Kevin J. Olival

Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia

The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Molecular investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.

A Strategy To Estimate Unknown Viral Diversity in Mammals

ABSTRACT The majority of emerging zoonoses originate in wildlife, and many are caused by viruses. However, there are no rigorous estimates of total viral diversity (here termed “virodiversity”) for any wildlife species, despite the utility of this to future surveillance and control of emerging zoonoses. In this case study, we repeatedly sampled a mammalian wildlife host known to harbor emerging zoonotic pathogens (the Indian Flying Fox, Pteropus giganteus) and used PCR with degenerate viral family-level primers to discover and analyze the occurrence patterns of 55 viruses from nine viral families. We then adapted statistical techniques used to estimate biodiversity in vertebrates and plants and estimated the total viral richness of these nine families in P. giganteus to be 58 viruses. Our analyses demonstrate proof-of-concept of a strategy for estimating viral richness and provide the first statistically supported estimate of the number of undiscovered viruses in a mammalian host. We used a simple extrapolation to estimate that there are a minimum of 320,000 mammalian viruses awaiting discovery within these nine families, assuming all species harbor a similar number of viruses, with minimal turnover between host species. We estimate the cost of discovering these viruses to be ~

Host and viral traits predict zoonotic spillover from mammals

6.3 billion (or ~

Ebola virus antibodies in fruit bats, bangladesh.

1.4 billion for 85% of the total diversity), which if annualized over a 10-year study time frame would represent a small fraction of the cost of many pandemic zoonoses. IMPORTANCE Recent years have seen a dramatic increase in viral discovery efforts. However, most lack rigorous systematic design, which limits our ability to understand viral diversity and its ecological drivers and reduces their value to public health intervention. Here, we present a new framework for the discovery of novel viruses in wildlife and use it to make the first-ever estimate of the number of viruses that exist in a mammalian host. As pathogens continue to emerge from wildlife, this estimate allows us to put preliminary bounds around the potential size of the total zoonotic pool and facilitates a better understanding of where best to allocate resources for the subsequent discovery of global viral diversity. Recent years have seen a dramatic increase in viral discovery efforts. However, most lack rigorous systematic design, which limits our ability to understand viral diversity and its ecological drivers and reduces their value to public health intervention. Here, we present a new framework for the discovery of novel viruses in wildlife and use it to make the first-ever estimate of the number of viruses that exist in a mammalian host. As pathogens continue to emerge from wildlife, this estimate allows us to put preliminary bounds around the potential size of the total zoonotic pool and facilitates a better understanding of where best to allocate resources for the subsequent discovery of global viral diversity.

Characterization of Nipah Virus from Naturally Infected Pteropus vampyrus Bats, Malaysia

The majority of human emerging infectious diseases are zoonotic, with viruses that originate in wild mammals of particular concern (for example, HIV, Ebola and SARS). Understanding patterns of viral diversity in wildlife and determinants of successful cross-species transmission, or spillover, are therefore key goals for pandemic surveillance programs. However, few analytical tools exist to identify which host species are likely to harbour the next human virus, or which viruses can cross species boundaries. Here we conduct a comprehensive analysis of mammalian host–virus relationships and show that both the total number of viruses that infect a given species and the proportion likely to be zoonotic are predictable. After controlling for research effort, the proportion of zoonotic viruses per species is predicted by phylogenetic relatedness to humans, host taxonomy and human population within a species range—which may reflect human–wildlife contact. We demonstrate that bats harbour a significantly higher proportion of zoonotic viruses than all other mammalian orders. We also identify the taxa and geographic regions with the largest estimated number of ‘missing viruses’ and ‘missing zoonoses’ and therefore of highest value for future surveillance. We then show that phylogenetic host breadth and other viral traits are significant predictors of zoonotic potential, providing a novel framework to assess if a newly discovered mammalian virus could infect people.

Correlates of Viral Richness in Bats (Order Chiroptera)

To determine geographic range for Ebola virus, we tested 276 bats in Bangladesh. Five (3.5%) bats were positive for antibodies against Ebola Zaire and Reston viruses; no virus was detected by PCR. These bats might be a reservoir for Ebola or Ebola-like viruses, and extend the range of filoviruses to mainland Asia.

Global distribution and genetic diversity of Bartonella in bat flies (Hippoboscoidea, Streblidae, Nycteribiidae)

We isolated and characterized Nipah virus (NiV) from Pteropus vampyrus bats, the putative reservoir for the 1998 outbreak in Malaysia, and provide evidence of viral recrudescence. This isolate is monophyletic with previous NiVs in combined analysis, and the nucleocapsid gene phylogeny suggests that similar strains of NiV are co-circulating in sympatric reservoir species.

Group C Betacoronavirus in Bat Guano Fertilizer, Thailand

Historic and contemporary host ecology and evolutionary dynamics have profound impacts on viral diversity, virulence, and associated disease emergence. Bats have been recognized as reservoirs for several emerging viral pathogens, and are unique among mammals in their vagility, potential for long-distance dispersal, and often very large, colonial populations. We investigate the relative influences of host ecology and population genetic structure for predictions of viral richness in relevant reservoir species. We test the hypothesis that host geographic range area, distribution, population genetic structure, migratory behavior, International Union for Conservation of Nature and Natural Resources (IUCN) threat status, body mass, and colony size, are associated with known viral richness in bats. We analyze host traits and viral richness in a generalized linear regression model framework, and include a correction for sampling effort and phylogeny. We find evidence that sampling effort, IUCN status, and population genetic structure correlate with observed viral species richness in bats, and that these associations are independent of phylogeny. This study is an important first step in understanding the mechanisms that promote viral richness in reservoir species, and may aid in predicting the emergence of viral zoonoses from bats.

Contrasting patterns in mammal-bacteria coevolution: bartonella and leptospira in bats and rodents.

Recently, a growing number Bartonella spp. have been identified as causative agents for a broadening spectrum of zoonotic diseases, emphasizing their medical importance. Many mammalian reservoirs and vectors however are still unknown, hindering our understanding of pathogen ecology and obscuring epidemiological connections. New Bartonella genotypes were detected in a global sampling of 19 species of blood-feeding bat flies (Diptera, Hippoboscoidea, Nycteribiidae, Streblidae) from 20 host bat species, suggesting an important role of bat flies in harboring bartonellae. Evolutionary relationships were explored in the context of currently known Bartonella species and genotypes. Phylogenetic and gene network analyses point to an early evolutionary association and subsequent radiation of bartonellae with bat flies and their hosts. The recovery of unique clades, uniting Bartonella genotypes from bat flies and bats, supports previous ideas of these flies potentially being vectors for Bartonella. Presence of bartonellae in some female bat flies and their pupae suggests vertical transmission across developmental stages. The specific function of bartonellae in bats and bat flies remains a subject of debate, but in addition to pathogenic interactions, parasitic, mutualistic, or reservoir functions need to be considered.

Molecular evidence of Ebola Reston virus infection in Philippine bats

To the Editor: Bats play a critical role in the transmission and origin of zoonotic diseases, primarily viral zoonoses associated with high case-fatality rates, including those caused by Nipah virus (NiV) and severe acute respiratory syndrome (SARS)–like coronavirus (CoV) infections (1). Recently, the World Health Organization (WHO) reported 44 confirmed cases of human infection with Middle East respiratory syndrome CoV, resulting in 22 deaths. Full-genome and phylogenetic analyses of these Middle East respiratory syndrome CoVs have been published elsewhere (2). The identified viruses from 2 patients (previously referred to as England/Qatar/2012 and EMC/2012) are genetically related and belong to group C betacoronavirus, which is most related to CoVs from Nycteris bats in Ghana and Pipistrellus bats in Europe (2,3). In addition, bat CoVs HKU4 and HKU5 originated from Tylonycteris pachypus and Pipistrellus abramus bats, respectively, in the People’s Republic of China (4). Bats are also known to harbor and transmit nonviral zoonotic pathogens, including the fungal pathogen Histoplasma capsulatum, which causes histoplasmosis in humans (5). Bat guano is sold for use as a fertilizer in several countries, including Thailand, Indonesia, Mexico, Cuba, and Jamaica. The practice of collecting and harvesting bat guano may pose a considerable health risk because guano miners have a high level of contact and potential exposure to bat-borne pathogens. To assess pathogens in bat guano, we examined bat guano from a cave in the Khao Chong Phran Non-hunting Area (KCP-NHA) in Ratchaburi Province, Thailand, where bat guano was sold as agricultural fertilizer, for the presence of NiV, CoV, and H. capsulatum fungi. Bats from 14 species in 7 families have been found roosting within this area. Tadarida plicata bats are the most abundant species (2,500,000 bats), and 3 other species of bats found at the site each had thousands of members: Taphozous melanopogon, Taphozous theobaldi, and Hipposideros larvatus. A random sample of dry bat guano, ≈100 g, was collected in a sterile plastic bag weekly from the main cave at KCP-NHA from September 2006 through August 2007. The specimens were sent for analysis by express mail (at room temperature within 2–3 days) to the WHO Collaborating Centre for Research and Training in the Viral Zoonoses Laboratory at Chulalongkorn University. Samples were frozen immediately at –80°C until nucleic acids were extracted and PCR assays were run. A total of 52 collected bat guano specimens were examined in this study. Two aliquots of feces from each weekly specimen (104 samples total) were screened for CoV, NiV, and H. capsulatum by PCR. RNA was extracted from 10 mg of fecal pellet by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). CoV RNA was detected by using nested reverse transcription PCR with the degenerated primers to amplify the RNA-dependent RNA polymerase (RdRp) gene (6). NiV RNA was detected by duplex nested reverse transcription PCR (7). To detect H. capsulatum and other fungi, we extracted genomic DNA directly from bat guano by using the silica-guanidine thiocyanate protocol, NucliSense Isolation Reagent (bioMerieux, Boxtel, the Netherlands), according to the manufacturer’s protocol. We tested for fungal ribosomal DNA (rDNA) in extracted total nucleic acid specimens by using the PCR protocol designed to amplify all rDNA from 4 major fungus phyla at the internal transcribed spacer 1 and 2 regions (8). Four (3.8%) of 104 samples were positive for CoV. They were collected on September 2, 2006 (KCP9), October 26, 2006 (KCP12), November 14, 2006 (KCP15), and March 4, 2007 (KCP31). Three of the 4 positive CoV sequences (KCP9, KCP12, and KCP15) were identical at 152 nt of the RdRp region (ATCGTGCTATGCCTAATATGTGTAGGATTTTTGCATCTCTCATATTAGCTCGTAAACACAATACTTGTTGTAGTGTTTCAGACCGCTtTtATAGACTTGCaAACGAGTGTGCGCAAGTCTTGAGTGAGTATGTGCTATGTGGTGGTGGCTAT) and phylogenetically clustered with the group C betacoronavirus (Figure), with 76%, 80%, and 77% nt identity to bat CoV HKU4, bat CoV HKU5, and human CoV EMC and England1_CoV, respectively. The other CoV sequence (KCP31: ATCGTGCACTTCCCAATATGATACGCATGATTTCCGCCATGATTTTGGGATCAAAGCATGTTACTTGCTGTGACACATCTGATAAGTATTACCGTCTTTGTAATGAGCTtGCACAAGTTTTGACAGAGGTTGTTTATTCTAATGGTGGTTTC) showed 82% nt identity with bat CoV HKU8, an alphacoronavirus. Although we recognize that longer sequences or full genomes may alter the topology of the phylogeny slightly and give stronger branch support, we expect that the overall topology and placement of these CoVs would remain consistent. Samples from particular bat species could not be identified because bats of different species roost in this cave, and samples were pooled during collection for bat guano fertilizer. The detection of CoVs in bat guano from the KCP-NHA cave in Ratchaburi was consistent with the previous finding of alphacoronavirus from Hipposideros armiger bats from the same province in 2007, but those researchers tested fresh bat feces (9). Figure Phylogenetic tree of 4 coronaviruses (CoVs) isolated from bat guano collected in this study (KCP9, KCP12, KCP15, and KCP31); 35 additional human and animal CoVs from the National Center for Biotechnology Information database are included. Construction ... All bat guano samples screened by PCR were negative for NiV and Histoplasma spp. but were positive for group C betacoronavirus. The natural reservoir and complete geographic distribution of this CoV are currently unknown. Although we did not isolate live virus from these samples, the detection of nucleic acid and previous isolation of viruses from bat feces and urine (10) warrants some concern that guano miners might be exposed to bat pathogens in fresh excreta as well as in soil substances. We suggest that guano miners use preventive measures of personal hygiene and improved barrier protection to reduce the possibility of exposure to zoonotic pathogens.