Geographical distribution and relative risk of Anjozorobe virus (Thailand orthohantavirus) infection in black rats (Rattus rattus) in Madagascar

Hantaviruses are zoonotic viruses whose main reservoir hosts are rodents, although these viruses have recently been detected in insectivores and bats [1, 2]. Human infections appear in several areas of the globe. Clinical symptoms and severity of disease vary between regions according to the species of hantavirus involved. Two diseases associated with hantavirus infections are described, the Hantavirus Cardiopulmonary Syndrome (HCPS) and the Hemorrhagic Fever with Renal Syndrome (HFSR). Nephropathia Epidemica (NE) is a mild form of HFSR. While NE disease appears to be mild with 0.1% of mortality rates, HCPS and HFRS, are more severe with a mortality that can reach up to 50 and 15% respectively [3]. HCPS are mainly found in the Americas, and HFRS and NE appears in the Old World [4, 5].

Hantaviruses are generally thought to be relatively host-specific [6], for example, Seoul orthohantavirus (SEOV) is associated with the rat, Rattus norvegicus, [7] and Puumala orthohantavirus (PUUV) is preferentially hosted by the bank vole, Clethrionomys glareolus [8]. However, studies have shown that spillover infection can occur between sympatric host species [9, 10]. For example, the common vole, Microtus arvalis, is the preferential host of Tula orthohantavirus (TULV), but the virus is also detected in other vole species such as Microtus agrestis and Arvicola spec. [11]. Hantaviruses can be found in saliva, urine and feces of infected animals and transmission between reservoirs is thought to be through bites, scratches or inhalation of contaminated aerosols [12, 13]. Humans usually contract hantavirus infection by direct contact with rodents or by inhalation of contaminated aerosols from excretion or secretion from infected animals [14]. Transmission inter-human is very uncommon, although it has been reported for some lineages of Andes orthohantavirus (ANDV) [15, 16].

Although relatively few hantavirus studies have been carried out in Africa and the surrounding regions, since 2006 eight hantaviruses have been [14] identified in rodents, insectivores and bats in mainland Africa [17‐24] whilst recently two hantaviruses were detected in the Indian Ocean region [25, 26]. The two hantaviruses found in the Indian Ocean region both belonged to Thailand orthohantavirus (THAIV) specie which was primarily found in Southeast Asia [27]. Mayotte virus (MAYOV) harbored by Rattus rattus in Mayotte island [26] and Anjozorobe virus (ANJOV) found in Rattus rattus and the endemic rodent Eliurus majori in Madagascar [25].

Studies have also documented exposure of human populations in Africa to hantavirus [28‐30], and recently Heinemann et al. have reported the presence of Bowé orthohantavirus associated with human disease in an Ivorian patient [31]. Moreover, as Thailand orthohantavirus is associated with disease [32], it is probable that hantavirus infection in Madagascar is also symptomatic. Nevertheless, the lack of studies in Madagascar prevents us from highlighting the potential threat that this virus presents for public health and from raising awareness amongst clinicians of clinical symptoms associated with hantavirus infection.

In areas of the globe where hantavirus infections in humans are better monitored, there is strong spatial and temporal variation in disease incidence and studies have demonstrated effects of environmental variables, including climate, habitat and land-use [6]. Climatic conditions may influence persistence of hantavirus in the environment and, therefore, transmission rates to humans [33]. However, these environmental effects on incidence are thought to be primarily mediated by the impacts on the abundance of infected reservoirs [34, 35]. For individual hantavirus, spatial variation in risk is likely to largely depend on the bioclimatic and habitat preferences of the principle host, whilst temporal variation in risk may be driven by inter-annual climatic driven changes in food availability and breeding rates [36]. Despite the tendency for close associations between individual mammal species and individual hantaviruses, several studies have also suggested that increased mammal diversity may reduce the prevalence of hantavirus infection in the principal host [37‐40], consistent with a “dilution effect”.

For areas where data on infection in humans may be under-recorded, examining the spatial distribution of infected reservoirs provides an important first-step in understanding the potential risk to humans, as well as allowing analyses to explore epidemiological processes in the reservoir populations by identifying environmental risk factors. The previous study in Madagascar that detected Anjozorobe virus was limited to one forest location, Anjozorobe-Angavo forest [25]. Here we sample terrestrial peridomestic small mammals trapped in urban and rural sites of representative regions of Madagascar to (i) establish the distribution of Anjozorobe virus and (ii) evaluate to what extent spatial variation in infection rates is related to environmental and host-related variables. Our results will be helpful to implement policies for future awareness and surveillance programs for human communities, as well as increasing our understanding of the epidemiology of hantavirus in a peridomestic African context.

Seven species of small mammal were trapped. R. rattus were trapped in nearly all (97%) of the sites (38/39), whilst R. norvegicus were primarily trapped in the large cities (31% of sites, 12/39). We tested specimens from 1242 mammals belonging to seven species: 897 black rats R. rattus, 125 house mouse Mus musculus, 124 Norway rats R. norvegicus and 96 insectivores (Suncus murinus, Setifer setosus, Tenrec ecaudatus, Hemicentetes semispinosus) (Table 1). The number of animals tested per site ranged from 11 to 62.

Overall 113 (9%) small mammals were PCR positive for hantaviruses, of which 111 were R. rattus and two M. musculus. It is to be noted that all PCR-positive samples were confirmed by sequencing. Hantavirus RNA was not detected in samples from R. norvegicus or insectivores. Genetic analysis of the partial the coding region of the L segment (301 nt) of rodent-borne hantaviruses detected in Madagascar show that these viruses cluster with previous hantavirus detected in Madagascar; Anjozorobe virus (Fig. 1).

Positive individuals showed a widespread geographical distribution, with infected individuals in 90% of districts (18/20) (Fig. 2) and 69% of sites (27/39). The prevalence of infected R. rattus at district level ranged from 0 to 29% (Table 1). The highest prevalences were obtained in the districts of Moramanga (29%) Fianarantsoa (27%), Toamasina (26%), Maevatanana (25%), Mandritsara (25%), Farafangana (24%) and Antananarivo-Atsimondrano (23%).

At the site level, prevalence in R. rattus ranged from 0 to 80%, with highest prevalences in Moramanga Urban (80%, n = 5), Mandritsara Rural (58%, n = 19), Fianarantsoa Urban (43%, n = 30) and Anjozorobe Urban (43%, n = 7). For the 19 sites where both urban and rural sites were tested there was no correlation between the prevalence in the urban and the rural site (Spearman rank, p = 0.9).

Our national-scale study demonstrates that THAIV is widespread in the peridomestic small mammal communities of Madagascar, primarily infecting R. rattus, the most common species in our study, in both rural and urban sites. Rats living outside houses are more likely to be infected than those inhabiting houses and hantavirus prevalence is higher in humid areas of the island. Weight also influenced infection probability, as several studies have shown before for this chronic infection [47, 48]. Our results are important for understanding the potential risk to humans from hantavirus infection in Madagascar.

We confirm R. rattus as the main reservoir of hantavirus in Madagascar. Sequencing results from a subset of samples form a distinct cluster with Anjozorobe virus. The detection of two infected M. musculus may suggest spillover infections. Spillover infections in sympatric hosts of closely related species or genera have been reported before for Dobrava orthohantavirus (DOBV) [49] and Tula orthohantavirus (TULV) [11]. Our results are consistent with the idea of that whilst hantaviruses have preferred host species, some spillover infections can occur [11]. However, the importance of such secondary spillover hosts for the persistence and dynamics of infection is unclear. For TULV, the spatial distribution of infected individuals from secondary host species and their apparent clearance of the virus suggest they play a minor role, if any [11].

We did not detect any hantavirus in two other introduced mammals, R. norvegicus and S. murinus. R. norvegicus is the main reservoir of Seoul orthohantavirus, a virus that is found predominantly in the Old World [50‐52], but thought to have a more widespread global distribution [2] and recently confirmed using molecular techniques as causing human infections in Europe [53, 54]. S. murinus has been found infected with Thottapalayam orthohantavirus in India, Thailand and Indonesia. As our sample size for R. norvegicus and S. murinus were relatively small (n = 124 and n = 76 respectively), we cannot exclude the possibility that they are infected with hantavirus in Madagascar.

The global prevalence of infection amongst R. rattus was 12.4% (111/897), which is similar to the prevalence of Mayotte virus observed in R. rattus populations on Mayotte Island (18.1%: 29/160) [26]. However, we detected variation in prevalence at two spatial scales. Large-scale spatial variation was not driven by any rural-urban split but was related to climate, whilst there was also within-site variation related to habitat type. Spatial prevalence patterns at both scales may be associated with variation in transmission rates due to changing contact rates between potential hosts and infective particles. Serological infection may have found more rats that have been exposed to hantaviruses, but using a more conservative test that detects current presence of DNA is arguably more relevant for the potential infection risk for humans as these individuals are actively infected, moreover uncertainty about specificity of serological testing and the potential for cross-reaction with antibodies against other viruses could have resulted in noise within the dataset (false positives). Furthermore, RT-PCR could allow the detection of new variant of ANJOV or a new orthohantavirus which was of importance for us since to date, hantavirus described in Madagascar, were detected from R. rattus collected in a small region of Madagascar [25].

At the national scale, sites in humid environments presented the highest rates of hantavirus infection. Climatic drivers, including increased precipitation, have been linked to inter-annual variation of hantavirus cases in humans in China and the Four Corners Region of the USA [33, 34, 55]. Various mechanisms for these relationships have been proposed, including increased vegetation growth leading to more abundant rodent populations and increased contact and viral transmission between rodents and between rodents and humans [34, 55]. A positive relationship between host abundance and infection prevalence has been found for Puumala orthohantavirus in a spatiotemporal study of bank vole populations [39]. However, some other studies have found only weak relationships between principal host abundance and prevalence [56], and in our study we did not observe any significant association between rat abundance and hantavirus prevalence. The absence of strong effects of host abundance may reflect difficulties in measuring abundance in a relevant way, for example current prevalence may depend on previous host abundance rather than current host abundance, or because the relationship between abundance and contact rates is not linear [57]. Alternatively, other factors may drive the association between high precipitation and high prevalence through impacts on contact rates.

Rather than absolute rat abundance, the positive association may instead be linked to variation in the seasonality of reproduction. As indicated by the PCA analysis, sites with high precipitation are also characterized by less seasonality. Rats inhabiting such environments, where there are sufficient food resources throughout the year to allow reproduction, may exhibit behaviors that are linked to hantavirus transmission throughout the year (e.g. aggressive behaviors or behaviors associated with maintenance of territories). As humidity can maintain the infectivity and the stability of virus in the ex-vivo environment [58], an alternative explanation could be that increased contact between rodents and infective particles increases hantavirus infection rates in humid areas.

Within sites, rats trapped outside were more likely to be infected than rats trapped inside houses. This variation could also be due to differences in rat contact structures and virus persistence. Contact structures between rats living in houses are likely to be very different from rats inhabiting outdoors. If rats typically inhabit a single house, each house may represent a single small and relatively isolated population with a much-reduced contact structure. A non-exclusive alternative hypothesis could be that persistence of hantavirus in the environment is reduced within houses.

Several studies of hantavirus in reservoir populations have found an apparent dilution effect, with lower prevalence in sites with increased mammal diversity [37‐39]. Such an effect can occur through a variety of mechanisms such as decreases in host density due to competition or reductions in encounter rates between infected and susceptible hosts. In studies of Sin Nombre virus there has been some evidence that increased mammal diversity leads to reduced intraspecific contact rates in the principal host species [37]. In our study we found no evidence of an effect of diversity, measured at the site-level, on hantavirus prevalence and it seems unlikely that community diversity explains the difference in infection probability between inside and outside rats, as all the species trapped were caught in both types of locations (apart from M. musculus, which could only be captured by Sherman traps which were restricted to houses, and the rarely trapped H. semispinosus). The lack of a dilution effect in our study may suggest that whilst an effect of community composition appears to be common for hantaviruses in reservoir populations inhabiting natural and semi-natural habitats located in several parts of the world [59], it is not a feature of hantaviruses in a peridomestic context. Alternatively, as for abundance, our measures of community diversity may be inappropriate to capture the underlying mechanism. For example, the effects of community composition on Puumala orthohantavirus were driven by abundance of a specific species, rather than overall community diversity [39, 60]. Thus, more detailed analyses, as well as data from more diverse sites, would further improve our understanding of the potential mechanisms behind the spatial variation in prevalence.