Vertebrate reservoir hosts

Globally, most pathogens of medical and veterinary importance can infect multiple host species [1]. Indeed, an estimated 60–75% of emerging infectious diseases are multi-host zoonoses [2]. Zoonotic arboviruses, such as Rift Valley fever virus, West Nile virus, and Japanese encephalitis virus, have complex transmission cycles that include multiple host and vector species in maintenance and spillover [2–4]. Identifying optimal approaches to mitigate spillover of multi-host pathogens requires an understanding of how the transmission cycles of zoonotic viruses and non-human hosts contribute to spatiotemporal changes in the patterns of human disease [1, 5, 6]. The challenge of understanding the complex population biology of multi-host pathogens comes not only from identifying potential reservoir host species, but in disentangling which species contribute most to transmission and pathogen pressure, and whether any species are crucial to persistence within the reservoir community [7, 8].

The definition of a “reservoir” in infectious disease epidemiology is not straightforward [7]. This is especially the case for arboviruses, where complex and novel transmission dynamics among arboviruses has resulted in multiple definitions for the key term “reservoir” [9]. Given the diversity of virus-vector-vertebrate host interactions, there is unlikely to be a single definition suitable for all systems or for all applications [9]. Here, we are concerned with identifying which vertebrate hosts contribute most to the pathogen pressure on humans (via infected vectors). We therefore adopt the notion that an arbovirus “reservoir” is a vertebrate host species which, if present in sufficient abundance, will contribute to the pathogen pressure on humans. This will require that it has frequent contact with vector populations, is attractive to a vector as a blood meal source, is susceptible to infection, and can produce sufficient viraemia to infect another vector [9–11]. Kuno and Chang [3] identified three commonly used criteria for classifying vertebrate reservoirs of arboviruses: (i) virus isolation from suspected animals; (ii) relatively high antibody prevalence in the animals captured in the field; and (iii) demonstration of viraemia (of high virus titre and duration) in the suspected animals typically obtained under lab conditions. Methods commonly adopted to address these criteria include virus isolation, serosurveys and experimental infection studies, respectively.

Ross River virus

Ross River virus (RRV) is a zoonotic alphavirus and human infection is nationally notifiable in Australia. It is responsible for the greatest number of mosquito-borne infections in humans across every state and territory of Australia [12]. On average there are 4800 cases of RRV notified per year Australia-wide, with the majority from Queensland [12]. There are occasional large outbreaks of RRV involving a significantly higher number of human cases. In 2015, there were 9800 notifications of RRV - almost double the national average, 6192 of which were reported from Queensland. In 2017, an outbreak occurred in Victoria, with some 1200 notifications reported in January and February alone, exceeding the state counts for the previous four years combined [12].

Ross River Virus is not usually fatal [13]. However, patients with disease caused by RRV infection present with symptoms that include polyarthritis, myalgia, and fever and chronic joint pain which may last several weeks and, in some cases, months [14–16]. The economic costs of illness were estimated to be A$1070 per case in 2002, averaging more than A$5 million each year [17]. This is likely a conservative estimate of RRV cost as it does not include broader implications of infection, such as the inability for an individual to work or care for children [13]. Presently, there is no treatment or commercially available vaccine for RRV and the best means to reduce the risk of infection is through mosquito management and avoidance of mosquito bites [14, 18].

More than 40 species of mosquitoes have yielded isolates of RRV (summarised in [19]), although many are likely to only have a minor role in transmission. Species most commonly associated with transmission include saltmarsh mosquitoes Aedes camptorhychus, presenting in southern Australia and is replaced by Ae. vigilax north of its range, and the freshwater mosquito (Culex annulirostris) that is present throughout Australia, excluding Tasmania [14].

A definitive description of the host-vector relationships in the transmission cycle of RRV is currently not available. Non-human reservoirs of RRV are thought to play a significant role in RRV endemicity [20–22]. While several authors have suggested that human-mosquito-human transmission of RRV may occur during epidemics [23–25], such transmission is not believed to be sufficient to account for the total number of reported cases each year in Australia [19], nor to be responsible for the long-term persistence of RRV.

Marsupials are generally considered better reservoirs of RRV than placental mammals, which in turn are better reservoirs than birds [13, 19, 26, 27]. This hypothesis first appeared in the literature in 1971 following epidemiological studies in northern Queensland where high rates of RRV seropositivity were detected in macropods (kangaroos and wallabies) [28]. However, the hypothesis deserves critical re-evaluation because there is evidence that RRV circulates in countries in the Pacific, where marsupials are absent [29–31].

This review aims to: (i) critically review the evidence supporting the hypothesis ‘marsupials are better reservoirs of RRV than placental mammals, which in turn are better reservoirs than birds’; (ii) characterise the limitations of that evidence; and (iii) identify research gaps with regards to RRV transmission cycles.

Statistical analysis

A meta-analysis of results across experimental infection studies was not possible as methods of infection and viral detection were highly variable. Instead, we conducted two one-way analysis of variance (ANOVA) for the one experimental infection study that assessed the greatest number of species (n = 10 species, [27]) to test the hypothesis that the duration of viraemia and peak viral titre differs between species groups (marsupial, placental mammal and bird).

For serosurveys, the seroprevalence range was calculated for each species group (marsupial, placental mammal or bird), and plotted as a boxplot. An ANOVA was used to test for differences in seroprevalence between species groups across different studies.

Experimental infection studies

Statistical analysis of the results from Kay et al. [27], the experimental infection study with the greatest number of species (n = 10) and largest number of individuals tested (n = 92), showed that although viraemia in grey kangaroos (Macropus giganteus) attained moderately high levels and lasted the longest duration (Fig. 1), there was no significant difference (between species groups (marsupial (n = 11 individuals, 2 species), placental mammals (n = 45 individuals, 5 species) and birds (n = 35 individuals, 3 species) for both duration of viraemia (F_{(2, 9)} = 2.312, P = 0.169) and peak titre level (F_{(2, 9)} = 3.177, P = 0.104).

For horses (Equus caballus) and little corellas (Cacatua sanguinea), Kay et al. [27] also used susceptible Cx annulirostris vectors to feed on infected hosts to determine the percentage of mosquitoes that became infected with RRV. Despite low titre and short duration viraemias (2.3 SMIC, 50 hours; Fig. 1), little corellas infected 14% of an unknown number of recipient vectors. Horses developed the highest titre viraemeia (6.3SMIC), of one of the longest durations (112 hours), and infected a comparable 11% of recipient vectors.

Virus isolation studies

Serosurvey studies

We identified a total of 30 serosurveys studies (Additional file 1: Table S2) that tested more than 17,000 serum samples from 77 host species. The majority of these studies were undertaken in Australia, with a small number in New Zealand, Fiji and Papua New Guinea (Table 1). Serosurveys for RRV in non-human species have spanned almost 50 years and, as such, the methods within these studies vary substantially. Studies were grouped by decade of publication to accommodate the different serological methods and species groups tested (Fig. 2). Earlier studies favoured haemoglobin inhibition. This technique has now largely been superseded in favour of assays with better sensitivity and specificity. Virus neutralisation, either through Plaque Reduction Neutralisation (PRNT) (the gold standard) or serum microneutralisation are highly specific [41] and have been used throughout the decades. These methods are generally considered more labour intensive, require trained personnel and a minimum of five days to perform. More recent serosurveys have used enzyme linked immunosorbent assay (ELISA) which can be purchased in commercial kits and are more commonly used in human diagnostic labs (Fig. 2a). In the first decade (1966–1975) of seroprevalence studies, 80% of all species sampled were birds (Fig. 2b). In the following decade (1976–1985) more than 82% of serosurveys were performed on placental mammal species. In the subsequent two decades (1986–2005) marsupial species were sampled most frequently (between 50–60% of serosurveys), followed by placental mammals (between 32–40% of serosurveys) and birds (between 0–17% of serosurveys).

Figure 3 shows the mean seropositivity in each of the three species groups. Half of all species sampled were marsupials (n = 39 species), followed by placental mammals (n = 27 species) and birds (n = 13 species). Placental mammals comprised the largest number of sera (n = 10,126), more than double that of marsupial sera (n = 4304) and quadruple that of bird sera (n = 2621). Within placental mammals, cattle have been sampled most frequently (28.9% of samples), closely followed by horses (28.8% of samples). For marsupials, 46% of serosurveys were from one study with a focus on western grey kangaroos, Macropus fuliginosus [42]. Within birds, chickens were the most sampled species, contributing 38% of all samples in this species group.

Overall, there was a significant difference in seroprevalence between species groups (F_(2, 76) = 7.091, P = 0.001). Across studies, the median seroprevalence in marsupials was greater when compared with placental mammals and birds (44%, 16% and 0%, respectively; Fig. 3). The interquartile range of seroprevalence was greatest in the marsupial group (5–75%) and smallest in the bird group (0–6%) (Fig. 3). Outliers in the bird seroprevalence results included black ducks (Anas superciliosa, 2 of 3 positive) and little corellas (Cacatua sanguinea, 6 of 12 positive). For placental mammals, the highest seroprevalence was observed in red foxes (Vulpes vulpes, 3 of 4 positive) followed by rabbits (Oryctolagus cuniculus, 6 of 10 positive). All of the following marsupial species have tested positive to RRV: the eastern barred bandicoot Perameles gunnii (n = 2/2), the eastern bettong Bettongia gaimardi (n = 1/1), the long-nosed potoroo Potorous tridactylus (n = 2/2), the northern nail-tail wallaby Onychogalea unguifera (n = 1/1), the Tasmanian devil Sarcophilus harrisii (n = 4/4) and the tiger quoll Dasyurus maculatus (n = 1/1).

The role of marsupials as reservoirs of RRV

Results from experimental infection, virus isolation and serosurvey studies on 31 marsupial species support the hypothesis that marsupials are competent reservoirs and likely contribute significantly to RRV transmission. However, the evidence is fragmentary and subject to sampling bias, which limits our ability to extrapolate across species, broad geographical areas, habitat and land use types.

Across experimental infection studies, marsupials generally developed high and long-lasting viraemia. This has previously been interpreted as evidence that marsupials are better reservoirs than other species groups, yet we found no significant difference between the mean duration of viraemia or peak viraemia of marsupials, placental mammals or birds. At least two factors must be considered when interpreting results of experimental infection studies. First, experimental infection studies are often constrained by small sample sizes both in the number of species and the number of studies that can be compared. Although we statistically analysed results from the RRV experimental infection study with the greatest number and diversity of species [27], sample sizes were still limited and likely influenced the statistical power of the results. In particular, the diversity of methods used limits comparisons and the effect of simultaneous co-infection with other viruses cannot be discounted. Secondly, while viraemia plays an important role in the maintenance and transmission of arboviruses, using this measure alone to identify potential reservoirs has limited value. For example, in experimental infection studies of West Nile virus, another zoonotic arbovirus, viraemia alone did not definitively identify vertebrate reservoirs: blue jays (Cyanocitta cristata), house finches (Haemorhous mexicanus) and house sparrows (Passer domesticus) were identified as the most competent reservoirs on the basis of viraemia profile [43]. Yet subsequent field investigations identified that American robins (Turdus migratorius), a less viraemic and relatively uncommon avian species, were responsible for the majority of WNV vector infections due to host feeding preferences [44].

The isolation of virus from naturally infected hosts is interpreted as evidence that the species can infect vector mosquitoes and, thus, infect humans. For marsupials, the isolation of RRV from two free-living agile wallabies (M. agilis) (from a total of 17 tested) demonstrates a vector-host relationship under natural conditions and suggests that this species is capable of infecting susceptible vectors, thereby supporting the argument for the species as reservoirs of RRV. Together with Kay et al.’s [27] observation of viraemia in grey kangaroos, this has led to the hypothesis that macropods are important RRV reservoirs within their range. However, the relative importance of this group of species as a reservoir is not clear, given that RRV has been isolated more frequently from horses and passerine birds and the majority of RRV cases in humans do not overlap with macropod home ranges [13].

Across all studies, marsupials had the highest RRV seroprevalence (44.3%), compared with placental mammals (22.7%) and birds (11.1%). Although informative, these data must be interpreted with caution because it is evident that marsupials were more likely to be targeted during sampling efforts in the decade 1986–1995 (Fig. 2b). This shift in targeted species group followed the results of experimental infection studies demonstrating marsupials as competent amplifiers of RRV in 1986. Further, without information on the age of individuals, seroprevalence data should be compared between studies with caution.

Sampling biases are likely to have arisen from the frequent use of convenience sampling or ‘active surveillance’ methods (where investigator-driven data collection is designed to meet specific information needs [45]). The focus on marsupials as hosts for RRV to the exclusion of other host species is premature, and is unlikely to be uniform across all marsupial species. For example, brushtail possums were hypothesised to be the urban reservoir of RRV, being both marsupials and living in close proximity to humans [35], resulting in a focus on this species. However, targeted surveillance of this species between February and December 2005 in Sydney failed to identify any seropositive individuals [35, 46] of the 10 possums sampled. This number of animals is insufficient to draw strong conclusions about the host status, and further studies are required [46]. Furthermore, it is interesting to note that whilst brushtail possums are an abundant urban marsupial, ringtail possums are more commonly reported in major metropolitan areas including Brisbane, Sydney, Perth, Adelaide and Hobart [47] but only two studies (testing four individuals in total) have been undertaken serological assessments of the species (50% seropositivity) [48, 49].

The role of placental mammals as reservoirs of RRV

Placental mammals comprise the greatest diversity of species tested, including ungulates, carnivorous and small urban species. While placental mammals meet the three criteria for arboviral reservoirs as a species group, there are significant differences among species.

The role of birds as reservoirs of RRV

Birds are the most common arboviral reservoir for zoonotic flaviviruses and alphaviruses globally [4]; however, their contribution as reservoirs of RRV has been largely overlooked. On the basis of experimental infection viraemia data alone, birds appear to be poor amplifiers of RRV. Four species of birds (chickens, pigeons, little corellas and black ducks) have been experimentally infected with RRV. Across experimental infection studies, birds had the lowest peak titre and the shortest duration of viraemia in comparison to marsupials and placental mammals (Table 3). Furthermore, pigeons were one of the only species that did not develop a detectable viraemia. However, little corellas (Cactua sanguinea) were capable of infecting 14% of susceptible Cx annuilostris mosquitoes, despite having a low and short viraemia. This is important because in the same study, horses developed the highest titre but only infected 11% of susceptible vectors. Possible reasons for this were not discussed in the original paper, but we suggest the capability of a vertebrate species to infect susceptible mosquito vectors with RRV may be a more relevant measure of reservoir capacity than viraemia.

The isolation of RRV from birds further supports their capacity as amplifiers. More than 750 virus isolation attempts, across 104 species, yielded the first 3 isolates of RRV from the heart muscle of passerine birds in Northern Queensland: a magpie lark, a flycatcher and a masked finch (Table 4). Passerine birds are recognised as important amplifiers of other arboviruses including flaviviruses such as West Nile virus [43], tick-borne pathogens such as Borrelia burgdorferi - the causative agent for Lyme disease [64] and an arthritic alphavirus closely related to RRV, Sindbis [65]. Indeed, the isolation of the alphavirus Sindbis from passerine birds, in combination with genetic studies and antibody prevalence investigations has implicated birds as the reservoir host of Sindbis [62].

Serological surveys have found low seroprevalence of RRV in birds. However almost 40% of bird sera tested has been from sentinel chickens. Chickens are considered appropriate sentinels for flaviviruses such as Murray valley encephalitis because they display a strong antibody response [66], however experimental infections suggest this is not the case for RRV [27, 37]. Notably, birds with positive serology for RRV were free-living native species: a Tawny frogmouth owl in NSW [48] and an Australasian gannet (Morus serrator) sampled in New Zealand [67]. Thus, the tendency towards sampling chickens in RRV serosurveys may underestimate the rates for birds as a whole, and future serosurveys would benefit from inclusion of greater bird species diversity.

Alternative evidence for non-human reservoirs

This review has focused on the intrinsic host variables important to reservoir capacity. There are other lines of evidence that can be important for investigating potential reservoirs such as blood meal analysis and modelling studies. Determining vector preferences, may indicate a higher feeding frequency, and thus if a capable reservoir, higher transmission rate. Blood meal analysis studies investigate the relationship between the vector and the host. Vector-host choice is a complicated matter, with factors such as host body size, carbon dioxide emission, olfaction, availability, abundance and vector genetics impacting feeding preferences [62, 68]. Blood meal studies are further complicated as they are easily confounded by the environment in which study took place, and as such these studies are best when accompanied with animal abundance and diversity measures. Of the 12 blood meal analysis papers in Australia, only one [69] has done this by asking the human residents to estimate numbers of animals. Further research is needed to investigate vector-host preferences in Australian urban, peri-urban and rural environments and determine the influence this may have on a species capacity to act as a reservoir.

Mathematical models are valuable way of describing and understanding complex disease systems such as RRV. Models can test assumptions of a disease system and generate predictions which can be used for management decisions. For RRV, five studies [20, 70–73] have utilised mechanistic modelling techniques (e.g. Susceptible-Infectious-Recovered models) to better understand the transmission dynamics underpinning the maintenance of the pathogen. Although the models differ in parameters, location and methods, all include a marsupial reservoir. Species that have been modelled as reservoirs are western grey kangaroos and brushtail possums. Overall these studies found that one host alone was insufficient to maintain virus in vector populations. Glass [72] concluded that although marsupials such as kangaroos and wallabies are generally assumed to be the most important reservoir hosts, the virus survived longed under all models when the marsupial host was replaced with one with a shorter infectious period and higher birth rate. Further, the same study reported that very large host populations (> 100,000 individuals) were required for the virus to survive for four years. Choi et. al. [70] similarly found that a kangaroo reservoir did not impact the number of human infections due to a small population size in the region. Carver et al. [20] reported a significant negative relationship between the abundance of a marsupial reservoir and RRV transmission. These findings are in contrast to the putative reservoir hypothesis. Tompkins & Slaney [73] noted that different species may be reservoirs in different environments, such as high-density urban areas and protected environmental habitats, which can result in different transmission cycles. These modelling studies highlight the importance of investigating alternative species as potential reservoirs of RRV. Ideally, the system should be explicitly modelled as a multihost system, but obtaining the necessary data to parameterise such models is challenging [74].

Ross River virus: a multi-host pathogen

Despite the evidence supporting marsupials as reservoirs of RRV, questions remain. Recent studies have found a high seroprevalence of RRV in the Pacific Islands in the absence of marsupial populations [29, 31], suggesting that marsupials are not the only species group capable of increasing the community infection for RRV. Studies modelling RRV reservoirs have suggested that the pathogen has a multi-host system [23, 72]. However, none of the studies reviewed in this paper specifically examined this hypothesis. Multi-host systems are not uncommon for arboviruses but quantifying these systems is challenging, requiring coordinated data collection over temporal and geographical scales for multiple species [3].

To understand RRV as a multi-host pathogen, two issues must be considered. First, as RRV has an international distribution spanning different environmental and social bounds it is important to define the ecological transmission of RRV across different ecosystems. Expansion of Claflin & Webb’s [14] categorisation of potential RRV vectors, habitats and reservoirs across inland, metropolitan and coastal regions to include transmission cycles is warranted. Secondly, to better understand the reservoir capacity between different host communities, identification of amplifying or diluting reservoir hosts is required. Given that humans are not considered significant maintenance reservoirs of RRV outside of epidemic periods in Australia, this may provide a benchmark for relative comparison of seroprevalence. For example, RRV seroprevalence in blood donors shows that the human IgG seroprevalence ranges from 8.38% in Australia in 2011 [75] to 34.4% in French Polynesia between 2011 and 2013 [29]. Whilst this only gives an indication of the number of people exposed and does not consider other contributing factors such as duration of antibody response, these data may be compared with animal serosurvey data to identify species with higher infection rates than humans. Vector-host preference may be key to understanding reservoir and transmission dynamics in other zoonotic arboviruses [3]. Overall, consideration of RRV as a multi-host pathogen may disentangle the complex ecological dynamics that may be taking place.