Phlebotomine sand flies (Diptera, Psychodidae) from Pha Tong cave, Northern Thailand with a description of two new species and taxonomical thoughts about Phlebotomus stantoni.

BACKGROUND: In South-East Asia, Thailand is the country with the highest number of human autochthonous cases of leishmaniases mostly due to Leishmania martiniquensis. Their transmission remains unresolved to date even though sand flies are known vectors of leishmaniases. As such, we focused a study on the sand fly fauna of a cave in Thailand to explore the biodiversity of potential Leishmania vectors. MAIN RESULTS: We carried out an inventory in Pha Tong cave. We caught and identified 570 Phlebotomine sand flies (452 females and 118 males) and identified 14 species belonging to the genera Phlebotomus, Idiophlebotomus, Chinius, Sergentomyia and Grassomyia. Among these 14 species, two could not be related to known sand fly species. Herein, we propose the description of two new sand fly species, previously unknown to science. The first new species, Phlebotomus shadenae n. sp. is a sand fly of the subgenus Anaphlebotomus. It is morphologically close to Ph. stantoni, a species widely distributed throughout Southeast Asia. However, it differs by the length of the genital filaments in males or by the length of the ducts of the spermathecae in females as well as the high divergence of cytochrome b sequences. Additionally, we revised the systematics of the subgenus Anaphlebotomus and reinstated, by examination of its holotype, the validity of Ph. maynei, an Indian wrongly considered as a synonym of Ph. stantoni in the past. The second new species, Sergentomyia maiae n. sp., differs from a species in the same group, Se. barraudi, by an original cibarial double row of vertical teeth as well as by molecular data. CONCLUSIONS: We propose the description of two new sand fly species for Science with morphological and molecular evidence. Ph. shadenae n. sp. was also found to be distributed in the south of Thailand and in Laos. Future studies need to determine whether these two species can play a role as vectors of Leishmania parasites, Trypanosomatids or Phlebovirus. Most of the species caught in the present study are strictly cavernicolous except Grassomyia sp. and a few Sergentomyia.

On the systematics of Phlebotomus betisi and two new related species from Laos with proposal of the new subgenus Lewisius.

Phlebotomus betisi was described from Malaysia and classified after its description in the subgenus Larroussius. It was the only species to have a pharyngeal armature composed of dot-like teeth and an annealed spermatheca whose head is carried by a neck in females. Males were characterized by having a style bearing five spines and a simple paramere. The study of sandflies originating from a cave in Laos enabled us to discover and describe two sympatric species close to Ph. betisi Lewis & Wharton, 1963 and new for Science: Ph. breyi Vongphayloth & Depaquit n. sp., and Ph. sinxayarami Vongphayloth & Depaquit n. sp. They were characterized morphologically, morphometrically, geomorphometrically, molecularly, and proteomically (MALDI-TOF). All approaches converged to validate the individualization of these species whose morphological differential characters lay in the two genders by the observation of the interocular suture and by the length of the last two segments of the maxillary palps. In males, the length of the genital filaments discriminates these species. Females are distinguished by the length of the ducts of the spermathecae as well as by the narrow or enlarged shape of the neck bearing their head. Lastly, the particular position of the spines of the gonostyle coupled with molecular phylogeny led us to remove these three species from the subgenus Larroussius Nizulescu, 1931 and to classify them in a new subgenus: Lewisius Depaquit & Vongphayloth n. subg.

Title: Sur la systématique de Phlebotomus betisi et de deux nouvelles espèces apparentées du Laos avec proposition du nouveau sous-genre Lewisius. Abstract: Phlebotomus betisi a été décrit de Malaisie et fut classé après sa description dans le sous-genre Larroussius. C'était la seule espèce à posséder chez la femelle une armature pharyngienne composée de dents en forme de points et à avoir une spermathèque annelée dont la tête est portée par un cou. Les mâles se caractérisaient par un style porteur de cinq épines et par un paramère simple. L'étude de Phlébotomes originaires d'une grotte du Laos nous a permis de découvrir et de décrire deux espèces sympatriques proches de Ph. betisi Lewis & Wharton, 1963 et nouvelles pour la Science : Ph. breyi Vongphayloth & Depaquit n. sp., et Ph. sinxayarami Vongphayloth & Depaquit n. sp. Elles ont été caractérisées morphologiquement, morphométriquement, géomorphométriquement, moléculairement et protéomiquement (MALDI-TOF). Toutes ces approches convergent pour valider l'individualisation de chacune de ces espèces dont les caractères morphologiques différentiels reposent dans les deux sexes par l'observation de la suture interoculaire et par la longueur des deux derniers segments des palpes maxillaires. Chez les mâles, la longueur des filaments génitaux discrimine ces espèces. Les femelles sont distinguées par la longueur des conduits des spermathèques ainsi que par la forme étroite ou élargie du cou portant la tête de ces spermathèques. Enfin, la position particulière des épines sur le gonostyle couplée à une phylogénie moléculaire nous amène à extraire ces trois espèces du sous genre Larroussius Nitzulescu, 1931 pour les classer dans un nouveau sous-genre : Lewisius Depaquit & Vongphayloth n. subg.

The Genus Haemaphysalis (Acari: Ixodidae) in Laos: An Update of Species Records and a Review of Taxonomic Challenges.

Haemaphysalis Koch, 1844, is the largest genus of ticks in Southeast Asia, but little information is available concerning the species present in Laos. Recent research has yielded records for 10 Haemaphysalis species in Laos, including 5 new records, as well as 3 morphological entities of uncertain status. Further morphological and molecular studies are needed to clarify our taxonomic understanding of this genus in Southeast Asia.

Bat coronaviruses related to SARS-CoV-2 and infectious for human cells.

The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1-4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7-9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10-12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

Low seroprevalence of COVID-19 in Lao PDR, late 2020.

BACKGROUND: In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure. METHODS: Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex. FINDINGS: In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative). INTERPRETATION: We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses. FUNDING: Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs ("PaReCIDS II").

Screening of ectoparasites from domesticated dogs for bacterial pathogens in Vientiane, Lao PDR.

Arthropod-borne diseases are widespread worldwide and are a complex interaction between animals, humans and ectoparasites. The understanding of the diversity and epidemiology of organisms transmitted by arthropod vectors, and the role of hosts and vectors in transmission of infections remain limited in Lao PDR. What knowledge does exist is primarily focused on more rural regions of the country. This study screened ectoparasites from domestic dogs in Vientiane city for the presence of bacterial pathogens of zoonotic importance. A total of 3,511 arthropod vectors were collected from 112 dogs. Vectors collected were Rhipicephalus sanguineus ticks, Ctenocephalides felis felis and Ctenocephalides felis orientis fleas and Heterodoxus spiniger lice. A sub-sample of vectors from each dog was analysed by PCR to identify the potential bacteria. From 129 vector pools, Rickettsia spp. was detected in 6.7% (7/105) pools of ticks, 86.4% (19/22) pools of fleas and both pools of lice. Sequencing analysis confirmed Rickettsia felis in 13 flea pools and one louse pool and Rickettsia asembonensis in six flea pools. Anaplasmataceae was identified in 14.3% (15/105) tick pools and 100% (22/22) flea pools. Sequencing revealed the presence of Anaplasma platys in ticks and Wolbachia pipientis in fleas. Leptospira spp. was detected in one tick and one louse pool, and Brucella spp. was detected in 12.4% (13/105) tick pools. All samples were negative for Bartonella spp., Coxiella burnetii and Borrelia burgdorferi. This is the first study providing evidence of R. asembonensis in fleas in Laos. Results from this study show arthropods are potential vectors to transmit zoonotic infection in Vientiane city, suggesting humans are at risk of zoonotic infections in the city.

Description of a new species of Dermacentor Koch, 1844 (Acari: Ixodidae) from the mountains of Laos and Thailand.

Dermacentor pasteuri n. sp. (Acari: Ixodidae) is described based on adults ex wild boar and vegetation from Laos and Thailand. Adults of D. pasteuri n. sp. are similar to those of D. compactus Neumann, 1901, but can be distinguished by the shape of conscutum in the male, development of dorsal cornua in the female, size and shape of spurs on coxae and coloration of leg segments in both sexes.

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.

Six Nearly Complete Genome Segments of a Novel Reovirus Identified in Laotian Batflies.

As part of the characterization of viral communities of Laotian batflies, we report here the sequencing of six nearly complete genome segments of a novel reovirus identified in Laotian batflies that is distantly related to reoviruses recently reported in various Diptera species.

Description of a new species of Dermacentor Koch, 1844 (Acari: Ixodidae) from Laos and Thailand.

Dermacentor laothaiensis n. sp. (Acari: Ixodidae) is described based on adults ex wild boar and vegetation from Laos and Thailand. Adults of D. laothaiensis n. sp. are similar to those of D. bellulus (Schulze, 1935) and D. steini (Schulze, 1933) but can be distinguished by the conscutum shape, colour pattern of the conscutum and scutum, the density of punctations on the pseudoscutum and scutum and the shape of female genital structures.

New records and updated checklist of mosquitoes (Diptera: Culicidae) from Lao People's Democratic Republic, with special emphasis on adult and larval surveillance in Khammuane Province.

A list of mosquitoes from the Nakai Nam Theun National Protected Area along the Nam Theun, Nam Mon, Nam Noy, and Nam On rivers, Nakai District, Khammuane Province, Lao People's Democratic Republic (Lao PDR) is presented. Fifty-four mosquito taxa were identified, including 15 new records in the Lao PDR. A fragment of the mtDNA cytochrome c oxidase subunit I (COI) gene, barcode region, was generated for 34 specimens, and together with four specimens already published, it represented 23 species in eight genera. In addition, an updated checklist of 170 mosquito taxa from Lao PDR is provided based on field collections from Khammuane Province, the literature, and specimens deposited in the Smithsonian Institution, National Museum of Natural History (SI-NMNH), Washington, DC, U.S.A. This paper provides additional information about the biodiversity of mosquito fauna in Lao PDR.

First Record of Aedes (Stegomyia) malayensis Colless (Diptera: Culicidae) in the Lao People's Democratic Republic, Based on Morphological Diagnosis and Molecular Analysis.

This is the first confirmed record of Aedes (Stegomyia) malayensis Colless from the Lao People's Democratic Republic. Its larvae were collected from rock pools and rock holes along the Nam Noy River in the Nakai Nam Theun National Protected Area, Khammuane Province. Larvae were reared in the laboratory and emerged adults were identified based on morphological characters and mitochondrial DNA analysis, using data from the mitochondrial cytochrome c oxidase subunit I. Detailed photographs of the morphological diagnostic characters and information on the bionomics of Ae. malayensis are included.

New Locality Records of Ixodes granulatus and Ixodes vespertilionis (Acari: Ixodidae) From Tree-Shrews (Scandentia: Tupaiidae) and Bats (Chiroptera: Hipposideridae) in Laos.

Between May and June 2015, 512 bats, six rodents, and six tree-shrews were examined for ectoparasites in Vientiane and Khammouane Provinces, Laos. Thirty-seven ticks (33 females and four males) identified as Ixodes granulatus Supino were collected from four individuals of the Northern tree-shrew, Tupaia belangeri (Wagner) (Scandentia:Tupaiidae), and one female of Ixodes vespertilionis Koch was collected from one individual of the Intermediate Leaf-nosed Bat, Hipposideros larvatus (Horsfield) (Chiroptera:Hipposideridae). This appears to be the first record of I. vespertilionis from Laos, as well as the first record of this host-parasite relationship in the Southeast Asia region.

The Genus Dermacentor (Acari: Ixodidae) in Laos: A Review and Update of Species Records.

Two species of the ixodid tick genus Dermacentor Koch, 1844 (Acari: Ixodidae) have previously been documented from Laos: D. auratus Supino, 1897 and D. steini (Schulze, 1933). Between 2012 and 2017, we recorded four additional Dermacentor species from this country: D. bellulus (Schulze, 1935); D. compactus Neumann, 1901; D. filippovae Apanaskevich & Apanaskevich, 2015; and D. tamokensisApanaskevich & Apanaskevich, 2016. In addition, seven specimens in the tick collection at the Institut Pasteur du Laos may represent new species and are currently under taxonomic investigation.

Large-Scale Survey for Tickborne Bacteria, Khammouan Province, Laos.

We screened 768 tick pools containing 6,962 ticks from Khammouan Province, Laos, by using quantitative real-time PCR and identified Rickettsia spp., Ehrlichia spp., and Borrelia spp. Sequencing of Rickettsia spp.-positive and Borrelia spp.-positive pools provided evidence for distinct genotypes. Our results identified bacteria with human disease potential in ticks in Laos.

Mosquito Fauna of Lao People's Democratic Republic, with Special Emphasis on the Adult and Larval Surveillance at Nakai District, Khammuane Province.

This article includes the distribution records and updated checklist of mosquitoes (Culicidae, Diptera) from the Lao People's Democratic Republic (PDR), based on the literature, specimens deposited at the US National Museum of Natural History mosquito collections, and our recent field collections from the Nakai District, Khammuane Province. Ten of 101 species in the updated checklist of mosquitoes are new records for the Lao PDR.

MALACOLOGICAL INVESTIGATION OF THE FULLY OPERATIONAL NAM THEUN 2 HYDROELECTRIC DAM PROJECT IN KHAMMOUANE PROVINCE, CENTRAL LAO PDR.

We conducted a malacological investigation in four districts of the Nam Theun 2 (NT2) hydroelectric dam project area, Khammouane Province, central Lao PDR (Nakai, Gnommalath, Mahaxai and Xe Bang Fai), after the first and second years of full operation in March 2010 and November 2011 to determine health risks for humans. A total 10,863 snail specimens (10 families/23 species) from 57 sampling stations and 12,902 snail specimens (eight families/21 species) from 66 sampling stations were collected in 2010 and 2011, respectively. Neotricula aperta (gamma race), the intermediate host for Schistosoma mekongi, was found in large numbers (5,853 specimens) in 2010 in Nam Gnom (downstream) at Station 25 (Mueang Gnommalath: Gnommalath District) and in fewer numbers (170 specimens) at Station 26 (Ban Thathod: Gnommalath District). In 2011, significantly fewer numbers (434 specimens) of N. aperta were found at Station 25. No snails were found to be infected with S. mekongi; however, 3.6% and 0.45% of Bithynia (D.). s. goniomphalos specimens collected were found to be infected with Opisthorchis viverrini (human liver fluke) during 2010 and 2011, respectively. Pomacea canaliculata, the rice crop pest, the intermediate host of Angiostrongylus (Parastrongylus) cantonensis, was found in the greatest numbers during 2010 and 2011; the prevalence increased significantly from 1.3% in 2010 to 53.3% in 2011. We also found seasonal variation in snail populations in terms of abundance and diversity. The snail fauna and risk for transmission of parasitic diseases need to be monitored continuously to evaluate the long-term impact of the dam project.

Risk factors for the presence of Aedes aegypti and Aedes albopictus in domestic water-holding containers in areas impacted by the Nam Theun 2 hydroelectric project, Laos.

We assessed risk factors for vectors of dengue and chikungunya viruses near a new hydroelectric project, Nam Theun 2, in Laos. Immature stages of Aedes aegypti were found only in sites within 40 km of the urban provincial capital, but Aedes albopictus was found throughout. Aedes aegypti pupae were most common in water storage jars (odds ratio [OR] = 4.72) and tires (OR = 2.99), and Ae. albopictus pupae were associated with tires in 2009 (OR = 10.87) and drums, tires, and jars in 2010 (drums OR = 3.05; tires OR = 3.45, jars OR = 6.59). Compared with water storage vessels, containers used for hygiene, cooking, and drinking were 80% less likely to harbor Ae. albopictus pupae in 2010 (OR = 0.20), and discarded waste was associated with a 3.64 increased odds of infestation. Vector control efforts should focus on source reduction of water storage containers, particularly concrete jars and tires.

Burkholderia pseudomallei detection in surface water in southern Laos using Moore's swabs.

The causal agent of melioidosis, Burkholderia pseudomallei, has been cultured from paddy fields in the Lao PDR. We carried out a pilot study to examine the relationship between bacterial soil contamination and that of nearby surface waters in Saravane Province. Soil sampling was conducted at a depth of 30 cm (100 holes in a 45 × 45 m grid) at two sites, East and West Saravane. Moore's swabs were used for water sampling of paddy fields, lakes, rivers, boreholes, and storage tanks within 2 km of the two soil sampling sites. B. pseudomallei from soil and water were cultured on Ashdown's agar. Thirty-six percent and 6% of water samples collected around East and West Saravane, respectively, were culture positive for B. pseudomallei. Low pH and high turbidity were independently associated with culture of B. pseudomallei. Most positive water samples were from the Sedone River, downstream of the East Saravane site. Moore's swabs are simple and inexpensive tools for detecting B. pseudomallei in surface waters.