Rodents and Risk in the Mekong Delta of Vietnam: Seroprevalence of Selected Zoonotic Viruses in Rodents and Humans

Introduction

In the Mekong Delta of Vietnam, rats are commonly trapped in rice fields before and after harvesting and sold live for food consumption. The rat trade is vigorous, involving up to 3300–3600 tonnes of meat sold per year (Khiem et al. 2003). Rat infestations in rice fields cause enormous crop damage, and trapping provides a means of pest control as well as augmenting local dietary sources of animal protein. Rodent parts (heads, tails, and innards) are also often fed to domestic livestock or reptiles reared in capitivity (frogs, snakes, crocodiles). Rodents are well-recognized vectors of a range of zoonotic viruses transmitted via close contact with saliva, urine, or feces (Meerburg 2010).

Given the frequency and intensity of human and livestock contact with rodents, we aimed to explore potential risks associated with these exposures through seroprevelance studies. We determined seroprevalence to five groups of known zoonotic viruses in rodents representing the common species sold in wet markets and present in peridomestic and farm settings. In parallel, we determined seroprevalence to the same set of viruses in a cohort of individuals with high levels of occupational and residential exposure to rodents and domestic livestock. The viruses of interest (namely flaviviruses, hantaviruses, poxviruses, arenaviruses, and rodent parechoviruses) were selected on the basis of their known or probable endemicity, their documented associations with rodent-borne disease, and considerations of feasibility and resources (i.e., access to reference strains and well-established diagnostic protocols). In addition to serological screening, we tested rodent liver and lung tissues by RT-PCR using generic primers for flaviviruses and hantaviruses).

Methods

Results

Human serology

Seropositivity was detected to all five virus groups, with high levels of seropositives to TBEV (116/245, 47.3%), moderate seropositivity to Ljungan virus (18.8%, 46/245), and lesser rates of seroposivity to cowpox virus (4.5%, 11/245), hantaviruses (3.7%, 9/245), and LCMV (0.8%, 2/245) (Table 3). Regarding the nine individuals with hantavirus antibodies, there were two positive only for DOBV, five positive only for SEOV, and two positive for both. Seropositivity to TBEV and poxviruses was associated with increased age, whereas seropositivity to Ljungan virus was higher in younger individuals (Fig. 1). There were no significant associations between seropositivity and occupation. TBEV antibody detection was higher in males (62.4%) versus females (36.8%) (p<0.001). Differential IFAT titrations against DENV and JEV were performed on a subset of 60 human sera, including 30 TBEV seropositives and 30 TBEV seronegatives. Of the 30 TBEV positive samples, 12 had equivalent titers to JEV and DENV, 17 had higher relative titers to JEV than DENV, and only one had a higher titer to TBEV than to the JEV and DENV.

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FIG. 1.

Human seroprevalence to flavivirus (TBEV), parechovirus (Ljungan virus), and poxvirus (cowpox virus) stratified by age group. Cohort age groups were 4–18 years (n=37), 19–38 years (n=86), 39–59 years (n=89), and 60–76 years (n=33). Bars indicate 95% confidence intervals.

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Table 3.

Human Seroprevalence to Five Groups of Rodent-Borne Viruses

		No. of positive
	No. of humans	TBEV	Hantavirus	Cowpox virus	LCMV	Ljungan virus
Cohort sex
Male	101	63 (62.4)	5 (4.9)	7 (6.9)	2 (1.9)	27 (26.7)
Female	144	53 (36.8)	4 (2.7)	4 (2.7)	0 (0)	19 (13.2)
Cohort occupation
Farmer	181	88 (48.6)	6 (3.3)	8 (4.4)	1 (0.6)	31 (17.1)
Animal health worker	29	14 (48.3)	1 (3.4)	1 (3.4)	1 (3.4)	5 (17.2)
Pig slaughterer	12	4 (33.3)	1 (8.3)	1 (8.3)	0 (0)	4 (33.3)
Poultry slaughterer	18	7 (38.8)	1 (5.6)	1 (5.6)	0 (0)	4 (22.2)
Rat trader	5	3 (60.0)	0 (0)	0 (0)	0 (0)	2 (40.0)
Total	245	116 (47.3)	9 (3.7)	11 (4.5)	2 (0.8)	46 (18.8)

Values within parentheses are percentages.

Discussion

Rodents are known to host a wide spectrum of human viral and bacterial pathogens; however, investigations of rodent-borne diseases are relatively rare, and this diverse group of infections is not frequently prioritized by biomedical or public health research communities. Hantaviruses (family Bunyaviridae) are arguably the rodent-borne viruses of greatest public health signficance in Asia, where they are associated with an estimated 150,000–200,000 cases of hemorrhagic fever with renal syndrome (HFRS) each year (Bi et al. 2008). The hantavirus genus comprises at least 24 recognized virus species, several of which are pathogenic to humans. Although hantaviruses are known to circulate in Vietnam, few clinical cases have been reported (Huong et al. 2010), and the relative scope of the associated disease burden remains poorly characterized.

Our study indicated an overall 8.3% hantavirus seroprevalence among the five rodent species tested from southern Vietnam. This prevalence is consistent with previous reports from Vietnam (approximately 2.8% and 8.6% seropositivity in rodents from the Ho Chi Minh City and northern provinces, respectively) (Truong et al. 2009, Luan et al. 2012). We found hantavirus antibodies in all rat species with the exception of R. exulans. Despite the relatively high seroprevalence for hantaviruses, and the current understanding that most reservoir hosts develop chronic persistent infections (Easterbrook et al. 2007, Vaheri et al. 2013), only one rat sample (R. argentiventer) was confirmed to be hantavirus positive, with conventional PCR indicating SEOV. R. argentiventer has not previously been identified as a host of hantaviruses, and the worldwide distribution of SEOV has been almost exclusively associated with R. norvegicus (Lin et al. 2012).

Murine hantaviruses are believed to cross-react strongly; however, to ensure detection of a broad spectrum of potential hantavirus antibodies, we used two types of antigens (SEOV and DOBV), representing rat or mouse hosts, respectively. (It should be noted, however, that hosts of DOBV, Apodemus mice, are not found in Vietnam.) Interestingly, most of the rodent sera revealed variable reactivity to DOBV and SEOV viruses, with only five of 19 (26.3%) sera testing positive for both, suggesting that multiple hantaviruses may be circulating in the area. It is known from other studies in southeast Asia that B. indica carries Thailand hantavirus (Hugot et al. 2006), R. tanezumi carries Serang virus (Plyusnina et al. 2009), and R. norvegicus carries SEOV.

Regarding the human cohort, we detected seropositivity to both SEOV (7/245, 2.9%) and DOBV (4/245, 1.6%), again suggesting that variable hantaviruses may circulate. The results are consistent with previous low seroprevalence rates detected from humans in northern Vietnam (Truong et al. 2009). Risk factor analysis did not suggest any significant associations between hantavirus seroprevelance and contact patterns with any particular animal species. Our results indicate that one or more hantavirus species is endemic in common rat species of Vietnam; however, despite relatively intense and common human exposures to rats, human seroconversions to hantavirus are not common.

The Flavivirus genus (family Flaviviridae) comprises approximately 70 virus species, including a number of important human pathogens and a clade of species presumed to be transmitted primarily by rodents, other small mammals, and bats (the so-called “no known vector” group) (Kuno et al. 1998). Within southeast Asia, the only two recognized human clinical diseases caused by flaviviruses are dengue and Japanese encephalitis, and infections with tick-borne encephalitis (TBEV) have not been reported. Although Ixodes spp. ticks have been described in Vietnam (Kolonin et al. 1995), direct observation of ectoparasites on rats suggests little prevalence of ticks on rats of the Mekong Delta region.

Our IFAT screening results indicated overall TBEV seropositivity in humans of 47% and lesser rates in the rodents of 5.4%. Given that flaviviruses induce life-long immunity, that dengue and JEV are both highly endemic in southern Vietnam (e.g., approximately 53% dengue seroconversion in humans by age 7, increasing to 88% by age 13) (Thai et al. 2005), and that JEV vaccination is now widely administered throughout the country (Yen et al. 2010), our observed TBEV immunological reactivity in the humans was not at all surprising and most likely reflects the notorious cross-reactivity of flaviviruses (Roehrig, 1986, Basile et al. 2013). In contrast, the rodent seroreactivity to TBEV (15/275, 5.4%) was more intriguing. Rodent TBEV positives are much less likely to be due to dengue, because the principal dengue mosquito vector, Aedes aegypti, is almost exclusively anthropophilic (Harrington et al. 2001). Rodents have never been suggested to play a role in JEV transmission; however, given the high levels of endemicity in farms and periurban areas of the Mekong (Lindahl et al. 2013), the observed seroreactivity in rodents could be explained by spillover infections from avian reservoirs vectored by various Culex species. Our failure to identify viral genomes in the liver tissues of any of the 275 rodents suggests that the observed seroconversions were caused by transient infections, and/or the tropism of the virus is nonhepatotropic. The present results leave open the possibility that non-DENV, non-JEV flaviviruses circulate endemically within rodents of the Mekong, perhaps involving tick vectors, as has been found in Malaysia (Smith 1956) and Thailand (Bancroft et al. 1976).

None of remaining virus groups included in our survey, namely cowpox virus, arenaviruses, and rodent parechoviruses, showed evidence of high seroreactivity in the rodents. Cowpox viruses belong to the genus Orthopoxvirus that is immunologically cross-reactive and cross-protective, and cowpox infection in rodents has been reported across Europe from a wide range of rodent species, although R. norvegicus and bank voles are thought to be the principal reservoirs (Essbauer et al. 2010). In the present study, poxvirus antibody detection in humans was 4.5%, and was notably higher among people over 60, which may be attributable to prior smallpox vaccination. In contrast, poxvirus seropositivity was only 1.1% in rodents; the positive rodents were B. indica and R. tanezumi. Notably, we found a significant association between presence of pox antibody among cohort members and exposure to bats (RR 8.07, 95% CI 1.46, 44.67; p<0.05). Seroprevalence to LCMV was much lower (human, 0.8%; rodent, 0.4%) than has been observed from Thailand, where overall LCMV seroprevalence among rodents in one study was 13.3%, mostly in Bandicota savilei (35.7%) and R. norvegicus (31.5%) (Nitatpattana et al. 2000).

Ljungan virus is a rodent-borne parechovirus and suspected human pathogen (Klitz and Niklasson 2013), and although relatively high seroprevalence in humans has been documented from northern Europe (Jääskeläinen et al. 2013), to date there have been no studies of Ljungan virus in Asia. None of the Vietnam rats were seroreactive to Ljungan virus; the lack of Ljungan virus seropositives among the Vietnamese rats may not be surprising, because Ljungan virus in Europe has previously been associated primarily with bank or field voles (members of the Arvicolinae) rather than Rattus spp. However, as many as 18.8% (46/245) of human cohort members tested positive to Ljungan virus antibodies. The human Ljungan seroreactivity did not appear to be linked to any particular animal exposures. The age distribution of seropositives declined with age, as has been observed from surveys of human parechoviruses (HPeV) in Europe (Benschop et al. 2008). This pattern of declining immunity with age may indicate human-to-human transmission via fecal–oral transmission, as is typical for the known HPeVs , rather than exposures to a zoonotic reservoir. It should be noted, however, that current knowledge of Ljungan immunology suggests that the virus does not cross-react with HPeV 1, 2, or 4–6 (Jääskeläinen et al. 2013). Our results provide intriguing suggestions of frequent human exposures to an as-yet-unidentified parechovirus.

In summary, our study is the first to document the common rodent species traded in wet markets and sold for human consumption in Vietnam (primarily R. argentiventer and B. indica). Our results confirm low-to-moderate levels of endemic circulation of hantaviruses in the region and suggest possible transmission of as yet unidentified rodent flavivirus and human transmission of a Ljungan-like parechovirus. Further explorations of pathogen diversity in rodent reservoirs using larger sample sizes and more sensitive molecular screening techniques (e.g., alternative degenerate primers of genus-specific studies, or metagenomic and deep sequencing approaches) will no doubt uncover substantial additional viral diversity. Given the scale and scope of rodent trading in the Mekong, and indications that rodent consumption may also be increasing in northern Vietnam, establishing the epidemiological relevance of human exposures to rat viruses is an important public health objective.