The role of gastropods in African swine fever virus ecology.

The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.

[Taxonomic status of the Artashat virus (ARTSV) (Bunyaviridae, Nairovirus) isolated from the ticks Ornithodoros alactagalis Issaakjan, 1936 and O. verrucosus Olenev, Sassuchin et Fenuk, 1934 (Argasidae Koch, 1844) collected in Transcaucasia].

The Artashat virus (ARTSV) was originally isolated fom the Ornithodoros alactagalis Issaakjan, 1936 (Argasidae Koch, 1844), which were collected in the burrow of small five-toed jerboa (Allactaga elater Lichtenstein, 1825) in Armenia in 1972. Later, the ARTSV was isolated from the O. verrucosus Olenev, Sassuchin et Fenuk, 1934 collected in the burrows of Persian gerbil (Meriones persicus Blanford, 1875) in Azerbaijan. Based on the virion morphology, the ARTSV was assigned to the Bunyaviridae viruses. In this work, the ARTSV genome was partially sequenced (GenBank ID: KF801650) and it was shown that the ARTSV is a new member of the Nairovirus genus. ARTSV has from 42% (Issyk-Kul virus) to 58% (Raza virus, Hughes group) similarity with the nairoviruses for nucleotide sequence of part of RNA-dependent RNA-polymerase (RdRp). The similarity on the amino acid level is 65-70%. Low level of homology and the equidistant position of the ARTSV on phylogenetic tree indicate that the ARTSV is a new prototype species of the Nairovirus genus (Bunyaviridae) forming a separate phylogenetic branch.

[Genetic characterization of the Geran virus (GERV, Bunyaviridae, Nairovirus, Qalyub group) isolated from the ticks Ornithodoros verrucosus Olenev, Zasukhin and Fenyuk, 1934 (Argasidae) collected in the burrow of Meriones erythrourus Grey, 1842 in Azerbaijan].

The full-length genome of the unclassified Geran virus (GERV, strain LEIV-10899Az) isolated from the ticks (Ornithodoros verrucosus Olenev, Zasukhin and Fenyuk, 1934 (Argasidae, Ornithodorinae)) collected in the burrow of the red-tailed gerbils (Meriones (Cricedidae) erythrourus Grey, 1842) near the Geran station (Azerbaijan) was sequenced using the next-generation approach (GenBank ID: KF801649). It was shown that the GERV is a new representative of the Nairovirus genus (family Bunyaviridae). The comparative analysis of the full-length genome sequences of the GERV with other nairoviruses showed that the highest level of homology (55.6% for N protein (S-segment) of 54.2% for the polyprotein Gn/Gc (M-segment) and 74.8% for the RNA-dependent RNA polymerase (L-segment)) GERV had with the Chim virus (CHIMV) that is also associated with the shelters biocenoses (rodent burrows) in Central Asia and was previously assigned to the Qalyub virus group (QYBV). Comparing the GERV with the QYBV sequences (partial sequence 413 n.o. of RdRp gene) revealed a high level of homology: 74.3 and 97.4% for the nucleotide and amino acid sequences, respectively. The data obtained in this work provided an opportunity to classify the GERV to the QYBV group; the Nairovirus genus, to the family Bunyaviridae.

Sialotranscriptomics of the argasid tick Ornithodoros moubata along the trophogonic cycle.

The argasid tick Ornithodoros moubata is the main vector of human relapsing fever (HRF) and African swine fever (ASF) in Africa. Salivary proteins are part of the host-tick interface and play vital roles in the tick feeding process and the host infection by tick-borne pathogens; they represent interesting targets for immune interventions aimed at tick control. The present work describes the transcriptome profile of salivary glands of O. moubata and assesses the gene expression dynamics along the trophogonic cycle using Illumina sequencing. De novo transcriptome assembling resulted in 71,194 transcript clusters and 41,011 annotated transcripts, which represent 57.6% of the annotation success. Most salivary gene expression takes place during the first 7 days after feeding (6,287 upregulated transcripts), while a minority of genes (203 upregulated transcripts) are differentially expressed between 7 and 14 days after feeding. The functional protein groups more abundantly overrepresented after blood feeding were lipocalins, proteases (especially metalloproteases), protease inhibitors including the Kunitz/BPTI-family, proteins with phospholipase A2 activity, acid tail proteins, basic tail proteins, vitellogenins, the 7DB family and proteins involved in tick immunity and defence. The complexity and functional redundancy observed in the sialotranscriptome of O. moubata are comparable to those of the sialomes of other argasid and ixodid ticks. This transcriptome provides a valuable reference database for ongoing proteomics studies of the salivary glands and saliva of O. moubata aimed at confirming and expanding previous data on the O. moubata sialoproteome.

Detection of African swine fever virus genotype XV in a sylvatic cycle in Saadani National Park, Tanzania.

African swine fever (ASF) is a severe haemorrhagic disease of domestic pigs caused by ASF virus (ASFV). ASFV is transmitted by soft ticks (Ornithodoros moubata complex group) and by direct transmission. In Africa, ASF is maintained in transmission cycles of asymptomatic infection involving wild suids, mainly warthogs (Phacochoerus africanus). ASF outbreaks have been reported in many parts of Tanzania; however, active surveillance has been limited to pig farms in a few geographical locations. There is an information gap on whether and where the sylvatic cycle may occur independently of domestic pigs. To explore the existence of a sylvatic cycle in Saadani National Park in Tanzania, blood and serum samples were collected from 19 warthogs selected using convenience sampling along vehicle-accessible transects within the national park. The ticks were sampled from warthog burrows. Blood samples and ticks were subjected to ASFV molecular diagnosis (PCR) and genotyping, and warthog sera were subjected to serological (indirect ELISA) testing for ASFV antibody detection. All warthog blood samples were PCR-negative, but 16/19 (84%) of the warthog sera were seropositive by ELISA confirming exposure of warthogs to ASFV. Of the ticks sampled, 20/111 (18%) were positive for ASFV by conventional PCR. Sequencing of the p72 virus gene fragments showed that ASF viruses detected in ticks belonged to genotype XV. The results confirm the existence of a sylvatic cycle of ASFV in Saadani National Park, Tanzania, that involves ticks and warthogs independent of domestic pigs. Our findings suggest that genotype XV previously reported in 2008 in Tanzania is likely to be widely distributed and involved in both wild and domestic infection cycles. Whole-genome sequencing and analysis of the ASFV genotype XV circulating in Tanzania is recommended to determine the phylogeny of the viruses.

Potential role of ticks as vectors of bluetongue virus.

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus.

Differential vector competence of Ornithodoros soft ticks for African swine fever virus: What if it involves more than just crossing organic barriers in ticks?

BACKGROUND: Several species of soft ticks in genus Ornithodoros are known vectors and reservoirs of African swine fever virus (ASFV). However, the underlying mechanisms of vector competence for ASFV across Ornithodoros species remain to be fully understood. To that end, this study compared ASFV replication and dissemination as well as virus vertical transmission to descendants between Ornithodoros moubata, O. erraticus, and O. verrucosus in relation to what is known about the ability of these soft tick species to transmit ASFV to pigs. To mimic the natural situation, a more realistic model was used where soft ticks were exposed to ASFV by allowing them to engorge on viremic pigs. METHODS: Ornithodoros moubata ticks were infected with the ASFV strains Liv13/33 (genotype I) or Georgia2007/1 (genotype II), O. erraticus with OurT88/1 (genotype I) or Georgia2007/1 (genotype II), and O. verrucosus with Ukr12/Zapo (genotype II), resulting in five different tick-virus pairs. Quantitative PCR (qPCR) assays targeting the VP72 ASFV gene was carried out over several months on crushed ticks to study viral replication kinetics. Viral titration assays were also carried out on crushed ticks 2 months post infection to confirm virus survival in soft ticks. Ticks were dissected. and DNA was individually extracted from the following organs to study ASFV dissemination: intestine, salivary glands, and reproductive organs. DNA extracts from each organ were tested by qPCR. Lastly, larval or first nymph-stage progeny emerging from hatching eggs were tested by qPCR to assess ASFV vertical transmission. RESULTS: Comparative analyses revealed higher rates of ASFV replication and dissemination in O. moubata infected with Liv13/33, while the opposite was observed for O. erraticus infected with Georgia2007/1 and for O. verrucosus with Ukr12/Zapo. Intermediate profiles were found for O. moubata infected with Georgia2007/1 and for O. erraticus with OurT88/1. Vertical transmission occurred efficiently in O. moubata infected with Liv13/33, and at very low rates in O. erraticus infected with OurT88/1. CONCLUSIONS: This study provides molecular data indicating that viral replication and dissemination in Ornithodoros ticks are major mechanisms underlying ASFV horizontal and vertical transmission. However, our results indicate that other determinants beyond viral replication also influence ASFV vector competence. Further research is required to fully understand this process in soft ticks.

A host species-informative internal control for molecular assessment of African swine fever virus infection rates in the African sylvatic cycle Ornithodoros vector.

African swine fever virus (ASFV) infection in adult Ornithodoros porcinus (Murry 1877, sensuWalton 1979) ticks collected from warthog burrows in southern and East Africa was assessed using a duplex genomic amplification approach that is informative with respect to the invertebrate host species and infecting sylvatic cycle virus. DNA extracted from individual ticks was used as template for the simultaneous amplification of a C-terminal 478-bp ASFV p72 gene region and a approximately 313-bp fragment of the tick mitochondrial 16S rRNA gene, under optimized reaction conditions. Within-warthog burrow infection rates ranged from 0% to 43% using this approach, and phylogenetic analysis of 16S gene sequences revealed the presence of three geographically discrete O. porcinus lineages, but no support for subspecies recognition. False negatives are precluded by the inclusion of host species-informative primers that ensure the DNA integrity of cytoplasmically located genome extracts. In addition, infection rate estimates are further improved as false positives arising from carry-over contamination when performing a two-step nested polymerase chain reaction are negated by the one-step approach. Phylogenetic comparison of full-length virus gene sequences with the partial C-terminal p72 gene target confirmed the epidemiological utility of the latter in a sylvatic setting. The method is therefore of particular value in studies assessing the prevalence and diversity of ASFV in relation to the African sylvatic tick vector and holds potential for investigating the role of alternative tick species in virus maintenance and transmission.

African swine fever.

African swine fever (ASF) is a devastating haemorrhagic fever of pigs that causes up to 100% mortality, for which there is no vaccine. It is caused by a unique DNA virus that is maintained in an ancient cycle between warthogs and argasid ticks, making it the only known DNA arbovirus. ASF has a high potential for transboundary spread, and has twice been transported from Africa to other continents--Europe and subsequently the Caribbean and Brazil (1957, 1959) and the Caucasus (2007). It is also a devastating constraint for pig production in Africa. Research at Onderstepoort Veterinary Institute has made and is making important contributions to knowledge of this disease, focusing on the cycle in warthogs and tampans and transmission from that cycle to domestic pigs, resistance to its effects in domestic pigs, and the molecular genetic characterisation and epidemiology of the virus.

Molecular monitoring of African swine fever virus using surveys targeted at adult ornithodoros ticks: a re-evaluation of Mkuze game reserve, South Africa.

The Mkuze Game Reserve (MGR), in north-eastern KwaZulu-Natal Province, South Africa is an African swine fever virus (ASF) controlled area. In a survey conducted in 1978, ASF prevalence in warthogs and Ornithodoros ticks in MGR was determined to be 2% and 0.06%, respectively. These values, acknowledged as being unusually low compared to other East and southern African ASF-positive sylvatic-cycle host populations, have not been assessed since. The availability of a sensitive PCR-based virus detection method, developed specifically for the sylvatic tampan host, prompted a re-evaluation of ASF virus (ASFV) prevalence in MGR ticks. Of the 98 warthog burrows inspected for Ornithodoros presence, 59 (60.2%) were found to contain tampans and tick sampling was significantly male-biased. Whilst gender sampling-bias is not unusual, the 27% increase in infestation rate of warthog burrows since the 1978 survey is noteworthy as it anticipates a concomitant increase in ASFV prevalence, particularly in light of the high proportion (75%) of adult ticks sampled. However, despite DNA integrity being confirmed by internal control amplification of the host 16S gene, PCR screening failed to detect ASFV. These results suggest that ASFV has either disappeared from MGR or if present, is localized, occurring at exceptionally low levels. Further extensive surveys are required to establish the ASFV status of sylvatic hosts in this controlled area.

Cell lines from the soft tick Ornithodoros moubata.

Primary cell cultures (n = 16) were initiated from tissues of embryonic and neonatal larval Ornithodoros moubata following methods developed for hard ticks. After maintenance for 20-25 months in vitro, cell multiplication commenced in surviving cultures, leading to the establishment of six cell lines designated OME/CTVM21, 22, 24, 25, 26 and 27. All lines are maintained at 28 degrees C, with subculture at 2-8 week intervals. The cultures comprise heterogeneous populations of large cells of 15-100 microm in diameter, often with finger-like protrusions and/or intracellular crystals, rarely attached, predominantly floating and forming clumps or hollow multicellular vesicles up to 1 mm in diameter. Attempts to cryopreserve the cells are described. Tick-borne encephalitis virus has been serially passaged ten times in OME/CTVM21 cells without significant decrease in virus production and with no change in its biological properties as shown by the size and morphology of plaques produced in porcine kidney cells.

Reviewing the Potential Vectors and Hosts of African Swine Fever Virus Transmission in the United States.

African swine fever virus (ASFV) continues to threaten global animal health and agricultural biosecurity. Mitigating the establishment of ASFV in the United States (U.S.) is contingent on (1) the identification of arthropod vectors and vertebrate hosts that are capable of viral maintenance and transmission in the U.S. and (2) knowledge of vector-host associations that may permit transmission. We aggregated data on vector competence, host competence and tick-host associations by systematic review of published articles and collection records to identify species that may support the invasion of ASFV in the U.S. Three species of competent soft ticks occur in the U.S., Ornithodoros coriaceus, Ornithodoros turicata, and Ornithodoros puertoricensis, however, vector competence for the majority of soft ticks in the U.S. remains unknown. Three species of competent vertebrate hosts currently occur in the U.S.: domestic pigs (Sus scrofa domesticus), feral hogs (Sus scrofa), and common warthogs (Phacochoerus africanus). Hierarchical hazard categories based on vector competence, tick-host contact rates, and vector abundance were used to semiquantitatively rank U.S. soft tick species by their relative risk for contributing to ASFV transmission to identify which soft tick species are a priority for future studies. High-risk vector and host species identified in this study can be used to focus ASFV risk assessments in the U.S., guide targeted surveillance and control strategies, and proactively prepare for an ASFV incursion event. Results indicate O. coriaceus, O. turicata, and O. puertoricensis demonstrate the highest relative risk for contributing to ASFV transmission in the U.S., however, many gaps in knowledge exist preventing the full evaluation of at least 30 soft tick species in the U.S. Further study is required to identify soft tick vectors that interact with feral swine populations, elucidate vector competence, and further understand the biology of soft tick species.

African swine fever.

The continuing spread of African swine fever (ASF) outside Africa in Europe, the Russian Federation, China and most recently to Mongolia and Vietnam, has heightened awareness of the threat posed by this devastating disease to the global pig industry and food security. In this review we summarise what we know about the African swine fever virus (ASFV), the disease it causes, how it spreads and the current global situation. We discuss current control methods in domestic and wild pigs and prospects for development of vaccines and other tools for control.

Comparative vector competence of the Afrotropical soft tick Ornithodoros moubata and Palearctic species, O. erraticus and O. verrucosus, for African swine fever virus strains circulating in Eurasia.

African swine fever (ASF) is a lethal hemorrhagic disease in domestic pigs and wild suids caused by African swine fever virus (ASFV), which threatens the swine industry globally. In its native African enzootic foci, ASFV is naturally circulating between soft ticks of the genus Ornithodoros, especially in the O. moubata group, and wild reservoir suids, such as warthogs (Phacochoerus spp.) that are bitten by infected soft ticks inhabiting their burrows. While the ability of some Afrotropical soft ticks to transmit and maintain ASFV is well established, the vector status of Palearctic soft tick species for ASFV strains currently circulating in Eurasia remains largely unknown. For example, the Iberian soft tick O. erraticus is a known vector and reservoir of ASFV, but its ability to transmit different ASFV strains has not been assessed since ASF re-emerged in Europe in 2007. Little is known about vector competence for ASFV in other species, such as O. verrucosus, which occurs in southern parts of Eastern Europe, including Ukraine and parts of Russia, and in the Caucasus. Therefore, we conducted transmission trials with two Palearctic soft tick species, O. erraticus and O. verrucosus, and the Afrotropical species O. moubata. We tested the ability of ticks to transmit virulent ASFV strains, including one of direct African origin (Liv13/33), and three from Eurasia that had been involved in previous (OurT88/1), and the current epizooties (Georgia2007/1 and Ukr12/Zapo). Our experimental results showed that O. moubata was able to transmit the African and Eurasian ASFV strains, whereas O. erraticus and O. verrucosus failed to transmit the Eurasian ASFV strains. However, naïve pigs showed clinical signs of ASF when inoculated with homogenates of crushed O. erraticus and O. verrucosus ticks that fed on viraemic pigs, which proved the infectiousness of ASFV contained in the ticks. These results documented that O. erraticus and O. verrucosus are unlikely to be capable vectors of ASFV strains currently circulating in Eurasia. Additionally, the persistence of infection in soft ticks for several months reaffirms that the infectious status of a given tick species is only part of the data required to assess its vector competence for ASFV.

Assay for and replication of Karshi (mammalian tick-borne flavivirus group) virus in mice.

Little is known about the replication of Karshi virus, a member of the mammalian tick-borne flavivirus group, in its rodent hosts. Therefore, we developed a quantitative real-time RT-PCR assay and measured the amount of viral RNA in selected tissues of infected Swiss Webster mice. Two-day-old mice were highly susceptible, with 100% fatality 9 to 12 days after infection, whereas infection in 9-day-old mice was less virulent, with death occurring only rarely. In nearly all cases, mice inoculated when 2 days old contained similar numbers of viral genome equivalents from blood and liver samples from any given mouse, with titers declining after day 7. In contrast, the amount of viral RNA in the brain began to rise rapidly 4 days after exposure, peaked at about 6 days after virus exposure (titer of > 10(13) genome equivalents/g), and remained at that level until euthanasia or death. Viral profiles were similar in needle-inoculated or tick-exposed mice.

The intracellular proteome of African swine fever virus.

African swine fever (ASF) is a viral disease that affects members of the Suidae family such as African bush pigs, warthogs, but also domestic pigs, and wild boar. It is transmitted by direct contact of naïve with infected animals, by soft ticks of the Ornithodoros genus, or indirectly by movement of infected animals, improper disposal of contaminated animal products or other sources related to human activity. The recent spread of ASF into Eastern and Central European countries is currently threatening the European pig industry. The situation is aggravated as to-date no efficient vaccine is available. African swine fever virus (ASFV) is a large enveloped ds DNA-virus encoding at least 150 open reading frames. Many of the deduced gene products have not been described, less functionally characterized. We have analysed ASFV protein expression in three susceptible mammalian cell lines representing a susceptible host (wild boar) and two non-susceptible species (human and green monkey) by mass spectrometry and provide first evidence for the expression of 23 so far uncharacterized ASFV ORFs. Expression levels of several newly identified ASFV proteins were remarkably high indicating importance in the viral replication cycle. Moreover, expression profiles of ASFV proteins in the three cell lines differed markedly.

African Swine Fever Virus: A Review.

African swine fever (ASF) is a highly contagious viral disease of swine which causes high mortality, approaching 100%, in domestic pigs. ASF is caused by a large, double stranded DNA virus, ASF virus (ASFV), which replicates predominantly in the cytoplasm of macrophages and is the only member of the Asfarviridae family, genus Asfivirus. The natural hosts of this virus include wild suids and arthropod vectors of the Ornithodoros genus. The infection of ASFV in its reservoir hosts is usually asymptomatic and develops a persistent infection. In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which there is no effective vaccine. Identification of ASFV genes involved in virulence and the characterization of mechanisms used by the virus to evade the immune response of the host are recognized as critical steps in the development of a vaccine. Moreover, the interplay of the viral products with host pathways, which are relevant for virus replication, provides the basic information needed for the identification of potential targets for the development of intervention strategies against this disease.

The African swine fever control zone in South Africa and its current relevance.

African swine fever (ASF) has been reported in South Africa since the early 20th century. The disease has been controlled and confined to northern South Africa over the past 80 years by means of a well-defined boundary line, with strict control measures and movement restrictions north of this line. In 2012, the first outbreak of ASF outside the ASF control zone since 1996 occurred. The objective of this study was to evaluate the current relevance of the ASF control line as a demarcation line between endemic ASF (north) areas and ASF-free (south) area and to determine whether there was a need to realign its trajectory, given the recent outbreaks of ASF, global climate changes and urban development since the line's inception. A study of ASF determinants was conducted in an area 20 km north and 20 km south of the ASF control line, in Limpopo, Mpumalanga, North West and Gauteng provinces between May 2008 and September 2012. The study confirmed that warthogs, warthog burrows and the soft tick reservoir, Ornithodoros moubata, are present south of the ASF control line, but no virus or viral DNA was detected in these ticks. There appears to be an increasing trend in the diurnal maximum temperature and a decrease in humidity along the line, but the impact of these changes is uncertain. No discernible changes in minimum temperatures and average rainfall along the disease control line were observed between 1992 and 2014. Even though the reservoirs were found south of the ASF boundary line, the study concluded that there was no need to realign the trajectory of the ASF disease control line, with the exception of Limpopo Province. However, the provincial surveillance programmes for the reservoir, vector and ASF virus south of this line needs to be maintained and intensified as changing farming practices may favour the spread of ASF virus beyond the control line.

No evidence for the involvement of the argasid tick Ornithodoros faini in the enzootic maintenance of marburgvirus within Egyptian rousette bats Rousettus aegyptiacus.

BACKGROUND: The cave-dwelling Egyptian rousette bat (ERB; Rousettus aegyptiacus) was recently identified as a natural reservoir host of marburgviruses. However, the mechanisms of transmission for the enzootic maintenance of marburgviruses within ERBs are unclear. Previous ecological investigations of large ERB colonies inhabiting Python Cave and Kitaka Mine, Uganda revealed that argasid ticks (Ornithodoros faini) are hematophagous ectoparasites of ERBs. Yet, their potential role as transmission vectors for marburgvirus has not been sufficiently assessed. FINDINGS: In the present study, 3,125 O. faini were collected during April 2013 from the rock crevices of Python Cave, Uganda. None of the ticks tested positive for marburgvirus-specific RNA by Q-RT-PCR. The probability of failure to detect marburgvirus at a conservative prevalence of 0.1 % was 0.05. CONCLUSIONS: The absence of marburgvirus RNA in O. faini suggests they do not play a significant role in the transmission and enzootic maintenance of marburgvirus within their natural reservoir host.