Detection, isolation, and genetic characterization of Rift Valley fever virus from Anopheles (Anopheles) coustani, Anopheles (Anopheles) squamosus, and Culex (Culex) antennatus of the Haute Matsiatra region, Madagascar

Following veterinary alerts of Rift Valley fever (RVF) in the districts of Fianarantsoa I and II in November 2008 and in the district of Ambalavao in April 2009, entomological and virological investigations were carried out to identify the mosquito species that could act as RVF virus (RVFV) vectors in the region. A total of 12,785 adult mosquitoes belonging to 5 genera and 21 species were collected. After identification, mosquitoes were pooled by species, sex, and female status (fed or unfed) and then stored at -80°C. Of 319 pools of unfed monospecific female mosquito tested by real-time RT-polymerase chain reaction, RVFV was detected in 1 pool of Anopheles coustani, 5 pools of An. squamosus, and 2 pools of Culex antennatus mosquitoes. The virus was isolated in mosquito cell lines from two of the five Real Time-RT-polymerase chain reaction (real time-RT-PCR) positive pools of An. squamosus mosquitoes. From the eight RVFV strains detected, partial S, M, and L genome segments sequences were obtained. The phylogenetic analysis of these sequences showed that the strains circulating in mosquitoes were genetically close to those that circulated in livestock and humans during RVF outbreaks in 2008 and 2009. This study, therefore, provides strong evidence that An. squamosus, An. coustani, and Cx. antennatus could play a role as vectors of the RVFV during the disease outbreaks in 2008-2009. Bioecological, genetic, and RVF transmission studies on these three mosquito species are needed to address this question and thus improve prevention and control of future RVF outbreaks in Madagascar, where these species are present.     

A Review of Mosquitoes Associated with Rift Valley Fever Virus in Madagascar

Rift Valley fever (RVF) is a viral zoonotic disease occurring throughout Africa, the Arabian Peninsula, and Madagascar. The disease is caused by a Phlebovirus (RVF virus [RVFV]) transmitted to vertebrate hosts through the bite of infected mosquitoes. In Madagascar, the first RVFV circulation was reported in 1979 based on detection in mosquitoes but without epidemic episode. Subsequently, two outbreaks occurred: the first along the east coast and in the central highlands in 1990 and 1991 and the most recent along the northern and eastern coasts and in the central highlands in 2008 and 2009. Despite the presence of 24 mosquitoes species potentially associated with RVFV transmission in Madagascar, little associated entomological information is available. In this review, we list the RVFV vector, Culex antennatus, as well as other taxa as candidate vector species. We discuss risk factors from an entomological perspective for the re-emergence of RVF in Madagascar.     

Virus isolations and high population density implicate Culex antennatus (Becker) (Diptera: Culicidae) as a vector of Rift Valley Fever virus during an outbreak in the Nile Delta of Egypt

In June, 2003, Egypt's hospital-based electronic disease surveillance system began to record increased cases of acute febrile illness from governorates in the Nile Delta. In response to a request for assistance from the Egyptian Ministry of Health and the World Health Organization (WHO), the U.S. Naval Medical Research Unit No. 3 (NAMRU-3) provided assistance in identifying the cause and extent of this outbreak. Testing of human clinical samples (n=375) from nine governorates in Egypt identified 29 cases of RVF viremia that spanned the period of June to October, and a particular focus of disease in Kafr el Sheikh governorate (7.7% RVF infection rate). Veterinary samples (n=101) collected during this time in Kafr el Sheikh and screened by immunoassay for RVFV-specific IgM identified probable recent infections in cattle (10.4%) and sheep (5%). Entomologic investigations that focused in rural, rice growing villages in the Sidi Salim District of Kafr el Sheikh during August-September, 2003, collected, identified, and tested host-seeking female mosquitoes for the presence of pathogenic viruses. Three isolates of RVF virus (RVFV) were obtained from 297 tested pools of female mosquitoes and all three RVFV isolates came from Cx. antennatus (Becker). While Cx. pipiens has been considered the primary vector of RVF virus in Egypt and is often the most common man-biting species found, Cx. antennatus was the dominant species captured at the 2003 outbreak location in Kafr el Sheikh governorate. This is the first time that Cx. antennatus has been found naturally infected with RVFV in Egypt.     

Prevalence of Rift Valley fever infection in ruminants in Madagascar after the 2008 outbreak

A Rift Valley fever (RVF) outbreak occurred in Madagascar from January to May 2008. The objectives of this study were (1) to assess the current and past circulation of RVF virus (RVFV) in livestock in Madagascar and (2) to evaluate the extent and magnitude of the 2008 RVF outbreak in livestock. The results of a country-wide serosurvey conducted in August 2008 on small and large ruminants are reported here. The study included 3437 cattle and 989 small ruminants (227 sheep and 762 goats) sampled in 30 of the 111 Malagasy districts, selected to be representative of the different ecozones and livestock density areas. Sera of animals were tested for the detection of immunoglobulins M (IgM) and G (IgG) against RVFV using commercial enzyme-linked immunosorbent assays kits. Recent infections (presence of IgM against RVFV) were detected in only 9 cattle (0.3% [0.1-0.4]) and 33 small ruminant (3.3% [2.2-4.5]) samples. Past infections (presence of IgG and absence of IgM against RVFV) were detected in 887 cattle (25.8% [24.3-27.3]) and 244 small ruminant (24.7% [22.0-27.4]) samples. Past infections were detected in all sampled sites. All ecozones were affected. In the southern and northwestern areas, the prevalence of cattle showing evidence of past infection with RVFV increased with the age of the animals. Our results suggest that there has been country-wide circulation of RVFV in 2008 in Madagascar, including in parts of the country where no clinical illness, either in animals or in humans, was reported. The data also suggest that the southern and northwestern areas may be endemic for RVFV, and that the virus may spread when ecological conditions are favorable for its amplification.     

Recombinant Rift Valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice

Rift Valley fever (RVF) is a zoonotic disease endemic in Africa and the Arabian Peninsula caused by the highly infectious Rift Valley fever virus (RVFV) that can be lethal to humans and animals and results in major losses in the livestock industry. RVF is exotic to the United States; however, mosquito species native to this region can serve as biological vectors for the virus. Thus, accidental or malicious introduction of this virus could result in RVFV becoming endemic in North America. Such an event would likely lead to significant morbidity and mortality in humans, and devastating economic effects on the livestock industry. Currently, there are no licensed vaccines for RVF that are both safe and efficacious. To address this issue, we developed two recombinant RVFV vaccines using vaccinia virus (VACV) as a vector for use in livestock. The first vaccine, vCOGnGc, was attenuated by the deletion of a VACV gene encoding an IFN-γ binding protein, insertional inactivation of the thymidine kinase gene, and expression of RVFV glycoproteins, Gn and Gc. The second vaccine, vCOGnGcγ, is identical to the first and also expresses the human IFN-γ gene to enhance safety. Both vaccines are extremely safe; neither resulted in weight loss nor death in severe combined immunodeficient mice, and pock lesions were smaller in baboons compared with the controls. Furthermore, both vaccines induced protective levels of antibody titers in vaccinated mice and baboons. Mice were protected from lethal RVFV challenge. Thus, we have developed two safe and efficacious recombinant vaccines for RVF.     

Genetic evidence for the origins of Venezuelan equine encephalitis virus subtype IAB outbreaks.

Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.     

Rift Valley fever virus infection in African buffalo (Syncerus caffer) herds in rural South Africa: evidence of interepidemic transmission

Rift Valley fever virus (RVFV) is an emerging biodefense pathogen that poses significant threats to human and livestock health. To date, the interepidemic reservoirs of RVFV are not well defined. In a longitudinal survey of infectious diseases among African buffalo during 2000-2006, 550 buffalo were tested for antibodies against RVFV in 820 capture events in 302 georeferenced locations in Kruger National Park, South Africa. Overall, 115 buffalo (21%) were seropositive. Seroprevalence of RVFV was highest (32%) in the first study year, and decreased progressively in subsequent years, but had no detectable impact on survival. Nine (7%) of 126 resampled, initially seronegative animals seroconverted during periods outside any reported regional RVFV outbreaks. Seroconversions for RVFV were detected in significant temporal clusters during 2001-2003 and in 2004. These findings highlight the potential importance of wildlife as reservoirs for RVFV and interepidemic RVFV transmission in perpetuating regional RVFV transmission risk.     

Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b in Wild Birds and Poultry in Botswana

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020–2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.     

Lineage II of Southeast Asian/American DENV-2 Is Associated with a Severe Dengue Outbreak in the Peruvian Amazon

During 2010 and 2011, the Loreto region of Peru experienced a dengue outbreak of unprecedented magnitude and severity for the region. This outbreak coincided with the reappearance of dengue virus-2 (DENV-2) in Loreto after almost 8 years. Whole-genome sequence indicated that DENV-2 from the outbreak belonged to lineage II of the southeast Asian/American genotype and was most closely related to viruses circulating in Brazil during 2007 and 2008, whereas DENV-2 previously circulating in Loreto grouped with lineage I (DENV-2 strains circulating in South America since 1990). One amino acid substitution (NS5 A811V) in the 2010 and 2011 isolates resulted from positive selection. However, the 2010 and 2011 DENV-2 did not replicate to higher titers in monocyte-derived dendritic cells and did not infect or disseminate in a higher proportion of Aedes aegypti than DENV-2 isolates previously circulating in Loreto. These results suggest that factors other than enhanced viral replication played a role in the severity of this outbreak.     

Detection of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by In Situ Hybridization (RNAscope®)

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope^® to detect RVFV infection within the mosquito species, Culex pipiens, derived from the United Kingdom was demonstrated. Extensive RVFV replication was detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles.     

Phylogenetic Characterisation of the Full Genome of a Bagaza Virus Isolate from Bird Fatalities in South Africa

Bagaza virus (BAGV), a member of the Ntaya serogroup in the Flavivirus genus of the Flaviviridae, was isolated from the brain tissue of a Himalayan monal pheasant that died following neurological signs in Pretoria, South Africa in 2016. Next-generation sequencing was carried out on this isolate resulting in a genome sequence of 10980nt. The full genome sequence of this isolate, designated ZRU96-16, shared 98% nucleotide identity with a BAGV isolate found in Culex univitattus mosquitoes from Namibia and 97% nucleotide identity with a Spanish BAGV sequence isolated from an infected partridge. In total, seven amino acid variations were unique to ZRU96-16 after alignment with other BAGV and Israel turkey meningoencephalomyelitis (ITV) genomes. The 3′UTR sequence of ZRU96-16 was resolved with sufficient detail to be able to annotate the variable and conserved sequence elements within this region. Multiple sequence alignment of the 3′UTR suggested that it could be useful in lineage designation as more similar viruses carried similar mutations across this region, while also retaining certain unique sites. Maximum likelihood phylogenetic analysis revealed two clusters containing both BAGV and ITVs from Europe, the Middle East and Africa. Broadly, temporal clustering separated isolates into two groups, with one cluster representing viruses from the 1960–2000’s and the other from 2010 onwards. This suggests that there is consistent exchange of BAGV and ITV between Europe and Africa. This investigation provides more information on the phylogenetics of an under-represented member of the Flaviviridae and provides an avenue for more extensive research on its pathogenesis and geographic expansion.     

Detection and Isolation of Sindbis Virus from Field Collected Mosquitoes in Timimoun,Algeria

Sindbis virus (SINV) is a zoonotic alphavirus (family Togaviridae, genus Alphavirus) that causes human diseases in Africa, Europe, Asia, and Australia. Occasionally, SINV outbreaks were reported in South Africa and northern Europe. Birds are the main amplifying hosts of SINV, while mosquitoes play the role of the primary vector. Culex mosquitoes were collected in Algeria and subsequently tested for SINV. SINV RNA was detected in 10 pools out of 40, from a total of 922 mosquitoes tested. A strain of SINV was isolated from a pool displaying high viral load. Whole-genome sequencing and phylogenetic analysis showed that the SINV Algeria isolate was most closely related to a Kenyan strain. This was the first record of SINV in Algeria and more broadly in northwestern Africa, which can be a potential risk for human health in the circulating area. Further studies are needed to measure the impact on public health through seroprevalence studies in Algeria.     

Prediction of a Rift Valley fever outbreak

El Niño/Southern Oscillation related climate anomalies were analyzed by using a combination of satellite measurements of elevated sea-surface temperatures and subsequent elevated rainfall and satellite-derived normalized difference vegetation index data. A Rift Valley fever (RVF) risk mapping model using these climate data predicted areas where outbreaks of RVF in humans and animals were expected and occurred in the Horn of Africa from December 2006 to May 2007. The predictions were subsequently confirmed by entomological and epidemiological field investigations of virus activity in the areas identified as at risk. Accurate spatial and temporal predictions of disease activity, as it occurred first in southern Somalia and then through much of Kenya before affecting northern Tanzania, provided a 2 to 6 week period of warning for the Horn of Africa that facilitated disease outbreak response and mitigation activities. To our knowledge, this is the first prospective prediction of a RVF outbreak.     

Phylogenetic Analysis of Dengue Virus Types 1 and 4 Circulating in Puerto Rico and Key West, Florida, during 2010 Epidemics

Abstract. We describe sequences of six strains of dengue virus (DENV): three DENV-1 isolates and two DENV-4 isolates from Puerto Rico, and a DENV-1 strain from Key West, Florida, obtained from blood donors during 2010 epidemics. Phylogenetic analysis revealed that the Puerto Rico DENV-1 strains constitute a new lineage within genotype V different from those that circulated in Puerto Rico during the past two decades. The newer Puerto Rico DENV-1 strains associated with strains from the Caribbean and South America. The DENV-1 strain from Key West, Florida clustered with a strain isolated from mosquito pools collected in that area and with a number of strains from Nicaragua and Mexico circulating during 2006-2009. The Puerto Rico DENV-4 isolates of genotype II associated with strains that have circulated on the island throughout the 1980s and 1990s and with strains from the Caribbean region and Central America. Introduction and circulation of novel DENV lineages in dengue-endemic regions have the potential to increase the severity of dengue cases.     

Universal pattern of RpoB gene mutations among multidrug-resistant isolates of Mycobacterium tuberculosis complex from Africa.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) presents an increasing burden in Southern Africa. Rapid diagnostic tests for drug resistance to rifampicin have been developed based on mutation analysis of the rpoB gene. However, geographic differences of underlying mutations have recently been suggested. OBJECTIVE: Drug-resistant strains of Mycobacterium tuberculosis complex from Africa were analysed for geographic differences in frequency and location of rpoB mutations. DESIGN: A random sample of rifampicin-resistant strains was collected from 87 patients with pulmonary MDR-TB treated in 12 hospitals from six different regions of South Africa. In addition, 18 isolates of M. tuberculosis complex from Namibia, Sierra Leone, and Uganda, including 13 isolates of M. africanum, were analyzed. Point mutations were detected by direct sequence analysis of the rpoB gene. RESULTS: Missense mutations were identified for 91 isolates (87%). Double mutations were present in eight (8%) MDR-TB isolates, two of which carried one mutation outside a previously described diagnostic region. We found no geographic differences regarding the frequency and pattern of single rpoB gene mutations. CONCLUSION: Our results confirm that molecular genetic analysis of rifampicin resistance based on a core region within the rpoB gene is universally applicable to strains of M. tuberculosis complex from different geographic regions.     

The pathogenesis of Rift Valley fever

Rift Valley fever (RVF) is an emerging zoonotic disease distributed in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV) of the family Bunyaviridae and the genus Phlebovirus. The virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. RVFV infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. This review describes the pathology of RVF in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect RVFV pathogenesis.     

Further Characterization of Rio Grande Virus and Potential for Cross Reactivity with Rift Valley Fever Virus Assays

Phleboviruses (genus Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses (Equus caballus) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.     

Description and analysis of the cattle trade network in the Madagascar highlands: Potential role in the diffusion of Rift Valley fever virus

In 2008–2009 a Rift Valley Fever (RVF) outbreak occurred in the Anjozorobe area, a temperate and mountainous area of the Madagascar highlands. The results of a serosurvey conducted in 2009 suggested recurrent circulation of RVF virus (RVFV) in this area and potential involvement of the cattle trade in RVFV circulation. The objective of this study was to describe the cattle trade network of the area and analyse the link between network structure and RVFV circulation. Five hundred and sixteen animals that tested negative in 2009 were sampled again in 2010. The 2009–2010 cattle-level seroconversion rate was estimated at 7% (95% CI: 5–10%). Trade data from 386 breeders of 48 villages were collected and analysed using social network analysis methodology, nodes being villages and ties being any movements of cattle connecting villages. The specific practice of cattle barter, known as kapsile, that involves frequent contacts between cattle of two breeders, was observed in addition to usual trade. Trade data were analysed using a logistic model, the occurrence of seroconversion at the village level being the outcome variable and the network centrality measures being the predictors. A negative association was observed between the occurrence of seroconversion in the village and introduction of cattle by trade (p = 0.03), as well as the distance to the nearest water point (p = 0.002). Conversely, the practice of kapsile, was a seroconversion risk factor (p = 0.007). The kapsile practice may be the support for inter-village RVFV circulation whereas the trade network is probably rather implicated in the introduction of RVFV to the area from other parts of Madagascar. The negative association of the distance to the nearest water point suggests that after RVFV introduction, a substantial part of transmission may be due to vectors.