Complete Genome Sequences of Dengue Virus Type 2 Epidemic Strains from Reunion Island and the Seychelles.

Dengue virus has recently reemerged in the southern Indian Ocean islands, causing outbreaks in Reunion Island and the Seychelles. In the present study, we determined the complete genome sequences of closely related clinical isolates of dengue virus type 2 circulating in the Seychelles in 2016 and Reunion Island in 2018.

Low seroprevalence of hepatitis E on Reunion island.

OBJECTIVE: Hepatitis E virus (HEV) has been scarcely investigated in the Indian Ocean. Following a nationwide serosurvey among blood donors, we conducted a population-based serosurvey to assess the magnitude of HEV exposure on Reunion Island. METHODS: Four hundred and sixty-six archived frozen human sera from the 2009 CoPanFlu-RUN cohort were analysed using the Wantai HEV IgG enzyme immunoassay. HEV seropositivity was defined as an IgG titre ≥5 UI/ml. Raw and weighted seroprevalences were assessed to account for the discrepancy between the CoPanFlu-RUN subset and the general community. Prevalence proportion ratios (PPR) were measured using log-binomial models. RESULTS: The raw and the weighted seroprevalences of HEV were 9.01% (95% CI 6.41-11.61) and 6.73% (95% CI 4.47-8.98), respectively. The presence of HEV IgG antibodies was associated with increasing age (P < 0.001). In a survey-adjusted model minimizing the sampling bias and adjusting for age, males were more likely to be seropositive than females (adjusted PPR 2.59, 95% CI 1.07-6.25). Seropositivity was spatially heterogeneous across the island (P < 0.01). Living in the neighbourhood of a pig farm within a low to intermediate slope area was associated with seropositivity in several models adjusting for age, gender, altitude of residency and interaction between slope and pig farms. CONCLUSION: Reunion Island is a low endemic area for HEV exposure. Despite limitations related to the retrospective study design, our findings confirm the roles of cumulative lifetime exposure and male gender in HEV exposure. The risk associated with neighbouring pig farms might also suggest environmental contamination in this setting.

Seroprevalence of typhus group and spotted fever group Rickettsia exposures on Reunion island.

OBJECTIVE: Murine typhus has been increasingly reported on Reunion island, Indian ocean, following documentation of eight autochthonous infections in 2012-2013. We conducted a serosurvey to assess the magnitude of the seroprevalence of rickettsioses in the population. Two hundred and forty-one stored frozen sera taken from the 2009 Copanflu-RUN cohort were analysed using an immunofluorescence assay allowing to distinguish typhus group (TGR) and spotted fever group Rickesttsiae (SFGR). Seropositivity was defined for a dilution titre of Rickettsia IgG antibodies ≥ 1:64. Seroprevalence was weighted to account for the discrepancy between the Copanflu-RUN subset and the general population, as to infer prevalence at community level. Prevalence proportion ratios (PPR) were measured using log-binomial models. RESULTS: The weighted seroprevalences of typhus group rickettsioses and spotted fever group rickettsioses were of 12.71% (95% CI 8.84-16.58%) and 17.68% (95% CI 13.25-22.11%), respectively. Pooled together, data suggested that a fifth of the population had been exposed at least to one Rickettsia group. Youths (< 20 years) were less likely seropositive than adults (adjusted PPR 0.13, 95% CI 0.01-0.91). People living in the western dryer part of the island were more exposed (adjusted PPR 2.53, 95% CI 1.07-5.97). Rickettsioses are endemic on Reunion island and circulated before their first identification as murine typhus in year 2011. Surprisingly, since isolation of Rickettsia africae from Amblyomma variegatum in year 2004 or isolation of Rickettsia felis from Amblyomma loculosum, no autochthonous cases of African tick-bite fever or flea-borne spotted fever has yet been diagnosed.

Seroprevalence of Coxiella burnetii (Q fever) Exposure in Humans on Reunion Island.

After the documentation of sporadic cases of Q fever endocarditis, we conducted a serosurvey to assess Coxiella burnetii exposure on Reunion Island. Two hundred forty-one stored frozen human sera were analyzed using an immunofluorescence assay. The weighted seroprevalence of Q fever was of 6.81% (95% confidence interval, 4.02%-9.59%). Despite the absence of infection in youths <20 years of age, exposure was not driven by age or by gender. There was a spatial disparity in exposure across the island, with higher prevalence being reported in regions where ruminant farms are present. The seroprevalence pattern suggests that Q fever is endemic on Reunion Island.

The epidemic of Dengue virus type-2 Cosmopolitan genotype on Reunion Island relates to its active circulation in the Southwestern Indian Ocean neighboring islands.

Reunion Island is currently experiencing an epidemic caused by Dengue virus type-2 (DENV-2) resulting in over 6,763 cases from austral summer 2017 to winter 2018. Phylogenetic analyses on two non-imported cases of dengue infection from Reunion Island highlight a regional circulation of DENV-2 Cosmopolitan lineage 1 virus on both Reunion Island and the Seychelles.

Insight into the global evolution of Rodentia associated Morbilli-related paramyxoviruses.

One portion of the family Paramyxoviridae is a group of Unclassified Morbilli-Related Viruses (UMRV) recently recognized in wild small mammals. At a global level, the evolutionary history of these viruses is not properly understood and the relationships between UMRV and their hosts still remain largely unstudied. The present study revealed, for the first time, that Rodentia associated UMRV emerged from a common ancestor in southern Africa more than 4000 years ago. Sequenced UMRV originating from different regions in the world, clustered into four well-supported viral lineages, which suggest that strain diversification occurred during host dispersal and associated exchanges, with purifying selection pressure as the principal evolutionary force. In addition, multi-introductions on different continents and islands of Rodentia associated UMRV and spillover between rodent species, most probably Rattus rattus, were detected and indicate that these animals are implicated in the vectoring and in the worldwide emergence of this virus group. The natural history and the evolution dynamics of these zoonotic viruses, originating from and hosted by wild animals, are most likely shaped by commensalism related to human activities.

Serological Evidence of Lyssaviruses among Bats on Southwestern Indian Ocean Islands.

We provide serological evidence of lyssavirus circulation among bats on southwestern Indian Ocean (SWIO) islands. A total of 572 bats belonging to 22 species were collected on Anjouan, Mayotte, La Réunion, Mauritius, Mahé and Madagascar and screened by the Rapid Fluorescent Focus Inhibition Test for the presence of neutralising antibodies against the two main rabies related lyssaviruses circulating on the African continent: Duvenhage lyssavirus (DUVV) and Lagos bat lyssavirus (LBV), representing phylogroups I and II, respectively. A total of 97 and 42 sera were able to neutralise DUVV and LBV, respectively. No serum neutralised both DUVV and LBV but most DUVV-seropositive bats (n = 32/220) also neutralised European bat lyssavirus 1 (EBLV-1) but not Rabies lyssavirus (RABV), the prototypic lyssavirus of phylogroup I. These results highlight that lyssaviruses belonging to phylogroups I and II circulate in regional bat populations and that the putative phylogroup I lyssavirus is antigenically closer to DUVV and EBLV-1 than to RABV. Variation between bat species, roost sites and bioclimatic regions were observed. All brain samples tested by RT-PCR specific for lyssavirus RNA were negative.

An eco-epidemiological study of Morbilli-related paramyxovirus infection in Madagascar bats reveals host-switching as the dominant macro-evolutionary mechanism.

An eco-epidemiological investigation was carried out on Madagascar bat communities to better understand the evolutionary mechanisms and environmental factors that affect virus transmission among bat species in closely related members of the genus Morbillivirus, currently referred to as Unclassified Morbilli-related paramyxoviruses (UMRVs). A total of 947 bats were investigated originating from 52 capture sites (22 caves, 18 buildings, and 12 outdoor sites) distributed over different bioclimatic zones of the island. Using RT-PCR targeting the L-polymerase gene of the Paramyxoviridae family, we found that 10.5% of sampled bats were infected, representing six out of seven families and 15 out of 31 species analyzed. Univariate analysis indicates that both abiotic and biotic factors may promote viral infection. Using generalized linear modeling of UMRV infection overlaid on biotic and abiotic variables, we demonstrate that sympatric occurrence of bats is a major factor for virus transmission. Phylogenetic analyses revealed that all paramyxoviruses infecting Malagasy bats are UMRVs and showed little host specificity. Analyses using the maximum parsimony reconciliation tool CoRe-PA, indicate that host-switching, rather than co-speciation, is the dominant macro-evolutionary mechanism of UMRVs among Malagasy bats.

The role of seabirds of the Iles Eparses as reservoirs and disseminators of parasites and pathogens.

The role of birds as reservoirs and disseminators of parasites and pathogens has received much attention over the past several years due to their high vagility. Seabirds are particularly interesting hosts in this respect. In addition to incredible long-distance movements during migration, foraging and prospecting, these birds are long-lived, site faithful and breed in dense aggregations in specific colony locations. These different characteristics can favor both the local maintenance and large-scale dissemination of parasites and pathogens. The Iles Eparses provide breeding and feeding grounds for more than 3 million breeding pairs of seabirds including at least 13 species. Breeding colonies on these islands are relatively undisturbed by human activities and represent natural metapopulations in which seabird population dynamics, movement and dispersal can be studied in relation to that of circulating parasites and pathogens. In this review, we summarize previous knowledge and recently-acquired data on the parasites and pathogens found in association with seabirds of the Iles Eparses. These studies have revealed the presence of a rich diversity of infectious agents (viruses, bacteria and parasites) carried by the birds and/or their local ectoparasites (ticks and louse flies). Many of these agents are widespread and found in other ecosystems confirming a role for seabirds in their large scale dissemination and maintenance. The heterogeneous distribution of parasites and infectious agents among islands and seabird species suggests that relatively independent metacommunities of interacting species may exist within the western Indian Ocean. In this context, we discuss how the patterns and determinants of seabird movements may alter parasite and pathogen circulation. We conclude by outlining key aspects for future research given the baseline data now available and current concerns in eco-epidemiology and biodiversity conservation.

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes.

BACKGROUND: Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. PRINCIPAL FINDINGS: We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7-15% of all reads were classified as "unknown", depending on the random amplification method. CONCLUSION: The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses.

Influenza A virus on oceanic islands: host and viral diversity in seabirds in the Western Indian Ocean.

Ducks and seabirds are natural hosts for influenza A viruses (IAV). On oceanic islands, the ecology of IAV could be affected by the relative diversity, abundance and density of seabirds and ducks. Seabirds are the most abundant and widespread avifauna in the Western Indian Ocean and, in this region, oceanic islands represent major breeding sites for a large diversity of potential IAV host species. Based on serological assays, we assessed the host range of IAV and the virus subtype diversity in terns of the islands of the Western Indian Ocean. We further investigated the spatial variation in virus transmission patterns between islands and identified the origin of circulating viruses using a molecular approach. Our findings indicate that terns represent a major host for IAV on oceanic islands, not only for seabird-related virus subtypes such as H16, but also for those commonly isolated in wild and domestic ducks (H3, H6, H9, H12 subtypes). We also identified strong species-associated variation in virus exposure that may be associated to differences in the ecology and behaviour of terns. We discuss the role of tern migrations in the spread of viruses to and between oceanic islands, in particular for the H2 and H9 IAV subtypes.

Highly diverse morbillivirus-related paramyxoviruses in wild fauna of the southwestern Indian Ocean Islands: evidence of exchange between introduced and endemic small mammals.

UNLABELLED: The Paramyxoviridae form an increasingly diverse viral family, infecting a wide variety of different hosts. In recent years, they have been linked to disease emergence in many different animal populations and in humans. Bats and rodents have been identified as major animal populations capable of harboring paramyxoviruses, and host shifting between these animals is likely to be an important driving factor in the underlying evolutionary processes that eventually lead to disease emergence. Here, we have studied paramyxovirus circulation within populations of endemic and introduced wild small mammals of the southwestern Indian Ocean region and belonging to four taxonomic orders: Rodentia, Afrosoricida, Soricomorpha, and Chiroptera. We report elevated infection levels as well as widespread paramyxovirus dispersal and frequent host exchange of a newly emerging genus of the Paramyxoviridae, currently referred to as the unclassified morbillivirus-related viruses (UMRVs). In contrast to other genera of the Paramyxoviridae, where bats have been shown to be a key host species, we show that rodents (and, in particular, Rattus rattus) are significant spreaders of UMRVs. We predict that the ecological particularities of the southwestern Indian Ocean, where small mammal species often live in densely packed, multispecies communities, in combination with the increasing invasion of R. rattus and perturbations of endemic animal communities by active anthropological development, will have a major influence on the dynamics of UMRV infection. IMPORTANCE: Identification of the infectious agents that circulate within wild animal reservoirs is essential for several reasons: (i) infectious disease outbreaks often originate from wild fauna; (ii) anthropological expansion increases the risk of contact between human and animal populations and, as a result, the risk of disease emergence; (iii) evaluation of pathogen reservoirs helps in elaborating preventive measures to limit the risk of disease emergence. Many paramyxoviruses for which bats and rodents serve as major reservoirs have demonstrated their potential to cause disease in humans and animals. In the context of the biodiversity hot spot of southwestern Indian Ocean islands and their rich endemic fauna, we show that highly diverse UMRVs exchange between various endemic animal species, and their dissemination likely is facilitated by the introduced Rattus rattus. Hence, many members of the Paramyxoviridae appear well adapted for the study of the viral phylodynamics that may be associated with disease emergence.

Rickettsia spp. in seabird ticks from western Indian Ocean islands, 2011-2012.

We found a diversity of Rickettsia spp. in seabird ticks from 6 tropical islands. The bacteria showed strong host specificity and sequence similarity with strains in other regions. Seabird ticks may be key reservoirs for pathogenic Rickettsia spp., and bird hosts may have a role in dispersing ticks and tick-associated infectious agents over large distances.

Influenza C virus high seroprevalence rates observed in 3 different population groups.

The epidemiology of Influenza C virus (FLUCV) infections remains poorly characterised. Here, we have examined the age- and location-specific seroprevalence of antibodies against FLUCV in 1441 sera from metropolitan continental France (Marseille), South-West Indian Ocean French territories (Reunion Island) and United-Kingdom (Edinburgh) using a combination of haemagglutination inhibition, virus neutralisation and ELISA assays. Our results show that immunity to FLUCV is common in all locations studied (global seroprevalence values >50%) and that the first immunising contacts generally occur early in life (i.e., in the 0-4 year-old age group). The latter item is further supported by the detection of FLUCV RNA by RT-PCR in naso-pharyngeal samples collected in patient attending the Emergency Room of the Public hospitals of Marseille, France with a large majority of children under 10 years-old: 17 (60.7%) in children ≤3 yo, 10 (35.7%) in the 4-10 yo age group and 1 (3.6%) in an adult (49yo). The temporal distribution of cases was atypical with regard to influenza (a large proportion of cases occurred in spring and summer) and the clinical presentation was diverse, including but being not limited to classical Influenza-like-Ilnesses. Altogether, our results indicate an intense circulation of FLUCV in the different study areas and an early occurrence of infection in human life. Flu C appears to be a widely under-diagnosed and under-studied human paediatric disease that obviously deserves further clinical and epidemiological characterisation.

Massive infection of seabird ticks with bacterial species related to Coxiella burnetii.

Seabird ticks are known reservoirs of bacterial pathogens of medical importance; however, ticks parasitizing tropical seabirds have received less attention than their counterparts from temperate and subpolar regions. Recently, Rickettsia africae was described to infect seabird ticks of the western Indian Ocean and New Caledonia, constituting the only available data on bacterial pathogens associated with tropical seabird tick species. Here, we combined a pyrosequencing-based approach with a classical molecular analysis targeting bacteria of potential medical importance in order to describe the bacterial community in two tropical seabird ticks, Amblyomma loculosum and Carios (Ornithodoros) capensis. We also investigated the patterns of prevalence and host specificity within the biogeographical context of the western Indian Ocean islands. The bacterial community of the two tick species was characterized by a strong dominance of Coxiella and Rickettsia. Our data support a strict Coxiella-host tick specificity, a pattern resembling the one found for Rickettsia spp. in the same two seabird tick species. Both the high prevalence and stringent host tick specificity suggest that these bacteria may be tick symbionts with probable vertical transmission. Detailed studies of the pathogenicity of these bacteria will now be required to determine whether horizontal transmission can occur and to clarify their status as potential human pathogens. More generally, our results show that the combination of next generation sequencing with targeted detection/genotyping approaches proves to be efficient in poorly investigated fields where research can be considered to be starting from scratch.

Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods.

Absence of coronaviruses, paramyxoviruses, and influenza A viruses in seabirds in the southwestern Indian Ocean.

We investigated circulation of coronaviruses, paramyxoviruses, and influenza A viruses in eight seabird species of the southwestern Indian Ocean. Viruses were not detected by real-time polymerase chain reactions in the 338 tested cloacal swab samples, supporting that they did not circulate in the studied colonies at the time of sampling.

High prevalence of hepatitis E in humans and pigs and evidence of genotype-3 virus in swine, Madagascar.

Hepatitis E virus (HEV) causes an orofecal disease transmitted through poor hygiene environments, contaminated food (mainly pork products), or by contacts with infected animals. Very little data are currently available regarding the disease in the Southwestern Indian Ocean Islands. We report the first sero- and viro-survey for HEV in human and swine in Madagascar. A seroprevalence rate of 14.1% (60 of 427) was measured in slaughterhouse workers. Seroprevalence to HEV in pigs was estimated to 71.2% (178 of 250), strongly suggesting the existence of a zoonotic cycle. Three out of 250 pig livers (1.2%) tested HEV RNA-positive by quantitative polymerase chain reaction. Phylogenetic analyses based on 1-kb sequences of the ORF 2-3 identified these viruses as HEV genotype 3. Sequences clustered in a distinct Malagasy sub-clade, possibly representative of a new sub-genotype, for which the date of emergence was estimated around 1989. Further studies are needed to confirm other transmission routes of HEV to humans, especially through non-zoonotic cycles.

Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean.

Bats are reservoirs for many emerging zoonotic viruses. In this study, we screened 197 animals from 15 different bat species of the Southwest Indian Ocean for paramyxovirus infection and identified paramyxoviruses in five insectivorous bat-species from the Union of the Comoros (3/66), Mauritius (1/55) and Madagascar (4/76). Viral isolation was possible via cell culture and phylogenetic analysis revealed these viruses clustered in a Morbillivirus-related lineage, with relatively high nucleotide sequence similarity to other recently discovered insectivorous-bat paramyxoviruses but distinct from those known to circulate in frugivorous bats.