The genetic diversity of Nipah virus across spatial scales.

BACKGROUND: Nipah virus (NiV), a highly lethal virus in humans, circulates in Pteropus bats throughout South and Southeast Asia. Difficulty in obtaining viral genomes from bats means we have a poor understanding of NiV diversity. METHODS: We develop phylogenetic approaches applied to the most comprehensive collection of genomes to date (N=257, 175 from bats, 73 from humans) from six countries over 22 years (1999-2020). We divide the four major NiV sublineages into 15 genetic clusters. Using Approximate Bayesian Computation fit to a spatial signature of viral diversity, we estimate the presence and the average size of genetic clusters per area. RESULTS: We find that, within any bat roost, there are an average of 2.4 co-circulating genetic clusters, rising to 5.5 clusters at areas of 1500-2000km2. We estimate that each genetic cluster occupies an average area of 1.3million km2 (95%CI: 0.6-2.3 million), with 14 clusters in an area of 100,000km2 (95%CI: 6-24). In the few sites in Bangladesh and Cambodia where genomic surveillance has been concentrated, we estimate that most clusters have been identified, but only ∼15% of overall NiV diversity has been uncovered. CONCLUSION: Our findings are consistent with entrenched co-circulation of distinct lineages, even within roosts, coupled with slow migration over larger spatial scales.

The genetic diversity of Nipah virus across spatial scales.

Nipah virus (NiV), a highly lethal virus in humans, circulates silently in Pteropus bats throughout South and Southeast Asia. Difficulty in obtaining genomes from bats means we have a poor understanding of NiV diversity, including how many lineages circulate within a roost and the spread of NiV over increasing spatial scales. Here we develop phylogenetic approaches applied to the most comprehensive collection of genomes to date (N=257, 175 from bats, 73 from humans) from six countries over 22 years (1999-2020). In Bangladesh, where most human infections occur, we find evidence of increased spillover risk from one of the two co-circulating sublineages. We divide the four major NiV sublineages into 15 genetic clusters (emerged 20-44 years ago). Within any bat roost, there are an average of 2.4 co-circulating genetic clusters, rising to 5.5 clusters at areas of 1,500-2,000 km2. Using Approximate Bayesian Computation fit to a spatial signature of viral diversity, we estimate that each genetic cluster occupies an average area of 1.3 million km2 (95%CI: 0.6-2.3 million), with 14 clusters in an area of 100,000 km2 (95%CI: 6-24). In the few sites in Bangladesh and Cambodia where genomic surveillance has been concentrated, we estimate that most of the genetic clusters have been identified, but only ~15% of overall NiV diversity has been uncovered. Our findings are consistent with entrenched co-circulation of distinct lineages, even within individual roosts, coupled with slow migration over larger spatial scales.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

Longitudinal monitoring in Cambodia suggests higher circulation of alpha and betacoronaviruses in juvenile and immature bats of three species.

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46-7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.

A novel SARS-CoV-2 related coronavirus in bats from Cambodia.

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses.

Epidemiology and pathological progression of erythematous lip lesions in captive sun bears (Helarctos malayanus).

This study investigates the occurrence of erythematous lip lesions in a captive sun bear population in Cambodia, including the progression of cheilitis to squamous cell carcinoma, and the presence of Ursid gammaherpesvirus 1. Visual assessment conducted in 2015 and 2016 recorded the prevalence and severity of lesions. Opportunistic sampling for disease testing was conducted on a subset of 39 sun bears, with histopathological examination of lip and tongue biopsies and PCR testing of oral swabs and tissue biopsies collected during health examinations. Lip lesions were similarly prevalent in 2015 (66.0%) and 2016 (68.3%). Degradation of lip lesion severity was seen between 2015 and 2016, and the odds of having lip lesions, having more severe lip lesions, and having lip lesion degradation over time, all increased with age. Cheilitis was found in all lip lesion biopsies, with histological confirmation of squamous cell carcinoma in 64.5% of cases. Single biopsies frequently showed progression from dysplasia to neoplasia. Eighteen of 31 sun bears (58.1%) had at least one sample positive for Ursid gammaherpesvirus 1. The virus was detected in sun bears with and without lip lesions, however due to case selection being strongly biased towards those showing lip lesions it was not possible to test for association between Ursid gammaherpesvirus 1 and lip squamous cell carcinoma. Given gammaherpesviruses can play a role in cancer development under certain conditions in other species, we believe further investigation into Ursid gammaherpesvirus 1 as one of a number of possible co-factors in the progression of lip lesions to squamous cell carcinoma is warranted. This study highlights the progressively neoplastic nature of this lip lesion syndrome in sun bears which has consequences for captive and re-release management. Similarly, the detection of Ursid gammaherpesvirus 1 should be considered in pre-release risk analyses, at least until data is available on the prevalence of the virus in wild sun bears.

Nipah virus circulation at human-bat interfaces, Cambodia.

OBJECTIVE: To better understand the potential risks of Nipah virus emergence in Cambodia by studying different components of the interface between humans and bats. METHODS: From 2012 to 2016, we conducted a study at two sites in Kandal and Battambang provinces where fruit bats (Pteropus lylei) roost. We combined research on: bat ecology (reproductive phenology, population dynamics and diet); human practices and perceptions (ethnographic research and a knowledge, attitude and practice study); and Nipah virus circulation in bat and human populations (virus monitoring in bat urine and anti-Nipah-virus antibody detection in human serum). FINDINGS: Our results confirmed circulation of Nipah virus in fruit bats (28 of 3930 urine samples positive by polymerase chain reaction testing). We identified clear potential routes for virus transmission to humans through local practices, including fruit consumed by bats and harvested by humans when Nipah virus is circulating, and palm juice production. Nevertheless, in the serological survey of 418 potentially exposed people, none of them were seropositive to Nipah virus. Differences in agricultural practices among the regions where Nipah virus has emerged may explain the situation in Cambodia and point to actions to limit the risks of virus transmission to humans. CONCLUSION: Human practices are key to understanding transmission risks associated with emerging infectious diseases. Social science disciplines such as anthropology need to be integrated in health programmes targeting emerging infectious diseases. As bats are hosts of major zoonotic pathogens, such integrated studies would likely also help to reduce the risk of emergence of other bat-borne diseases.

Whole genome sequencing and phylogenetic characterization of a novel bat-associated picornavirus-like virus with an unusual genome organization.

The order Picornavirales is one of the most important viral orders in terms of virus diversity and genome organizations, ranging from a mono- or bi-cistronic expression strategies to the recently described poly-cistronic Polycipiviridae viruses. We report here the description and characterization of a novel picorna-like virus identified in rectal swabs of frugivorous bats in Cambodia that presents an unusual genome organization. Kandabadicivirus presents a unique genome architecture and distant phylogenetic relationship to the proposed Badiciviridae family. These findings highlight a high mosaicism of genome organizations among the Picornavirales.

Insights into the Host Range, Genetic Diversity, and Geographical Distribution of Jingmenviruses.

Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low.IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure.

A Novel Polycipiviridae Virus Identified in Pteropus lylei Stools.

Polycipiviridae is a recently recognized viral family within the order Picornavirales with unusual genome organization and phylogenetic placement. Viruses belonging to this family were only reported from arthropod hosts. We describe here the first full genome of a distant polycipivirus-related virus identified in frugivorous bat stools in Cambodia.

Pteropus lylei primarily forages in residential areas in Kandal, Cambodia.

Bats are the second most species-rich Mammalian order and provide a wide range of ecologically important and economically significant ecosystem services. Nipah virus is a zoonotic emerging infectious disease for which pteropodid bats have been identified as a natural reservoir. In Cambodia, Nipah virus circulation has been reported in Pteropus lylei, but little is known about the spatial distribution of the species and the associated implications for conservation and public health.We deployed Global Positioning System (GPS) collars on 14 P. lylei to study their movements and foraging behavior in Cambodia in 2016. All of the flying foxes were captured from the same roost, and GPS locations were collected for 1 month. The habitats used by each bat were characterized through ground-truthing, and a spatial distribution model was developed of foraging sites.A total of 13,643 valid locations were collected during the study. Our study bats flew approximately 20 km from the roost each night to forage. The maximum distance traveled per night ranged from 6.88-105 km and averaged 28.3 km. Six of the 14 bats visited another roost for at least one night during the study, including one roost located 105 km away.Most foraging locations were in residential areas (53.7%) followed by plantations (26.6%). Our spatial distribution model confirmed that residential areas were the preferred foraging habitat for P. lylei, although our results should be interpreted with caution due to the limited number of individuals studied. Synthesis and applications: Our findings suggest that the use of residential and agricultural habitats by P. lylei may create opportunities for bats to interact with humans and livestock. They also suggest the importance of anthropogenic habitats for conservation of this vulnerable and ecologically important group in Cambodia. Our mapping of the probability of occurrence of foraging sites will help identification of areas where public awareness should be promoted regarding the ecosystem services provided by flying foxes and potential for disease transmission through indirect contact.

Extreme temperature event and mass mortality of insectivorous bats.

A mass mortality event involving Chaerephon plicatus and Taphozous theobaldi bats occurred during a heat wave in April 2016 in Cambodia. This was investigated to clarify the causes of the die-off and assess the risk to public health. Field evidences, clinical signs, and gross pathology findings were consistent with a heat stress hypothesis. However, the detection of a novel bat paramyxovirus raises questions about its role as a contributing factor or a coincidental finding. Systematic documentation of bat die-offs related to extreme weather events is necessary to improve understanding of the effect of changing weather patterns on bat populations and the ecosystem services they provide.

Detection of Low Pathogenicity Influenza A(H7N3) Virus during Duck Mortality Event, Cambodia, 2017.

In January 2017, an estimated 3,700 (93%) of 4,000 Khaki Campbell ducks (Anas platyrhynchos domesticus) died in Kampong Thom Province, Cambodia. We detected low pathogenicity avian influenza A(H7N3) virus and anatid herpesvirus 1 (duck plague) in the affected flock; however, the exact cause of the mortality event remains unclear.

Effects of mammarenavirus infection (Wenzhou virus) on the morphology of Rattus exulans.

The circulation of mammarenaviruses in rodent populations of the Mekong region has recently been established, with a genetic variant of Wenzhou virus, Cardamones virus, detected in two Rattus species. This study tests the potential teratogenic effects of Wenzhou infection on the development of a Murid rodent, Rattus exulans. Using direct virus detection, morphological records and comparative analyses, a link was demonstrated between host infection status and host morphologies (the spleen irrespective of weight, the skull shape and the cranial cavity volume) at the level of the individual (females only). This study demonstrates that mammarenavirus infections can impact natural host physiology and/or affect developmental processes. The presence of an infecting micro-parasite during the development of the rat may lead to a physiological trade-off between immunity and brain size. Alternatively, replication of virus in specialized organs can result in selective morphologic abnormalities and lesions.

Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Diversity of bat astroviruses in Lao PDR and Cambodia.

Astroviruses are known to infect humans and a wide range of animal species, and can cause gastroenteritis in their hosts. Recent studies have reported astroviruses in bats in Europe and in several locations in China. We sampled 1876 bats from 17 genera at 45 sites from 14 and 13 provinces in Cambodia and Lao PDR respectively, and tested them for astroviruses. Our study revealed a high diversity of astroviruses among various Yangochiroptera and Yinpterochiroptera bats. Evidence for varying degrees of host restriction for astroviruses in bats was found. Furthermore, additional Pteropodid hosts were detected. The astroviruses formed distinct phylogenetic clusters within the genus Mamastrovirus, most closely related to other known bat astroviruses. The astrovirus sequences were found to be highly saturated indicating that phylogenetic relationships should be interpreted carefully. An astrovirus clustering in a group with other viruses from diverse hosts, including from ungulates and porcupines, was found in a Rousettus bat. These findings suggest that diverse astroviruses can be found in many species of mammals, including bats.

Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (ßCoV), respectively. The ßCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat ßCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of ßCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii.

Evidence of human infection by a new mammarenavirus endemic to Southeastern Asia.

Southeastern Asia is a recognised hotspot for emerging infectious diseases, many of which have an animal origin. Mammarenavirus infections contribute significantly to the human disease burden in both Africa and the Americas, but little data exists for Asia. To date only two mammarenaviruses, the widely spread lymphocytic choriomeningitis virus and the recently described Wenzhou virus have been identified in this region, but the zoonotic impact in Asia remains unknown. Here we report the presence of a novel mammarenavirus and of a genetic variant of the Wenzhou virus and provide evidence of mammarenavirus-associated human infection in Asia. The association of these viruses with widely distributed mammals of diverse species, commonly found in human dwellings and in peridomestic habitats, illustrates the potential for widespread zoonotic transmission and adds to the known aetiologies of infectious diseases for this region.