Genetic diversity of Nipah virus in Bangladesh.

BACKGROUND: Nipah virus (NiV) infection, often fatal in humans, is primarily transmitted in Bangladesh through the consumption of date palm sap contaminated by Pteropus bats. Person-to-person transmission is also common and increases the concern of large outbreaks. This study aimed to characterize the molecular epidemiology, phylogenetic relationship, and the evolution of the nucleocapsid gene (N gene) of NiV. METHODS: We conducted molecular detection, genetic characterization, and Bayesian time-scale evolution analyses of NiV using pooled Pteropid bat roost urine samples from an outbreak area in 2012 and archived RNA samples from NiV case patients identified during 2012-2018 in Bangladesh. RESULTS: NiV-RNA was detected in 19% (38/456) of bat roost urine samples and among them; nine N gene sequences were recovered. We also retrieved sequences from 53% (21 out of 39) of archived RNA samples from patients. Phylogenetic analysis revealed that all Bangladeshi strains belonged to NiV-BD genotype and had an evolutionary rate of 4.64 × 10-4 substitutions/site/year. The analyses suggested that the strains of NiV-BD genotype diverged during 1995 and formed two sublineages. CONCLUSION: This analysis provides further evidence that the NiV strains of the Malaysian and Bangladesh genotypes diverged recently and continue to evolve. More extensive surveillance of NiV in bats and human will be helpful to explore strain diversity and virulence potential to infect humans through direct or person-to-person virus transmission.

Summarizing US Wildlife Trade with an Eye Toward Assessing the Risk of Infectious Disease Introduction.

The aim of this study was to characterize the role of the USA in the global exchange of wildlife and describe high volume trade with an eye toward prioritizing health risk assessment questions for further analysis. Here we summarize nearly 14 years (2000-2013) of the most comprehensive data available (USFWS LEMIS system), involving 11 billion individual specimens and an additional 977 million kilograms of wildlife. The majority of shipments contained mammals (27%), while the majority of specimens imported were shells (57%) and tropical fish (25%). Most imports were facilitated by the aquatic and pet industry, resulting in one-third of all shipments containing live animals. The importer reported origin of wildlife was 77.7% wild-caught and 17.7% captive-reared. Indonesia was the leading exporter of legal shipments, while Mexico was the leading source reported for illegal shipments. At the specimen level, China was the leading exporter of legal and illegal wildlife imports. The number of annual declared shipments doubled during the period examined, illustrating continually increasing demand, which reinforces the need to scale up capacity for border inspections, risk management protocols and disease surveillance. Most regulatory oversight of wildlife trade is aimed at conservation, rather than prevention of disease introduction.

Efficiency of the Clinical Veterinary Diagnostic Practices and Drug Choices for Infectious Diseases in Livestock in Bangladesh.

As in most low-income countries, adequate laboratory facilities are not available in Bangladesh to assist veterinarians in diagnosing animal diseases. We aimed to determine the efficiency of veterinary diagnoses for two common ruminant diseases in Bangladesh: Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD). We conducted the study from May 2009 to August 2010 in three government veterinary hospitals where veterinarians collected samples from sick livestock and recorded the presumptive diagnosis on the basis of clinical presentations. Samples were tested for PPR and FMD using real-time RT-PCR. We estimated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the presumptive diagnoses when compared to laboratory tests. We tested 539 goats for PPR and 340 cattle and goats for FMD. Our results indicate that the veterinarians' presumptive diagnoses were different from laboratory findings for both PPR (P < 0.05) and FMD (P < 0.05). The overall sensitivity of the presumptive clinical diagnoses was 54% (95% CI: 47-61%) while specificity was 81% (95% CI: 78-84%) compared to real-time RT-PCR tests. The kappa value obtained in our validation process for PPR (kappa: 0.25) and FMD (kappa 0.36) indicated a poor performance of the presumptive diagnoses. Most of the animals (93%) were treated with antibiotics. Our findings indicate that veterinarians can detect animals not infected with FMD or PPR but miss the true cases. The clinical competency of these veterinarians needs to be improved and access to laboratory diagnostic facilities could help veterinarians to improve the diagnostics and outcomes. The rational use of antibiotics by veterinarians in animals must be ensured.

Bat Hunting and Bat-Human Interactions in Bangladeshi Villages: Implications for Zoonotic Disease Transmission and Bat Conservation.

Bats are an important reservoir for emerging zoonotic pathogens. Close human-bat interactions, including the sharing of living spaces and hunting and butchering of bats for food and medicines, may lead to spillover of zoonotic disease into human populations. We used bat exposure and environmental data gathered from 207 Bangladeshi villages to characterize bat exposures and hunting in Bangladesh. Eleven percent of households reported having a bat roost near their homes, 65% reported seeing bats flying over their households at dusk, and 31% reported seeing bats inside their compounds or courtyard areas. Twenty percent of households reported that members had at least daily exposure to bats. Bat hunting occurred in 49% of the villages surveyed and was more likely to occur in households that reported nearby bat roosts (adjusted prevalence ratio [aPR] 2.3, 95% CI 1.1-4.9) and villages located in north-west (aPR 7.5, 95% CI 2.5-23.0) and south-west (aPR 6.8, 95% CI 2.1-21.6) regions. Our results suggest high exposure to bats and widespread hunting throughout Bangladesh. This has implications for both zoonotic disease spillover and bat conservation.

Evolving epidemiology of Nipah virus infection in Bangladesh: evidence from outbreaks during 2010-2011.

Drinking raw date palm sap is the primary route of Nipah virus (NiV) transmission from bats to people in Bangladesh; subsequent person-to-person transmission is common. During December 2010 to March 2011, we investigated NiV epidemiology by interviewing cases using structured questionnaires, in-depth interviews, and group discussions to collect clinical and exposure histories. We conducted a case-control study to identify risk factors for transmission. We identified 43 cases; 23 were laboratory-confirmed and 20 probable. Thirty-eight (88%) cases died. Drinking raw date palm sap and contact with an infected person were major risk factors; one healthcare worker was infected and for another case transmission apparently occurred through contact with a corpse. In absence of these risk factors, apparent routes of transmission included drinking fermented date palm sap. For the first time, a case was detected in eastern Bangladesh. Identification of new epidemiological characteristics emphasizes the importance of continued NiV surveillance and case investigation.

Integrated cluster- and case-based surveillance for detecting stage III zoonotic pathogens: an example of Nipah virus surveillance in Bangladesh.

This paper explores the utility of cluster- and case-based surveillance established in government hospitals in Bangladesh to detect Nipah virus, a stage III zoonotic pathogen. Physicians listed meningo-encephalitis cases in the 10 surveillance hospitals and identified a cluster when ⩾2 cases who lived within 30 min walking distance of one another developed symptoms within 3 weeks of each other. Physicians collected blood samples from the clustered cases. As part of case-based surveillance, blood was collected from all listed meningo-encephalitis cases in three hospitals during the Nipah season (January-March). An investigation team visited clustered cases' communities to collect epidemiological information and blood from the living cases. We tested serum using Nipah-specific IgM ELISA. Up to September 2011, in 5887 listed cases, we identified 62 clusters comprising 176 encephalitis cases. We collected blood from 127 of these cases. In 10 clusters, we identified a total of 62 Nipah cases: 18 laboratory-confirmed and 34 probable. We identified person-to-person transmission of Nipah virus in four clusters. From case-based surveillance, we identified 23 (4%) Nipah cases. Faced with thousands of encephalitis cases, integrated cluster surveillance allows targeted deployment of investigative resources to detect outbreaks by stage III zoonotic pathogens in resource-limited settings.

Identification and epidemiology of a rare HoBi-like pestivirus strain in Bangladesh.

The genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (BVDV-1), bovine viral diarrhoea virus 2 (BVDV-2), classical swine fever virus and border disease virus. A new putative pestivirus species tentatively named as either 'HoBi-like pestivirus' or BVDV-3 has recently been identified in Brazil, Italy and Thailand. Despite reports of serological evidence of BVDV in Bangladesh, the types of the virus circulating in cattle have not been identified. We conducted surveillance in cattle from May 2009 to August 2010 in three government veterinary hospitals to characterize BVDV in cattle of Bangladesh. We tested serum for BVDV using an antigen-capture ELISA. Of 638 cattle samples, 3% (16/638) tested positive for BVDV antigen. The ELISA-positive samples were selected for further molecular detection and characterization of BVDV. Molecular analysis of the partial 5' untranslated region (UTR) nucleotide sequences of BVDV-positive samples identified the rare HoBi-like pestivirus or BVDV-3 virus circulating in cattle of Bangladesh. The identification of this rare HoBi-like pestivirus or BVDV-3 strain in Bangladesh warrants further surveillance to evaluate its impact on livestock production.

West Nile virus in the British Virgin Islands.

West Nile virus (WNV) first emerged in the US in 1999 and has since spread across the Americas. Here, we report the continued expansion of WNV to the British Virgin Islands following its emergence in a flock of free-roaming flamingos. Histologic review of a single chick revealed lesions consistent with WNV infection, subsequently confirmed with PCR, immunohistochemistry and in situ hybridization. Full genome analysis revealed 99% sequence homology to strains circulating in the US over the past decade. This study highlights the need for rapid necropsy of wild bird carcasses to fully understand the impact of WNV on wild populations.

Coronaviruses in bats from Mexico.

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (ß)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were ß-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A ß-CoV with 96.5 % amino acid identity to the ß-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Emergence of fatal avian influenza in New England harbor seals.

UNLABELLED: From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission. IMPORTANCE: The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.

Evidence for Nipah virus recrudescence and serological patterns of captive Pteropus vampyrus.

This study aimed to describe the transmission dynamics, the serological and virus excretion patterns of Nipah virus (NiV) in Pteropus vampyrus bats. Bats in captivity were sampled every 7-21 days over a 1-year period. The data revealed five NiV serological patterns categorized as high and low positives, waning, decreasing and increasing, and negative in these individuals. The findings strongly suggest that NiV circulates in wild bat populations and that antibody could be maintained for long periods. The study also found that pup and juvenile bats from seropositive dams tested seropositive, indicating that maternal antibodies against NiV are transmitted passively, and in this study population may last up to 14 months. NiV was isolated from the urine of one bat, and within a few weeks, two other seronegative bats seroconverted. Based on the temporal cluster of seroconversion, we strongly believe that the NiV isolated was recrudesced and then transmitted horizontally between bats during the study period.

Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007.

In February 2007 an outbreak of Nipah virus (NiV) encephalitis in Thakurgaon District of northwest Bangladesh affected seven people, three of whom died. All subsequent cases developed illness 7-14 days after close physical contact with the index case while he was ill. Cases were more likely than controls to have been in the same room (100% vs. 9.5%, OR undefined, P<0.001) and to have touched him (83% vs. 0%, OR undefined, P<0.001). Although the source of infection for the index case was not identified, 50% of Pteropus bats sampled from near the outbreak area 1 month after the outbreak had antibodies to NiV confirming the presence of the virus in the area. The outbreak was spread by person-to-person transmission. Risk of NiV infection in family caregivers highlights the need for infection control practices to limit transmission of potentially infectious body secretions.

Henipaviruses: emerging paramyxoviruses associated with fruit bats.

Two related, novel, zoonotic paramyxoviruses have been described recently. Hendra virus was first reported in horses and thence humans in Australia in 1994; Nipah virus was first reported in pigs and thence humans in Malaysia in 1998. Human cases of Nipah virus infection, apparently unassociated with infection in livestock, have been reported in Bangladesh since 2001. Species of fruit bats (genus Pteropus) have been identified as natural hosts of both agents. Anthropogenic changes (habitat loss, hunting) that have impacted the population dynamics of Pteropus species across much of their range are hypothesised to have facilitated emergence. Current strategies for the management of henipaviruses are directed at minimising contact with the natural hosts, monitoring identified intermediate hosts, improving biosecurity on farms, and better disease recognition and diagnosis. Investigation of the emergence and ecology of henipaviruses warrants a broad, cross-disciplinary ecosystem health approach that recognises the critical linkages between human activity, ecological change, and livestock and human health.

Collaborative research approaches to the role of wildlife in zoonotic disease emergence.

Emerging infectious diseases are a key threat to public health and the majority are caused by zoonotic pathogens. Here we discuss new collaborative approaches to understanding the process of zoonotic disease emergence that link veterinary medicine, public health, and ecological approaches: conservation medicine and one health. We demonstrate how studies on the underlying drivers of disease emergence (bushmeat hunting, wildlife trade, and deforestation) can provide ways to model, predict, and ultimately prevent zoonotic disease emergence and spread.

Novel viral encephalitides associated with bats (Chiroptera)--host management strategies.

Several novel viruses recently described in bats of the genus Pteropus (sub-order Megachiroptera) in Australia and southeast Asia cause encephalitic disease in animals and humans. These viruses include Hendra virus and Nipah virus (genus Henipavirus, family Paramyxoviridae) and Australian bat lyssavirus (ABLV; genus Lyssavirus, family Rhabdoviridae). Broadly, strategies for disease prevention and control in the spillover host are directed at minimising direct or indirect contact with the natural host, improving farm-gate and on-farm biosecurity, and better disease recognition and diagnosis. Additional strategies for ABLV include the use of rabies vaccine for effective pre- and post-exposure prophylaxis in humans. Effective management strategies in the natural host are predicated on an understanding of the ecology of the disease in the natural host, and the identification and avoidance of factors putatively associated with emergence, such as habitat loss, land use change and demographic shifts. A possible future management strategy for ABLV in reservoir populations is immunisation using bait or plant-derived vaccination.

Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes.

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80 degrees C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies.

Parasitism by Dermocystidium ranae in a population of Rana esculenta complex in Central Italy and description of Amphibiocystidium n. gen.

We report the enigmatic parasite Dermocystidium ranae in a green frog population (Solomeo, Umbria, Italy) of the Rana esculenta complex, consisting of the parental species R. lessonae (L) and hybrid form R. esculenta (E). In this population a rapid 50% decline of the parental form L was observed. Large dermal U-shaped cysts of D. ranae were found primarily on the ventral aspect of infected individuals, with a significantly higher incidence of infection in the parental species compared to the clonal hybrid. In each form, however, there was little pathological change associated with infection, and the cause of the recent declines of R. lessonae at this site remains unknown. In this paper we present the first ultrastructural description of an amphibian Dermocystidium sp. and we review the taxonomy of Dermocystidium, Dermosporidium and Dermomycoides spp. from amphibians. We conclude that Dermosporidium multigranulare Broz & Kulda, 1954 is synonymous with Dermocystidium ranae Guyénot & Naville, 1922 and, due to lack of sufficient differences between genera and significant dissimilarities with fish Dermocystidium spp., the 3 amphibian genera are synonymous. We propose that they should be designated to a new genus, Amphibiocystidium n. gen., and Dermocystidium retained for those species parasitic in fish.

Five new species of coccidia (Apicomplexa: Eimeriidae) from Madagascan chameleons (Sauria: Chamaeleonidae).

Coprological examination of 19 Madagascan chameleons of the genera Furcifer and Brookesia revealed the presence of five new coccidian species. Isospora brygooi n. sp. from Furcifer pardalis has spherical to subspherical oöcysts with a slightly pitted wall, 20.7 (17-24.5) x 19.3 (16-23) microm and broadly ellipsoidal sporocysts, 12.2 (11.5-13) x 8.1 (8-8.5) microm, with Stieda and substieda bodies. Oöcysts of Eimeria glawi n. sp. from Furcifer pardalis are cylindrical to ellipsoidal, 27.7 (26-29.5) x 18.4 (17-19) microm, with ellipsoidal sporocysts, 7.3 (6.5-8) x 5.2 (5-5.5) microm. E. vencesi n. sp. described from F. pardalis has spherical to subspherical oöcysts, 14.3 (13-15.5) x 13.0 (12-13) microm, with small granules, one to three globular polar granules and ellipsoidal sporocysts, 7.3 (6.5-8) x 5.2 (5-5.5) microm. E. worthi n. sp., described from Furcifer oustaleti has spherical oöcysts, 17.9 (17.5-19.0) x 15.0 (14.5-16.0) microm without a polar granule and ellipsoidal to cylindroidal sporocysts, 8.2 (7.0-9.5) x 5.8 (5.0-6.5) microm. Oöcysts of E. brookesiae n. sp. from Brookesia decaryi are cylindrical, 25.6 (23-27) x 15.0 (13-16) microm with ellipsoidal sporocysts, 10.1 (9-11) x 6.9 (6-7) microm. Endogenous development of E. vencesi is confined to the intestine, while that of E. glawi occurs in the gall-bladder.