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Pteropine orthoreovirus (PRV) causes respiratory tract illness (RTI) in humans. PRVs were isolated from throat swabs collected from 9 of 91 wild bats captured on the Mindanao Islands, The Philippines, in 2013. The nucleic acid sequence of the whole genome of each of these isolates was determined. Phylogenetic analysis based on predicted amino acid sequences indicated that the isolated PRVs were novel strains in which re-assortment events had occurred in the viral genome. Serum specimens collected from 76 of 84 bats were positive for PRV-neutralizing antibodies suggesting a high prevalence of PRV in wild bats in the Philippines. The bat-borne PRVs isolated in the Philippines were characterized in comparison to an Indonesian PRV isolate, Miyazaki-Bali/2007 strain, recovered from a human patient, revealing that the Philippine bat-borne PRVs had similar characteristics in terms of antigenicity to those of the Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro). The impact of the Philippine bat-borne PRVs should be studied in human RTI cases in the Philippines.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles (order Eulipotyphla, families Soricidae and Talpidae) prompted a further exploration of their host diversification and geographic distribution by analyzing lung tissues from 376 fruit bats representing six genera (order Chiroptera, suborder Yinpterochiroptera, family Pteropodidae), collected in the Republic of the Philippines during 2008 to 2013. Hantavirus RNA was detected by RT-PCR in one of 15 Geoffroy's rousettes (Rousettus amplexicaudatus), captured in Quezon Memorial National Park on Luzon Island in 2009. Phylogenetic analyses of the S, M and L segments, using maximum-likelihood and Bayesian methods, showed that the newfound hantavirus, designated Quezon virus (QZNV), shared a common ancestry with hantaviruses hosted by insectivorous bats, in keeping with their evolutionary relationships and suggests that ancestral bats may have served as the early or original mammalian hosts of primordial hantaviruses. As the first hantavirus detected in a megabat or flying fox species, QZNV extends our knowledge about the reservoir host range., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bats are the second diversity species of mammals and widely distributed in the world. They are thought to be reservoir and vectors of zoonotic pathogens. However, there is scarce report of the evidence of pathogenic bacteria kept in bats. The precise knowledge of the pathogenic bacteria in bat microbiota is important for zoonosis control. Thus, metagenomic analysis targeting the V3-V4 region of the 16S rRNA of the rectal microbiota in Rousettus amplexicaudatus was performed using high throughput sequencing. The results revealed that 103 genera of bacteria including Camplyobacter were detected. Campylobacter was second predominant genus, and Campylobacter coli and Campylobacter jejuni were identified in microbiome of R. amplexicaudatus. Campylobacteriosis is one of the serious bacterial diarrhea in human, and the most often implicated species as the causative agent of campylobacteriosis is C. jejuni. Therefore, we investigated the prevalence of C. jejuni in 91 wild bats with PCR. As a result of PCR assay targeted on 16S-23S intergenic spacer, partial genome of C. jejuni was detected only in five R. amplexicaudatus. This is the first report that C. jejuni was detected in bat rectal swab samples. C. jejuni is the most common cause of campylobacteriosis in humans, transmitted through water and contact with livestock animals. This result indicated that R. amplexicaudatus may be a carrier of C. jejuni.

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Bats are naturally infected with zoonotic pathogens; thus, they are potential reservoirs of parasites. We investigated the species and genotype distribution as well as prevalence of Cryptosporidium and Eimeria in Philippine bats. We captured and examined 45 bats; four were positive for Cryptosporidium spp. and seven were positive for Eimeria spp. We detected Cryptosporidium bat genotype II from Ptenochirus jagori. Three other Cryptosporidium sequences, detected from Rhinolophus inops, Cynopterus brachyotis, and Eonycteris spelaea, could not be classified as any known species or genotype; we therefore propose the novel genotype Cryptosporidium bat genotypes V, VI, and VII. Bat genotype V is associated with human cryptosporidiosis clade, and therefore, this genotype may be transmissible to humans. Among the Eimeria sequences, BE3 detected from Scotophilus kuhlii was classified with known bat and rodent clades; however, other sequences detected from C. brachyotis, E. spelaea, Rousettus amplexicaudatus, and R. inops could not be classified with known Eimeria species. These isolates might represent a new genotype. Our findings demonstrate that the bats of the Philippines represent a reservoir of multiple Cryptosporidium and Eimeria spp.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

We present a complete sequence of mitochondrial genome of echolocating megabat Rousettus leschenaulti (16,704 nt, GenBank record KC702803) and provide its annotation. We also show that phylogenetic analysis based on mitochondrial genomes of eighteen bat species, including R. leschenaulti, supports division of Chiroptera into Yinpterochiroptera and Yangochiroptera.

Bat coronavirus (BtCoV) is assumed to be a progenitor of severe acute respiratory syndrome (SARS)-related coronaviruses. To explore the distribution of BtCoVs in the Philippines, we collected 179 bats and detected viral RNA from intestinal or fecal samples by RT-PCR. The overall prevalence of BtCoVs among bats was 29.6 %. Phylogenetic analysis of the partial RNA-dependent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others belonged to the genus Betacoronavirus. Western blotting revealed that 66.5 % of bat sera had antibodies to BtCoV. These surveys suggested the endemic presence of BtCoVs in the Philippines.

Bats have great potential as reservoirs for emerging viruses such as severe acute respiratory syndrome-coronavirus. In this study, bat coronaviruses (BtCoVs) were detected by RT-PCR from intestinal and fecal specimens of Miniopterus fuliginosus breeding colonies in Wakayama Prefecture caves, where we previously identified bat betaherpesvirus 2. Two primer sets were used for the detection of BtCoV: one was for the RNA-dependent RNA polymerase (RdRp) region and the other was for the spike (S) protein region. Eleven and 73% of intestinal and fecal specimens, respectively, were positive for RdRp region, and 2 and 40% of those were positive for S protein region. Sequencing and phylogenetic analysis showed that the detected BtCoV belonged to the group 1 (alpha) coronaviruses. These data suggest that BtCoV is endemic in M. fuliginosus in Japan.

Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription-PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9-1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.

Bats are now known as the source of several diseases in humans, but few studies regarding immune responses and factors associated with bats have so far been reported. In this study, we focused on STAT1, one of the critical components in interferon (IFN)-signaling and antiviral activity, which is often targeted by viral proteins to reduce antiviral activity and increase viral replication. We found that Rousettus aegyptiacus STAT1 (bat STAT1) is phosphorylatable and translocates to the nucleus when stimulated with human IFN-alpha (hIFN-alpha). Furthermore, phosphorylation of bat STAT1 and inhibition of nuclear translocation was observed in IFN-stimulated cells infected with the HEP-Flury strain of rabies virus, in the same manner as in other mammals. Additionally, quantitative real-time RT-PCR revealed that bat STAT1 mRNA was highly expressed in the liver, while low in muscle and spleen., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

In this study, cDNA of Toll-like receptors (TLR) 3, 7 and 9 were synthesized and completely sequenced. The coding regions of cDNA for bat TLR3, TLR7 and TLR9 were 2,718, 3,150 and 3,090 bp in length, respectively. The open reading frames encoded 905, 1,049 and 1,029 amino acids for TLR3, TLR7 and TLR9, respectively. The nucleotide sequences, predicted amino acid sequences and predicted domain structures of the three bat TLRs had high homology with those of other mammals. In addition, the expression profiles of each TLR in main organs were analyzed. Expression of TLR3 was highest in the liver, whereas the expressions of TLR7 and TLR9 were highest in the spleen.

This is the first report on the cDNA sequences of bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 p40, and tumor necrosis factor (TNF)-alpha. The cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha comprise 459, 405, 624, 537, 990, and 699 base pairs respectively. Moreover, each of the cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-alpha contain a single open reading frames encoding 152, 134, 207, 178, 329, and 232 amino acids, respectively. The comparison of bat cytokines with Perrissodactyla (horse), Carnivora (dog and cat), and Cetartiodactyla (cattle and pig) orthologs revealed a high degree of homology. Although the N-terminal amino acids and cysteine residues are highly conserved in each mature cytokine, the deduced N-linked glycosylation sites vary across species.

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3' part of the gB gene and the 5' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Bats are considered to be natural reservoirs for several viruses of clinical importance, including rabies virus, Nipah virus, and Hendra virus. Type I interferons (IFNs) is an important part of the immune system in the defense against viral infection. To investigate the function of type I IFNs upon viral infection in bats, the nucleic acid, and amino acid sequences of Egyptian Rousette (Rousettus aegyptiacus) IFN-alpha and -beta were characterized. Sequence data indicated that bat IFN-alpha consists of 562-bp encoded 187-aa, and IFN-beta consisted of 558-bp encoded 186-aa. Phylogenetic analysis of the overall identity of IFN-beta shared the highest sequence homology with pig IFN-beta in both nucleotide and amino acid level. Stimulation of bat primary kidney cells (BPKCs) and bat lung cell lines, Tb-1 Lu, with polyinosinic-polycytidylic acid (poly(I:C)) or exogenous bat type I IFNs resulted in increased type I IFNs mRNA expression in BPKCs, but not in Tb-1 Lu. Characterization of the bat IFN-alpha and -beta genes allows understanding of the immune responses upon stimulation in different tissues, thus providing practical strategies for control and treatment of clinically important diseases. These results are important especially for the virus infection, and suggest that future molecular studies on virus infection experiment of bats in vitro will require careful consideration of the differences of type I IFN expression patterns in different cell types.

Chiroptera is thought to be a vector or a natural reservoir of various pathogenic microbes. However, there are few basic studies on the subject of chiroptera immune systems. This is the first report to determine the sequence of bat CD4 cDNA. Comparison with other animals' CD4 and phylogenetic analysis have shown that bat CD4 had a higher homology to cat and dog CD4 than to human and mouse CD4. Moreover, from the analysis of the structure of the CD4 Ig-like C-type 1 region, in bat CD4 there was an insertion of 18 extra amino acids. In addition, bat CD4 lacked cystein, which suggested that the disulfide bond could not be formed. Human, monkey and mouse CD4 have the cystein and the disulfide bond, but pig, cat, whale and dog CD4, like that of the bat, lacked the cystein. We conducted the present study in order to help elucidate the infectious diseases derived from the bat as well as bat immune systems.

We examined the relationships between Megachiroptera and Microchiroptera, and between Chiroptera and other closely related species by the cross-reactivity of immunoglobulin epitopes. Rabbit polyclonal antibody to bat IgG was used for determining the cross-reactivity by a competitive ELISA method. Megachiroptera and Microchiroptera showed high cross-reactivity, over 95.3%, with each other. However, primates and insectivores showed very low cross-reactivity, 8.6 to 20.2% and 5.3 to 12.7%, respectively. These results suggest that suborders of Chiroptera are monophyletic and Chiroptera have a relatively closer relationship to primates than to insectivores.