Use of Infrared Camera to Understand Bats’ Access to Date Palm Sap: Implications for Preventing Nipah Virus Transmission

Pteropus bats are commonly infected with Nipah virus, but show no signs of illness. Human Nipah outbreaks in Bangladesh coincide with the date palm sap harvesting season. In epidemiologic studies, drinking raw date palm sap is a risk factor for human Nipah infection. We conducted a study to evaluate bats’ access to date palm sap. We mounted infrared cameras that silently captured images upon detection of motion on date palm trees from 5:00 pm to 6:00 am. Additionally, we placed two locally used preventative techniques, bamboo skirts and lime (CaCO₃) smeared on date palm trees to assess their effectiveness in preventing bats access to sap. Out of 20 camera-nights of observations, 14 identified 132 visits of bats around the tree, 91 to the shaved surface of the tree where the sap flow originates, 4 at the stream of sap moving toward the collection pot, and no bats at the tap or on the collection pots; the remaining 6 camera-nights recorded no visits. Of the preventative techniques, the bamboo skirt placed for four camera-nights prevented bats access to sap. This study confirmed that bats commonly visited date palm trees and physically contacted the sap collected for human consumption. This is further evidence that date palm sap is an important link between Nipah virus in bats and Nipah virus in humans. Efforts that prevent bat access to the shaved surface and the sap stream of the tree could reduce Nipah spillovers to the human population.     

Date Palm Sap Collection: Exploring Opportunities to Prevent Nipah Transmission

Nipah virus (NiV) infection is a seasonal disease in Bangladesh that coincides with the date palm sap collection season. Raw date palm sap is a delicacy to drink in Bengali culture. If fruit bats that are infected with NiV gain access to the sap for drinking, they might occasionally contaminate the sap through saliva and urine. In February 2007, we conducted a qualitative study in six villages, interviewing 27 date palm sap collectors (gachhis) within the geographical area where NiV outbreaks have occurred since 2001. Gachhis reported that bats pose a challenge to successful collection of quality sap, because bats drink and defecate into the sap which markedly reduces its value. They know some methods to prevent access by bats and other pests but do not use them consistently, because of lack of time and resources. Further studies to explore the effectiveness of these methods and to motivate gachhis to invest their time and money to use them could reduce the risk of human Nipah infection in Bangladesh.     

Raw Sap Consumption Habits and Its Association with Knowledge of Nipah Virus in Two Endemic Districts in Bangladesh

Human Nipah virus (NiV) infection in Bangladesh is a fatal disease that can be transmitted from bats to humans who drink contaminated raw date palm sap collected overnight during the cold season. Our study aimed to understand date palm sap consumption habits of rural residents and factors associated with consumption. In November-December 2012 the field team interviewed adult respondents from randomly selected villages from Rajbari and Kushtia Districts in Bangladesh. We calculated the proportion of people who consumed raw sap and had heard about a disease from raw sap consumption. We assessed the factors associated with raw sap consumption by calculating prevalence ratios (PR) adjusted for village level clustering effects. Among the 1,777 respondents interviewed, half (50%) reported drinking raw sap during the previous sap collection season and 37% consumed raw sap at least once per month. Few respondents (5%) heard about NiV. Thirty-seven percent of respondents reported hearing about a disease transmitted through raw sap consumption, inclusive of a 10% who related it with milder illness like diarrhea, vomiting or indigestion rather than NiV. Respondents who harvested date palm trees in their household were more likely to drink sap than those who did not own date palm trees (79% vs. 65% PR 1.2, 95% CI 1.1–1.3, p<0.001). When sap was available, respondents who heard about a disease from raw sap consumption were just as likely to drink it as those who did not hear about a disease (69% vs. 67%, PR 1.0, 95% CI 0.9–1.1, p = 0.512). Respondents’ knowledge of NiV was low. They might not have properly understood the risk of NiV, and were likely to drink sap when it was available. Implementing strategies to increase awareness about the risks of NiV and protect sap from bats might reduce the risk of NiV transmission.     

Nipah virus transmission in a hamster model

Based on epidemiological data, it is believed that human-to-human transmission plays an important role in Nipah virus outbreaks. No experimental data are currently available on the potential routes of human-to-human transmission of Nipah virus. In a first dose-finding experiment in Syrian hamsters, it was shown that Nipah virus was predominantly shed via the respiratory tract within nasal and oropharyngeal secretions. Although Nipah viral RNA was detected in urogenital and rectal swabs, no infectious virus was recovered from these samples, suggesting no viable virus was shed via these routes. In addition, hamsters inoculated with high doses shed significantly higher amounts of viable Nipah virus particles in comparison with hamsters infected with lower inoculum doses. Using the highest inoculum dose, three potential routes of Nipah virus transmission were investigated in the hamster model: transmission via fomites, transmission via direct contact and transmission via aerosols. It was demonstrated that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. These findings are in line with epidemiological data which suggest that direct contact with nasal and oropharyngeal secretions of Nipah virus infected individuals resulted in greater risk of Nipah virus infection. The data provide new and much-needed insights into the modes and efficiency of Nipah virus transmission and have important public health implications with regards to the risk assessment and management of future Nipah virus outbreaks.     

A Controlled Trial to Reduce the Risk of Human Nipah Virus Exposure in Bangladesh

Human Nipah virus (NiV) infection, often fatal in Bangladesh, is primarily transmitted by drinking raw date palm sap contaminated by Pteropus bats. We assessed the impact of a behavior change communication intervention on reducing consumption of potentially NiV-contaminated raw sap. During the 2012–2014 sap harvesting seasons, we implemented interventions in two areas and compared results with a control area. In one area, we disseminated a “do not drink raw sap” message and, in the other area, encouraged only drinking sap if it had been protected from bat contamination by a barrier (“only safe sap”). Post-intervention, 40% more respondents in both intervention areas reported knowing about a disease contracted through raw sap consumption compared with control. Reported raw sap consumption decreased in all areas. The reductions in the intervention areas were not significantly greater compared to the control. Respondents directly exposed to the “only safe sap” message were more likely to report consuming raw sap from a protected source than those with no exposure (25 vs. 15%, OR 2.0, 95% CI 1.5–2.6, P < 0.001). While the intervention increased knowledge in both intervention areas, the “only safe sap” intervention reduced exposure to potentially NiV-contaminated sap and should be considered for future dissemination.     

A randomized controlled trial of interventions to impede date palm sap contamination by bats to prevent nipah virus transmission in bangladesh

Background

Drinking raw date palm sap is a risk factor for human Nipah virus (NiV) infection. Fruit bats, the natural reservoir of NiV, commonly contaminate raw sap with saliva by licking date palm’s sap producing surface. We evaluated four types of physical barriers that may prevent bats from contacting sap.

Methods

During 2009, we used a crossover design and randomly selected 20 date palm sap producing trees and observed each tree for 2 nights: one night with a bamboo skirt intervention applied and one night without the intervention. During 2010, we selected 120 trees and randomly assigned four types of interventions to 15 trees each: bamboo, dhoincha (local plant), jute stick and polythene skirts covering the shaved part, sap stream, tap and collection pot. We enrolled the remaining 60 trees as controls. We used motion sensor activated infrared cameras to examine bat contact with sap.

Results

During 2009 bats contacted date palm sap in 85% of observation nights when no intervention was used compared with 35% of nights when the intervention was used [p<0.001]. Bats were able to contact the sap when the skirt did not entirely cover the sap producing surface. Therefore, in 2010 we requested the sap harvesters to use larger skirts. During 2010 bats contacted date palm sap [2% vs. 83%, p<0.001] less frequently in trees protected with skirts compared to control trees. No bats contacted sap in trees with bamboo (p<0.001 compared to control), dhoincha skirt (p<0.001) or polythene covering (p<0.001), but bats did contact sap during one night (7%) with the jute stick skirt (p<0.001).

Conclusion

Bamboo, dhoincha, jute stick and polythene skirts covering the sap producing areas of a tree effectively prevented bat-sap contact. Community interventions should promote applying these skirts to prevent occasional Nipah spillovers to human.     

Serological Evidence of Henipavirus Exposure in Cattle,Goats and Pigs in Bangladesh

Background

Nipah virus (NiV) is an emerging disease that causes severe encephalitis and respiratory illness in humans. Pigs were identified as an intermediate host for NiV transmission in Malaysia. In Bangladesh, NiV has caused recognized human outbreaks since 2001 and three outbreak investigations identified an epidemiological association between close contact with sick or dead animals and human illness.

Methodology

We examined cattle and goats reared around Pteropus bat roosts in human NiV outbreak areas. We also tested pig sera collected under another study focused on Japanese encephalitis.

Principal Findings

We detected antibodies against NiV glycoprotein in 26 (6.5%) cattle, 17 (4.3%) goats and 138 (44.2%) pigs by a Luminex-based multiplexed microsphere assay; however, these antibodies did not neutralize NiV. Cattle and goats with NiVsG antibodies were more likely to have a history of feeding on fruits partially eaten by bats or birds (PR = 3.1, 95% CI 1.6–5.7) and drinking palmyra palm juice (PR = 3.9, 95% CI 1.5–10.2).

Conclusions

This difference in test results may be due to the exposure of animals to one or more novel viruses with antigenic similarity to NiV. Further research may identify a novel organism of public health importance.     

Cluster of Nipah virus infection, Kushtia District, Bangladesh, 2007

Objective

In March 2007, we investigated a cluster of Nipah encephalitis to identify risk factors for Nipah infection in Bangladesh.

Methods

We defined confirmed Nipah cases by the presence of IgM and IgG antibodies against Nipah virus in serum. Case-patients, who resided in the same village during the outbreak period but died before serum could be collected, were classified as probable cases.

Results

We identified three confirmed and five probable Nipah cases. There was a single index case. Five of the secondary cases came in close physical contact to the index case when she was ill. Case-patients were more likely to have physical contact with the index case (71% cases versus 0% controls, p = <0.001). The index case, on her third day of illness, and all the subsequent cases attended the same religious gathering. For three probable cases including the index case, we could not identify any known risk factors for Nipah infection such as physical contact with Nipah case-patients, consumption of raw date palm juice, or contact with sick animals or fruit bats.

Conclusion

Though person-to-person transmission remains an important mode of transmission for Nipah infection, we could not confirm the source of infection for three of the probable Nipah case-patients. Continued surveillance and outbreak investigations will help better understand the transmission of Nipah virus and develop preventive strategies.     

Mapping Risk of Nipah Virus Transmission from Bats to Humans in Thailand

Nipah virus (NiV) is a zoonotic virus that can pose a serious threat to human and livestock health. Old-world fruit bats (Pteropus spp.) are the natural reservoir hosts for NiV, and Pteropus lylei, Lyle’s flying fox, is an important host of NiV in mainland Southeast Asia. NiV can be transmitted from bats to humans directly via bat-contaminated foods (i.e., date palm sap or fruit) or indirectly via livestock or other intermediate animal hosts. Here we construct risk maps for NiV spillover and transmission by combining ecological niche models for the P. lylei bat reservoir with other spatial data related to direct or indirect NiV transmission (livestock density, foodborne sources including fruit production, and human population). We predict the current and future (2050 and 2070) distribution of P. lylei across Thailand, Cambodia, and Vietnam. Our best-fit model predicted that central and western regions of Thailand and small areas in Cambodia are currently the most suitable habitats for P. lylei. However, due to climate change, the species range is predicted to expand to include lower northern, northeastern, eastern, and upper southern Thailand and almost all of Cambodia and lower southern Vietnam. This expansion will create additional risk areas for human infection from P. lylei in Thailand. Our combined predictive risk maps showed that central Thailand, inhabited by 2.3 million people, is considered highly suitable for the zoonotic transmission of NiV from P. lylei. These current and future NiV transmission risk maps can be used to prioritize sites for active virus surveillance and developing awareness and prevention programs to reduce the risk of NiV spillover and spread in Thailand.

     

Population genetics of fruit bat reservoir informs the dynamics,distribution and diversity of Nipah virus

The structure and connectivity of wildlife host populations may influence zoonotic disease dynamics, evolution and therefore spillover risk to people. Fruit bats in the genus Pteropus, or flying foxes, are the primary natural reservoir for henipaviruses—a group of emerging paramyxoviruses that threaten livestock and public health. In Bangladesh, Pteropus medius is the reservoir for Nipah virus—and viral spillover has led to human fatalities nearly every year since 2001. Here, we use mitochondrial DNA and nuclear microsatellite markers to measure the population structure, demographic history and phylogeography of P. medius in Bangladesh. We combine this with a phylogeographic analysis of all known Nipah virus sequences and strains currently available to better inform the dynamics, distribution and evolutionary history of Nipah virus. We show that P. medius is primarily panmictic, but combined analysis of microsatellite and morphological data shows evidence for differentiation of two populations in eastern Bangladesh, corresponding to a divergent strain of Nipah virus also found in bats from eastern Bangladesh. Our demographic analyses indicate that a large, expanding population of flying foxes has existed in Bangladesh since the Late Pleistocene, coinciding with human population expansion in South Asia, suggesting repeated historical spillover of Nipah virus likely occurred. We present the first evidence of mitochondrial introgression, or hybridization, between P. medius and flying fox species found in South‐East Asia (P. vampyrus and P. hypomelanus), which may help to explain the distribution of Nipah virus strains across the region.     

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters

In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.     

A Neutralizing Human Monoclonal Antibody Protects against Lethal Disease in a New Ferret Model of Acute Nipah Virus Infection

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID₅₀ within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.     

Nipah virus infection

Nipah virus (NiV), emerged in Peninsular Malaysia, caused an outbreak of severe febrile encephalitis in humans and respiratory diseases in pigs between 1998 and 1999. By May of 1999, the death of 105 humans and the culling of about 1.1 million pigs were reported. Fruitbats of Pteropid species were identified as the natural reservoir hosts. The epidemiological studies suggested that NiV was introduced into pig farms by fruitbats, and was than transmitted to humans (mainly pig farmers) and other animals such as dogs, cats and horses. In 2004, NiV reappeared in Bangladesh with greater lethality. In contrast to the Malaysia case, epidemiologic characteristics of this outbreak suggested the possibility of fruitbats-to-person, or person-to-person transmission. In this article, the epidemiological comparison between two outbreaks in Malaysia and Bangladesh, and the new-trends of virological studies of NiV will be discussed.     

Recombinant Measles Virus Vaccine Expressing the Nipah Virus Glycoprotein Protects against Lethal Nipah Virus Challenge

Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.     

Advances in diagnostics,vaccines and therapeutics for Nipah virus

Nipah virus is an emerging zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. The virus was first discovered after an outbreak of encephalitis in pig farmers in Malaysia and Singapore with subsequent outbreaks in Bangladesh or India occurring almost annually. Due to the highly pathogenic nature of NiV, its pandemic potential, and the lack of licensed vaccines or therapeutics, there is a requirement for research and development into highly sensitive and specific diagnostic tools as well as antivirals and vaccines to help prevent and control future outbreak situations.     

Nipah virus: vaccination and passive protection studies in a hamster model

Nipah virus, a member of the paramyxovirus family, was first isolated and identified in 1999 when the virus crossed the species barrier from fruit bats to pigs and then infected humans, inducing an encephalitis with up to 40% mortality. At present there is no prophylaxis for Nipah virus. We investigated the possibility of vaccination and passive transfer of antibodies as interventions against this disease. We show that both of the Nipah virus glycoproteins (G and F) when expressed as vaccinia virus recombinants induced an immune response in hamsters which protected against a lethal challenge by Nipah virus. Similarly, passive transfer of antibody induced by either of the glycoproteins protected the animals. In both the active and passive immunization studies, however, the challenge virus was capable of hyperimmunizing the vaccinated animals, suggesting that although the virus replicates under these conditions, the immune system can eventually control the infection.     

Ephrin-B2 and ephrin-B3 as functional henipavirus receptors

Members of the ephrin cell-surface protein family interact with the Eph receptors, the largest family of receptor tyrosine kinases, mediating bi-directional signaling during tumorogenesis and various developmental events. Surprisingly, ephrin-B2 and -B3 were recently identified as entry receptors for henipaviruses, emerging zoonotic paramyxoviruses responsible for repeated outbreaks in humans and animals in Australia, Southeast Asia, India and Bangladesh. Nipah virus (NiV) and Hendra virus (HeV) are the only two identified members in the henipavirus genus. While the initial human infection cases came from contact with infected pigs (NiV) or horses (HeV), in the more recent outbreaks of NiV both food-borne and human-to-human transmission were reported. These characteristics, together with high mortality and morbidity rates and lack of effective anti-viral therapies, make the henipaviruses a potential biological-agent threat. Viral entry is an important target for the development of anti-viral drugs. The entry of henipavirus is initiated by the attachment of the viral G envelope glycoprotein to the host cell receptors ephrin-B2 and/or -B3, followed by activation of the F fusion protein, which triggers fusion between the viral envelop and the host membrane. We review recent progress in the study of henipavirus entry, particularly the identification of ephrins as their entry receptors, and the structural characterization of the ephrin/Henipa-G interactions.     

Hendra and Nipah viruses: pathogenesis and therapeutics

Within the past decade a number of new zoonotic paramyxoviruses emerged from flying foxes to cause serious disease outbreaks in man and livestock. Hendra virus was the cause of fatal infections of horses and man in Australia in 1994, 1999 and 2004. Nipah virus caused encephalitis in humans both in Malaysia in 1998/99, following silent spread of the virus in the pig population, and in Bangladesh from 2001 to 2004 probably as a result of direct bat to human transmission and spread within the human population. Hendra and Nipah viruses are highly pathogenic in humans with case fatality rates of 40% to 70%. Their genetic constitution, virulence and wide host range make them unique paramyxoviruses and they have been given Biosecurity Level 4 status in a new genus Henipavirus within the family Paramyxoviridae. Recent studies on the virulence, host range and cell tropisms of henipaviruses provide insights into the unique biological properties of these emerging human pathogens and suggest approaches for vaccine development and therapeutic countermeasures.     

Contamination of Foods by Food Handlers: Experiments on Hepatitis A Virus Transfer to Food and Its Interruption

Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm² resulted in transfer of 9.2% ± 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% ± 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.     

Nipah virus uses leukocytes for efficient dissemination within a host

Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.