Abstract
After 12 serial Nipah virus outbreaks in humans since 1998, it has been noted that all except the initial event in Malaysia occurred during the first 5 months of the year. Increasingly higher morbidity and mortality have been observed in subsequent outbreaks in India and Bangladesh. This may have been related to different virus strains and transmission capability from bat to human without the need for an amplifying host and direct human-to-human transmission. A survey of virus strains in Pteropus lylei and seasonal preference for spillover of these viruses was completed in seven provinces of Central Thailand between May 2005 and June 2007. Nipah virus RNA sequences, which belonged to those of the Malaysian and Bangladesh strains, were detected in the urine of these bats, with the Bangladesh strain being dominant. Highest recovery of Nipah virus RNA was observed in May. Of two provincial sites where monthly surveys were done, the Bangladesh strain was almost exclusively detected during April to June. The Malaysian strain was found dispersed during December to June. Although direct contact during breeding (in December to April) was believed to be an important transmission factor, our results may not entirely support the role of breeding activities in spillage of virus. Greater virus shedding over extended periods in the case of the Malaysian strain and the highest peak of virus detection in May in the case of the Bangladesh strain when offspring started to separate may suggest that there may be responsible mechanisms other than direct contact during breeding in the same roost. Knowledge of seasonal preferences of Nipah virus shedding in P. lylei will help us to better understand the dynamics of Nipah virus transmission and have implications for disease management.
Introduction
Human infections with Nipah virus continued in Bangladesh in 2001 and 2003 to 2008 with approximately 70% mortality (Table 1) (Record of WHO Regional Office for South East Asia [WHO/SEARO]). Nipah virus–associated encephalitis outbreaks were also reported from India in 2001 and 2007 (Chadha et al. 2006, WHO/SEARO record). As of February 2008, a total of 475 cases were recognized with 247 deaths. The approximate case fatality rate was 52%. Comparing case fatality rate in Malaysian and the Indian-Bangladesh incidents, that of the latter was much higher (40% vs. 70.8%).
During the Malaysian outbreak, pigs were the apparent source for infection of humans. Domestic dogs and cats were also infected from eating pig carcasses (Yob et al. 2001). Studies of the prevalence of Nipah virus in bats showed that Pteropus lylei as well as other bats belonging to the same or other families (Table 2) could be infected and become reservoirs. In Malaysia, the highest seroprevalence was found in Megachiropteran bats (Pteropodidae family) (Pteropus hypomelanus [31%], Eonycteris spelaea [5%], and Cynopterus brachyotis [4%]), whereas only 3% of Scotophilus kuhlii (suborder of Microchiroptera) was antibody positive (Yob et al. 2001). Subsequent studies confirmed Pteropus bats as the main reservoir of this virus (Wacharapluesadee et al. 2005). Relatively large amounts of urine after the morning meal (which may include the virus), type of feeding (same fruits eaten by humans and other animals), and their roosting sites (trees), which are usually close to a human community, may also be factors in enhancing viral transmission. Virus and its RNA were demonstrated in the urine and serum of flying foxes (Chua et al. 2002, Reynes et al. 2005, Wacharapluesadee et al. 2005, 2006).
Boldface indicates countries where Nipah virus infection in bats was demonstated by antibody detection method.
Countries where Nipah virus infection in bats was confirmed by isolation.
Countries where Nipah virus infection in bats was confirmed by RNA detection.
Subsequent outbreaks in India and Bangladesh (Fig. 1), where pig farming was uncommon, suggested other intermediary sources or direct transmission from bats to humans. No cluster of ill animals, such as pigs, dogs, shrews, rodents, and birds, was observed or reported in Mehepur and Naogaon districts of Bangladesh (Hsu et al. 2004). Antibodies reactive to Nipah virus antigen were detected in Pteropus giganteus adult females. Investigations of different Nipah outbreaks in Bangladesh have identified different routes of transmission such as climbing trees, contact with sick persons, and contact with sick animals (Hsu et al. 2004, Gurley et al. 2007). Drinking of fresh date palm sap, possibly contaminated by fruit bats (P. giganteus) during the winter season, may have been responsible for indirect transmission of Nipah virus to humans (Luby et al. 2006).
Map shows field sampling locations in Thailand (detail in inset demonstrating all seven samplings sites: 1 = Chonburi, 2 = Chachoengsao, 3 = Prachinburi, 4 = Saraburi, 5 = Ayutthaya, 6 = Angthong, and 7 = Singburi). Historic outbreak sites (designated by ★stars) in Malaysia, Singapore, India, and Bangladesh. Range of Pteropus lylei (shown as shaded area).
Periodic excretion of Nipah virus from bat urine was suggested in one study from Malaysia (Chua et al. 2002). No virus was isolated from the urine of P. hypomelanus on the first two attempts in August 17, 1999, and August 23, 1999, but it was successfully isolated on the third trip (June 11, 2000). Here we report a systematic surveillance for Nipah virus infection in P. lylei bats in Thailand to determine whether there is a seasonal preference for virus transmission and whether this correlates to bat behavior(s). Population dynamics of P. lylei by bounded count method were also studied to determine the correlation between the number of bats and breeding activities.
Materials and Methods
Study sites
All of the study sites were at temples (wat). Two sites at Wat Luang, Chonburi province, and Wat Pho, Chachoengsao province, and other five roosting sites at Singburi, Ayutthaya, Saraburi, Angthong, and Prachinburi provinces (Fig. 1; Table 3) were chosen as study sites for evidence of Nipah virus shedding. P. lylei bats in Wat Luang were studied monthly from May 2005 to June 2007 and at Wat Pho monthly from June 2005 to June 2007 (Table 4). Those in the remaining sites were studied three times in February, May, and October of 2006 (Table 5). The criteria for selecting the sites were based on information from the Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment. They included bat populations of greater than 1000, whether it was possible to spread plastic sheets, and areas containing only one species (P. lylei). Direct observation by bat ecologists both before and during the process of bat urine collection was done to ensure that P. lylei was the only species. However, one of seven survey sites, Wat Chantaram, Angthong, was also found to contain two bird species (Phalacrocorax niger and Casmerodius albus) as well. Birds and bats were found together on the same tree only in October 2006. In such cases, areas where only bats were found were selected for urine collection. The plastic sheets were spread at 5 a.m., and urine was collected at 6 a.m.
asl, Above sea level.
Underlined numbers indicate Malaysian strain, and those without, Bangladesh strain.
Represents two of Bangladesh and two of Malaysian strains.
ND, not done due to flooding or technical problems.
ND, not done due to technical problems.
Population dynamics and behavioral studies
We used the bounded count method (Choudhary 1987) to completely enumerate all P. lylei bats at Wat Luang between October 2005 and September 2006. Roost areas were divided into 10 sections. Bats in all sections were concurrently counted within 1 day. This process involved 10 visits by 10 skilled forest officials. Monthly population numbers were obtained based on calculations according to a formula previously described (Choudhary 1987). Behavior of bats related to breeding activities was observed during each survey to determine when the breeding season would commence and when the offspring would appear, which should coincide with an increase in population.
Urine sample collection
Bat urine samples were collected using a plastic sheet as reported previously (Chua 2003). At each site, plastic sheets were laid at 26 spots under the trees where the urine and feces of fruit bats were expected to be deposited as indicated by the presence of previous droppings. Each sheet was 1.5 × 1.5 m. Sterile cotton swabs were used to soak up the urine on the plastic sheet. These were immersed immediately into 9 mL of Nuclisence Lysis buffer containing guanidine thiocyanate and Triton X-100 (Biomerieux, Boxtel, The Netherlands). Two cotton swabs containing approximately 1.2 mL of bat urine were pooled in each Lysis buffer tube. The tubes were kept cold by placing them in a cooled box and transporting back to the laboratory within 24 h.
Nipah virus genome detection
Pooled bat urine samples were tested for Nipah virus using the duplex-nested RT-PCR method as described (Wacharapluesadee and Hemachudha 2007). After vortex, the urine swabs were removed from the Lysis buffer tube. Total RNA was extracted from urine specimens by using the silica-guanidine thiocyanate method according to manufacturer's protocol (Biomerieux). The extracted nucleic acid was stored at −70°C until analysis. An RNA plasmid was introduced as an internal control in the duplex RT-PCR as previously described (Wacharapluesadee and Hemachudha 2007). Five microliters of extracted samples and 2 μL of internal control RNA were added to the One Step RT-PCR reagents (Qiagen, Valencia, CA) for first-round amplification. For nested PCR, 1 μL of the first amplification products were added to a new PCR mixture. The 10 μL of the PCR product was sized by gel electrophoresis in 2% agarose containing 0.5 μg/mL of ethidium bromide in Tris-borate-EDTA buffer and viewed under UV light. The positive control used in this study was the constructed RNA plasmid with 29 bp insertion. The negative control (water) was included in all runs.
Analysis of the sequences
To identify the strains detected by PCR, heminested amplification was performed from the first-round PCR product as described previously (Wacharapluesadee and Hemachudha 2007). Nucleotide sequences were aligned using CLUSTAL_X computer software version 1.81 (Thompson et al. 1997). The nucleotide sequences of N-gene at position 1197-1553 (according to GenBank accession no. NC_002728) were analyzed. The viral strain was defined by comparing similarities with other sequences deposited in the GenBank database (GenBank accession nos. NC_002728, AY988601, and AY858110 are the isolates from Malaysia, Bangladesh, and Cambodia, respectively).
Results
Bat population and behavior
During October 2005 and September 2006, bat populations at Wat Luang fluctuated between 8309 and 12,765 individuals (Table 6). With the exception of population numbers in August, during which time some trees were removed and some were cut back by pruning to remove branches, the lowest number was found in March and the highest in May. The breeding season commenced in December. Deliveries began in February, and pup separation from the mother started in May. The population decreased until August, and then increased as the next breeding season.
Observation times were during 10 a.m.–2 p.m. in all trips.
At level of significance α (p = 0.05).
Some roost trees were removed, and some were cut back by pruning to remove branches.
A 95% confidence interval for a population estimate varied most in May and November. Two sharp declines in the population occurred in the Wat Luang colony. The first incident appeared from February to March and the second in June and July (Table 6).
Temporal dynamics of virus detection
Wat Luang was found to have a higher urine positivity for the presence of Nipah virus RNA than Wat Pho (27/1137[2.4%] vs. 6/981[0.6%]). At Wat Luang, between May 2005 and June 2007, a total of 1137 pooled urine samples were collected. Nipah virus RNA could be detected in urine in May and June of 2005 and in January, April, May, and June of 2006 and February and May of 2007 (Table 4). Detection of Nipah virus RNA in urine was constantly found in May (positive rates of 6/48, 4/51, and 4/99 in 2005–2007).
At Wat Pho, during a similar time interval, a total of 981 pooled urine samples were collected. Virus RNA was detected in urine in December 2005 (1/25); March (1/33), June (1/48), and October (1/56) of 2006; and in May 2007 (2/42) (Table 4).
At the remaining sites where surveys were done in February, May, and October 2006, virus RNA was detected only in May at all five sites (3/48, 1/50, 7/58, 1/50, and 3/53) (Table 5).
Temporal–spatial relationship and Nipah virus strain identification
At Wat Luang, 16 out of 23 viruses were identical to those from Bangladesh, and 7 out of 23 to the Malaysian strains. Different patterns of virus shedding were found. The Bangladesh strain was found exclusively during a 3-month interval from April to June. May was the month when virus RNA was most detectable in urine (Table 4). A more dispersed pattern (January, February, May, June, and December) was found in the case of the Malaysian strain.
At Wat Pho, four out of six viruses belonged to the Bangladesh strains, and two out of six to the Malaysian strains were found in December and March (Table 4). At the remaining five sites where the surveys were done in February, May, and October, only the Bangladesh stain was identified in May (Table 5).
Forty-four PCR-positive specimens were sequenced to confirm strain identity and true-positive results. Direct sequencing of the 41 heminested PCR-amplified products of the 357-nucleotide coding region of the nucleoprotein and subsequent sequence analyses resulted in a 92–100% homology between isolates and a 93–100%, 92–99%, and 93–98% similarity with isolates from Malaysia (NC_002728), Bangladesh, (AY988601), and Cambodia (AY858110), respectively (GenBank accession nos. EF070185–EF070190, EU603724–EU603751, EU603753–EU603758, and EU620498). A Bangladesh strain like Nipah virus with 98–99% similarity was found in 35 of 41 positive specimens. Malaysian strain–like Nipah virus with 99–100% similarity was found in 6 of 41 positive specimens. From another three samples, only nested PCR amplicons of 181-nucleotide sequences were obtained, and this resulted in 99–100% similarity to the isolate from Malaysia (GenBank accession nos. EU624735–EU624737).
Relationship between bat behavior and time of viral transmission
Courting activity was observed starting in November followed by pregnancy. This continued until January. Delivery started in February, and separation from the mother started in May. Shedding of the Bangladesh strain peaked when offspring separated. Dispersal of all the Malaysian strain was not related to the time of breeding, which was between December and April.
Temperature and amounts of rainfall in Chonburi and Chachoengsao provinces
The temperature and amounts of rainfall for each of the months of the year that samples were collected at two study sites are shown in Figure 2a and b. They were Chonburi and Chachoengsao provinces, where Wat Luang and Wat Pho are located. Both areas showed similar patterns of rainfall and temperature.
Diagram showing temperature, amount of rainfall, and months of the year samples were collected in (
Discussion
This study confirms our earlier findings that P. lylei bats in Thailand have been infected by Nipah virus (Wacharapluesadee et al. 2005). Previous serosurveys in nine provinces in Central Thailand between March 2002 and February 2004 revealed evidence of Nipah virus infection in P. hypomelanus (4/26), P. lylei (76/813), and Pteropus vampyrus (1/39). Only urine of P. lylei was found to be Nipah virus RNA positive. Nevertheless, this does not underestimate the significance of other pteropid species because the nature of virus shedding is intermittent (Chua et al. 2002). P. hypomelanus and P. vampyrus roost on islands and forests, whereas P. lylei live in proximity to human households and temples, where either direct or indirect contact with humans is possible. P. lylei, therefore, is the focus of this study. Moreover, the finding of unusually high antibody titers in P. lylei suggests that Nipah virus circulates mainly in this bat species (Wacharapluesadee et al. 2005). P. lylei was the only bat species that was Nipah virus infected among 14 species tested in Cambodia (Reynes et al. 2005).
Wat Luang and Wat Pho were chosen as sites to determine the seasonal preference of virus transmission. These two temples were visited monthly. Wat Luang had previously been surveyed twice in February of 2003 and 2004 (antibody positive in both occasions and RNA positive only in 2004) (unpublished data). Wat Pho had been visited once in January 2003. Nipah virus antibodies and RNA had been detected (Wacharapluesadee et al. 2005).
Three other sites (Table 5) had been visited: once in 2002 (site A, Singburi, in April and site B, Ayutthaya, in October) and once in 2003 (site E, Prachinburi in April). Nipah virus antibodies by enzyme immunoassay were demonstrated in sites B and E (Wacharapluesadee et al. 2005). Nipah virus RNA was not detected in urine from sites A, B, and E. These sites were surveyed to confirm whether excretion patterns would be similar to those of Wat Luang and Wat Pho.
Low-level viral RNA shedding was detected, suggesting that herd immunity may play a role. This is less plausible, but cannot be totally excluded. Low levels of seroprevalence (9.3% [76/813]) were found in the P. lylei population in Thailand (Wacharapluesadee et al. 2005). An increase of seroprevalence from 7.8% in February 2003 to 18% in February 2004 and viral RNA prevalence (as detected in urine) from 0% to only 2.7%, respectively, at Wat Luang (unpublished data) may favor the role of herd immunity. In this study, RNA was taken from pooled samples, and serum antibody was not determined.
The amount of virus and/or number of shedding bats fluctuate with the season. Virus was found more frequently during the first 5 months, which nearly coincided with the reproductive cycle. Both direct and vertical transmission may play a role. Vertical transmission has been demonstrated by the findings of Hendra virus in placental and fetal tissues (Williamson et al. 2000) and demonstration of Nipah virus in the uterus (Middleton et al. 2007). Direct contact during the reproductive cycle is facilitated by mutual grooming, copulation, and parturition. Pregnant and lactating females of Pteropus scapulatus had a significant risk of Hendra virus infection, which may be transmitted via faeces, urine, and saliva (Plowright et al. 2008). Finding of age-specific seroprevalence in Hendra virus–infected bats also suggests horizontal transmission (Plowright et al. 2008). In our study, we found no correlation between viral shedding and the amount of rainfall, temperature, roost sites, parturition period, and amount of food (Fig. 2a, b, and observational data since 2001 of the Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources).
Although it was noted that viral shedding was mainly observed during the first 5 months, the peak of viral shedding (particularly of the Bangladesh strain) was detected in May at all sites (Tables 4 and 5). Physical separation from the mother was at a peak in May. This was associated with a fluctuation of population numbers that was observed only in May and correlated with juveniles in practicing flight. It has been observed that transient viral shedding in urine (1 of 6 bats) developed shortly after infection in one experiment, and virus could remain in the kidney and uterus in another experiment (2 of 11 bats) (Middleton et al. 2007). Passive transfer of Nipah virus antibody could be demonstrated from lactating females to pups (Epstein et al. 2008). Whether or not passive maternal immunity can delay viral shedding in the pup remains unclear.
Similar fluctuation of viral shedding with the seasons may be applicable to outbreaks that occurred in more northern areas of the Nipah outbreak range like India and Bangladesh. Most outbreaks were reported during the winter in Bangladesh and India, which covers the reproductive cycle of P. giganteus (between November and April) (Dr. Gongal, personal communication).
Viral shedding also appeared in two different patterns depending on viral strains. Some bats escaped shedding during the first 5 months of the reproduction cycle (Table 4). The Bangladesh strain showed a trend of clustering pattern of viral shedding (April, May, and June at Wat Luang; May, June, and October at Wat Pho; and only in May at the remaining five sites). Whereas with the Malaysian strain, shedding was in a dispersed pattern (January, February, May, June, and December at Wat Luang, and December and March at Wat Pho). Why there was this difference as well as why more of the RNA-positive urine samples were found in Wat Luang than in Wat Pho, though they have comparable numbers of P. lylei (approximately 10,000) and are only 24 km apart, cannot be answered by presently available data. It has not been known whether bats with preexisting immunity can be repeatedly infected.
Thailand as well as other countries in this region remain free of human outbreaks. This may be explained by the relatively low level of Nipah virus RNA excretion in urine (this study, Chua et al. 2002, Reynes et al. 2005, Middleton et al. 2007). However, understanding the dynamics of pteropid bat populations and of Nipah virus infection is important for risk management strategies in the countries where a natural bat habitat is found.
Footnotes
Acknowledgments
This work was supported by a grant from the Thailand Research Fund (Grant DGB4880001) and the Thai Red Cross Society. S.W. and T.H. received funding support from the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development, Thailand. We also acknowledge help from the Division of Research Affairs, Faculty of Medicine, Chulalongkorn University in the preparation of this manuscript.
Disclosure Statement
No competing financial interests exist.