Yun-Zhi Zhang

Metagenomic Analysis of Viruses from Bat Fecal Samples Reveals Many Novel Viruses in Insectivorous Bats in China

ABSTRACT Increasing data indicate that bats harbor diverse viruses, some of which cause severe human diseases. In this study, sequence-independent amplification and high-throughput sequencing (Solexa) were applied to the metagenomic analysis of viruses in bat fecal samples collected from 6 locations in China. A total of 8,746,417 reads with a length of 306,124,595 bp were obtained. Among these reads, 13,541 (0.15%) had similarity to phage sequences and 9,170 (0.1%) had similarity to eukaryotic virus sequences. A total of 129 assembled contigs (>100 nucleotides) were constructed and compared with GenBank: 32 contigs were related to phages, and 97 were related to eukaryotic viruses. The most frequent reads and contigs related to eukaryotic viruses were homologous to densoviruses, dicistroviruses, coronaviruses, parvoviruses, and tobamoviruses, a range that includes viruses from invertebrates, vertebrates, and plants. Most of the contigs had low identities to known viral genomic or protein sequences, suggesting that a large number of novel and genetically diverse insect viruses as well as putative mammalian viruses are transmitted by bats in China. This study provides the first preliminary understanding of the virome of some bat populations in China, which may guide the discovery and isolation of novel viruses in the future.

Antibodies to Nipah or Nipah-like Viruses in Bats, China

To the Editor: Hendra virus (HeV) and Nipah virus (NiV), the only known members of the genus Henipavirus, are 2 emerging paramyxoviruses that are highly pathogenic in a variety of vertebrate animals, including humans (1). Since the initial discovery of the viruses in Australia and Malaysia (2,3), sporadic HeV outbreaks have been reported from 1995 to 2007 in Australia (4), and regular NiV outbreaks have occurred in Bangladesh (5) and India (6). Numerous frugivorous bat species (genus Pteropus), and some insectivous bat species have been found to be reservoir hosts of henipaviruses in Australia and Asian countries (7–9). In this study conducted during 2004–2007, bats were trapped within their natural habitat from 10 provinces in mainland People’s Republic of China. Serum, pharyngeal, and fecal swab samples were collected and stored as described previously (10). An ELISA was developed to detect antibodies to the NiV nucleocapsid (N) and attachment glycoprotein (G) proteins. For confirmation, ELISA-positive samples were tested by using Western blot against a recombinant NiV G fragment (aa 71–193) fused with the maltose-binding protein. Virus neutralization tests were conducted with live NiV and HeV under Biosaftey Level 4 containment in Australia. In addition, a surrogate neutralization test was developed by using recombinant env– HIV-1, pseudotyped with NiV G and F. RNA was extracted by using the QIA amp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription–PCR (RT-PCR) was performed by using primers against the NiV N gene as described previously (3). In total, 692 bat serum specimens were screened for antibody to NiV N or G protein (or both) by ELISA, and 33 were positive (Appendix Table). These specimens were from 9 of the 23 bat species examined in this study. Of the 33 serum samples reactive in ELISA, 25 with sufficient quantity left were further tested by Western blot, and 17 of 25 serum samples were reactive with MBP-NiV G fusion fragment, but not with the control MBP. None of the samples inhibited entry of NiV F/G-pseudotyped virus or neutralized either HeV or NiV. No NiV-specific RNA was detected by RT-PCR among 479 fecal swab samples and 67 throat swab samples tested; therefore, virus isolation was not attempted. This study systematically investigated NiV presence among bats in China. The detection of henipavirus antibody suggests that several bat species have been exposed to NiV or a closely related virus. The prevalence of antibody was especially prominent among Myotis species from Yunnan Province. Antibody was detected in samples from 3 of 4 Myotis species captured in the same location in 2006 and 2007. A relatively high prevalence of henipavirus antibody was also found among Rousettus leschenaultia samples from Hainan Province in 2007. Notably, Yunnan and Hainan are both located in southern China. Although pteropid bats are not found in China, these data suggest henipaviruses could be introduced into China by other susceptible bat species that overlap their habitat with pteropid bats in neighboring countries. Several possibilities may explain the failure to detect neutralizing antibodies. One might be the unique immune response among those nonpteropid bats, which results in a low level of neutralizing antibodies that are difficult to detect in the current assay systems. Alternatively, and perhaps more likely, >1 Nipah-like viruses could be circulating among the bat populations sampled in this study, producing antibodies that cross-react with, but do not neutralize, the prototype Malaysian NiV virus isolate. This phenomenon has been observed previously by our group for severe acute respiratory syndrome (SARS)–like viruses in horseshoe bats, whose sera cross-reacted with, but did not neutralize, the SARS virus in humans (10). Obtaining serologic evidence of viruses in bat populations is typically more successful as a screening tool than either nucleic acid based assays or virus isolation; this is likely attributable to the often low-level of virus replication, the transient nature of the infection in bats, or both. The inability to amplify NiV sequences may have been attributable to the viral RNA present among these samples being below the threshold of detection in our assay or to the absence of infection in the population at the time of sampling. In addition, the primers used in the PCR may target regions of the NiV N protein that exhibit substantial sequence divergence in a Nipah-like virus. Bat species in the genera Rousettus, Myotis, Miniopterus, and Hipposideros naturally reside in trees, buildings, and caves that can be in close proximity to human residential areas, which increases the potential of transmission of zoonotic pathogens from bats to humans. The increased risk for these zoonotic infections to spread from bats to humans in areas of cohabitation is best illustrated by the repeated spillover events involving NiV in Bangladesh (5). Given the present initial evidence of exposure among bats in mainland China shown here, there is an urgent need to continue and expand surveillance studies for henipaviruses in China and elsewhere on the Asian continent.

Host range, prevalence, and genetic diversity of adenoviruses in bats.

ABSTRACT Bats are the second largest group of mammals on earth and act as reservoirs of many emerging viruses. In this study, a novel bat adenovirus (AdV) (BtAdV-TJM) was isolated from bat fecal samples by using a bat primary kidney cell line. Infection studies indicated that most animal and human cell lines are susceptible to BtAdV-TJM, suggesting a possible wide host range. Genome analysis revealed 30 putative genes encoding proteins homologous to their counterparts in most known AdVs. Phylogenetic analysis placed BtAdV-TJM within the genus Mastadenovirus, most closely related to tree shrew and canine AdVs. PCR analysis of 350 bat fecal samples, collected from 19 species in five Chinese provinces during 2007 and 2008, indicated that 28 (or 8%) samples were positive for AdVs. The samples were from five bat species, Hipposideros armiger, Myotis horsfieldii, M. ricketti, Myotis spp., and Scotophilus kuhlii. The prevalence ranged from 6.25% (H. armiger in 2007) to 40% (M. ricketti in 2007). Comparison studies based on available partial sequences of the pol gene demonstrated a great genetic diversity among bat AdVs infecting different bat species as well as those infecting the same bat species. This is the first report of a genetically diverse group of DNA viruses in bats. Our results support the notion, derived from previous studies based on RNA viruses (especially coronaviruses and astroviruses), that bats seem to have the unusual ability to harbor a large number of genetically diverse viruses within a geographic location and/or within a taxonomic group.

Genetic diversity of novel circular ssDNA viruses in bats in China

Novel circular ssDNA genomes have recently been detected in animals and in the environment using metagenomic and high-throughput sequencing approaches. In this study, five full-length circular ssDNA genomes were recovered from bat faecal samples using inverse PCR with sequences designed based on circovirus-related sequences obtained from Solexa sequencing data derived from a random amplification method. These five sequences shared a similar genomic organization to circovirus or the recently proposed cyclovirus of the family Circoviridae. The newly obtained circovirus/cyclovirus-like genomes ranged from 1741 to 2177 bp, and each consisted of two major ORFs, ORF1 and ORF2, encoding putative replicase (Rep) and capsid (Cap) proteins, respectively. The potential stem-loop region was predicted in all five genomes, and three of them had the typical conserved nonanucleotide motif of cycloviruses. A set of primers targeting the conserved Rep region was designed and used to detect the prevalence of circovirus/cyclovirus sequences in individual bats. Among 199 samples tested, 47 were positive (23.6%) for the circovirus genome and two (1.0%) were positive for the cyclovirus genome. In total, 48 partial Rep sequences plus the five full-length genomes were obtained in this study. Detailed analysis indicated that these sequences are distantly related to known circovirus/cyclovirus genomes and may represent 22 novel species that belong to the family Circoviridae.

Serological evidence of ebolavirus infection in bats, China

BackgroundThe genus Ebolavirus of the family Filoviridae currently consists of five species. All species, with the exception of Reston ebolavirus, have been found in Africa and caused severe human diseases. Bats have been implicated as reservoirs for ebolavirus. Reston ebolavirus, discovered in the Philippines, is the only ebolavirus species identified in Asia to date. Whether this virus is prevalent in China is unknown.FindingsIn this study, we developed an enzyme linked immunosorbent assay (ELISA) for ebolavirus using the recombinant nucleocapsid protein and performed sero-surveillance for the virus among Chinese bat populations. Our results revealed the presence of antibodies to ebolavirus in 32 of 843 bat sera samples and 10 of 16 were further confirmed by western blot analysis.ConclusionTo our knowledge, this is the first report of any filovirus infection in China.

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.

Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT We report the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.

Effects of ozone on membrane permeability and ultrastructure in Pseudomonas aeruginosa

Aims:  To examine the mechanism of ozone‐induced damage to cytoplasmic membrane and cell ultrastructure of Pseudomonas aeruginosa ATCC27853.

Detection of diverse novel astroviruses from small mammals in China

Astroviruses infect humans and many animal species and cause gastroenteritis. To extensively understand the distribution and genetic diversity of astrovirus in small mammals, we tested 968 anal swabs from 39 animal species, most of which were bats and rodents. We detected diverse astroviruses in 10 bat species, including known bat astroviruses and a large number of novel viruses. Meanwhile, novel groups of astroviruses were identified in three wild rodent species and a remarkably high genetic diversity of astrovirus was revealed in Eothenomys cachinus. We detected astroviruses in captive-bred porcupines and a nearly full-length genome sequence was determined for one strain. Phylogenetic analysis of the complete ORF2 sequence suggested that this strain may share a common ancestor with porcine astrovirus type 2. Moreover, to our knowledge, this study reports the first discovery of astroviruses in shrews and pikas. Our results provide new insights for understanding these small mammals as natural reservoirs of astroviruses.