-DH04 strain is likely a novel bat-borne filovirus in Asia and provide evidence that bats in Asia harbor more divergent filoviruses than previously thought. n nFruit bats in the genus Rousettus are widely distributed throughout Southeast Asia, South China, and the entire Indian subcontinent and have had positive serologic results for Ebola viruses in these regions (7–9), indicating that these bats play a role in the circulation of filoviruses in Asia. The possibility of new emerging filovirus-associated diseases in the continent emphasizes the need for further investigation of these animals. n nTechnical Appendix: nSample collection, preparation, and outcomes of testing for filoviral RNA in fruit bats, China. n nClick here to view.(200K, pdf)

Characterization of a novel orthoreovirus isolated from fruit bat, China

BackgroundIn recent years novel human respiratory disease agents have been described for Southeast Asia and Australia. The causative pathogens were classified as pteropine orthoreoviruses with a strong phylogenetic relationship to orthoreoviruses of bat origin.ResultsIn this report, we isolated a novel Melaka-like reovirus (named “Cangyuan virus”) from intestinal content samples of one fruit bat residing in China’s Yunnan province. Phylogenetic analysis of the whole Cangyuan virus genome sequences of segments L, M and S demonstrated the genetic diversity of the Cangyuan virus. In contrast to the L and M segments, the phylogenetic trees for the S segments of Cangyuan virus demonstrated a greater degree of heterogeneity.ConclusionsPhylogenetic analysis indicated that the Cangyuan virus was a novel orthoreovirus and substantially different from currently known members of Pteropine orthoreovirus (PRV) species group.

Prevalence and genetic characterization of Toxoplasma gondii in bats in Myanmar.

ABSTRACT We detected Toxoplasma gondii in 29.3% (95% confidence interval [CI], 25.5% to 33.1%) of 550 insectivorous bats collected in Myanmar. The genotyping of these positive samples revealed they were closely related to or belong to clonal type I, which is highly virulent in mice, showing that these bats are potential reservoirs for T. gondii transmission.

An outbreak of pig rabies in Hunan province, China

The first dog-associated outbreak of rabies in swine in China (Hunan province) has been diagnosed and the related virus isolated. Sequence analysis showed that the pig isolate was a genotype 1 rabies virus with a very high nucleotide identity to local dog isolates.

Genetic diversity of subgenotype 2.1 isolates of classical swine fever virus.

As the causative agent of classical swine fever, the economically devastating swine disease worldwide, classical swine fever virus (CSFV) is currently classified into the 11 subgenotypes, of which subgenotype 2.1 is distributed worldwide and showing more genetic diversity than other subgenotypes. Prior to this report, subgenotype 2.1 was divided into three sub-subgenotypes (2.1a-2.1c). To further analyze the genetic diversity of CSFV isolates in China, 39 CSFV isolates collected between 2004 and 2012 in two Chinese provinces Guangxi and Guangdong were sequenced and subjected to phylogenetic analysis together with reference sequences retrieved from GenBank. Phylogenetic analyses based on the 190-nt and/or 1119-nt full length E2 gene fragments showed that current CSFV subgenotype 2.1 virus isolates in the world could be divided into 10 sub-subgenotypes (2.1a-2.1j) and the 39 isolates collected in this study were grouped into 7 of them (2.1a-2.1c and 2.1g-2.1j). Among the 10 sub-subgenotypes, 2.1d-2.1j were newly identified. Sub-subgenotype 2.1d isolates were circulated only in India, however the rest 9 sub-subgenotypes were from China with some of them closely related to isolates from European and neighboring Asian countries. According to the temporal and spatial distribution of CSFV subgenotype 2.1 isolates, the newly classified 10 sub-subgenotypes were further categorized into three groups: dominant sub-subgenotype, minor sub-subgenotype and silent sub-subgenotype, and each sub-subgenotype can be found only in certain geographical areas. Taken together, this study reveals the complex genetic diversity of CSFV subgenotype 2.1 and improves our understanding about the epidemiological trends of CSFV subgenotype 2.1 in the world, particularly in China.