Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas.     

Insights into the western Bantu dispersal: mtDNA lineage analysis in Angola

Africa is the homeland of humankind and it is known to harbour the highest levels of human genetic diversity. However, many continental regions, especially in the sub-Saharan side, still remain largely uncharacterized (i.e. southwest and central Africa). Here, we examine the mitochondrial DNA (mtDNA) variation in a sample from Angola. The two mtDNA hypervariable segments as well as the 9-bp tandem repeat on the COII/tRNA^lys intergenic region have allowed us to allocate mtDNAs to common African haplogroups. Angola lies in the southern end of the putative western branch of the Bantu expansion, where it met the local Khoisan populations. Angolan mtDNA lineages show basically a Bantu substrate with no traces of Khoisan lineages. Roughly, more than half of the southwestern mtDNA pool can be assigned to west Africa, ~25% to central Africa and a significant 16% to east Africa, which points to the western gene pool having contributed most to the mtDNA lineages in Angola. We have also detected signals of extensive gene flow from southeast Africa. Our results suggest that eastern and western Bantu expansion routes were not independent from each other, and were connected south of the rainforest and along the southern African savannah. In agreement with historical documentation, the analysis also showed that the Angola mtDNA genetic pool shows affinities with the African lineages from Brazil, the main American destination of the slaves from Angola, although not all lineages in Brazil can be accounted for by the Angolan mtDNA pool.     

Ebola and Marburg virus diseases in Africa: Increased risk of outbreaks in previously unaffected areas?

Filoviral hemorrhagic fever (FHF) is caused by ebolaviruses and marburgviruses, which both belong to the family Filoviridae. Egyptian fruit bats (Rousettus aegyptiacus) are the most likely natural reservoir for marburgviruses and entry into caves and mines that they stay in has often been associated with outbreaks of MVD. On the other hand, the natural reservoir for ebola viruses remains elusive; however, handling of wild animal carcasses has been associated with some outbreaks of EVD. In the last two decades, there has been an increase in the incidence of FHF outbreaks in Africa, some being caused by a newly found virus and some occurring in previously unaffected areas such as Guinea, Liberia and Sierra Leone, in which the most recent EVD outbreak occurred in 2014. Indeed, the predicted geographic distribution of filoviruses and their potential reservoirs in Africa includes many countries in which FHF has not been reported. To minimize the risk of virus dissemination in previously unaffected areas, there is a need for increased investment in health infrastructure in African countries, policies to facilitate collaboration between health authorities from different countries, implementation of outbreak control measures by relevant multi‐disciplinary teams and education of the populations at risk.     

Phylogenetic and Evolutionary Relationships among Yellow Fever Virus Isolates in Africa

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.     

Genetic relationships and evolution of genotypes of yellow fever virus and other members of the yellow fever virus group within the Flavivirus genus based on the 3' noncoding region

Genetic relationships among flaviviruses within the yellow fever (YF) virus genetic group were investigated by comparing nucleotide sequences of the 3' noncoding region (3'NCR). Size heterogeneity was observed between members and even among strains of the same viral species. Size variation between YF strains was due to duplications and/or deletions of repeated nucleotide sequence elements (RYF). West African genotypes had three copies of the RYF (RYF1, RYF2, and RYF3); the Angola and the East and Central African genotypes had two copies (RYF1 and RYF3); and South American genotypes had only a single copy (RYF3). Nucleotide sequence analyses suggest a deletion within the 3'NCR of South American genotypes, including RYF1 and RYF2. Based on studies with the French neurotropic vaccine strain, passage of a YF virus strain in cell culture can result in deletion of RYF1 and RYF2. Taken together, these observations suggest that South American genotypes of YF virus evolved from West African genotypes and that the South American genotypes lost RYF1 and RYF2, possibly in a single event. Repeated sequence elements were found within the 3'NCR of other members of the YF virus genetic group, suggesting that it is probably characteristic for members of the YF virus genetic group. A core sequence of 15 nucleotides, containing two stem-loops, was found within the 3'NCR of all members of the YF genetic group and may represent the progenitor repeat sequence. Secondary structure predictions of the 3'NCR showed very similar structures for viruses that were closely related phylogenetically.     

Genetic evidence for Rift Valley fever outbreaks in Madagascar resulting from virus introductions from the East African mainland rather than enzootic maintenance

Rift Valley fever virus (RVFV), a mosquito-borne phlebovirus, has been detected in Madagascar since 1979, with occasional outbreaks. In 2008 to 2009, a large RVFV outbreak was detected in Malagasy livestock and humans during two successive rainy seasons. To determine whether cases were due to enzootic maintenance of the virus within Madagascar or to importation from the East African mainland, nine RVFV whole genomic sequences were generated for viruses from the 1991 and 2008 Malagasy outbreaks. Bayesian coalescent analyses of available whole S, M, and L segment sequences were used to estimate the time to the most recent common ancestor for the RVFVs. The 1979 Madagascar isolate shared a common ancestor with strains on the mainland around 1972. The 1991 Madagascar isolates were in a clade distinct from that of the 1979 isolate and shared a common ancestor around 1987. Finally, the 2008 Madagascar viruses were embedded within a large clade of RVFVs from the 2006-2007 outbreak in East Africa and shared a common ancestor around 2003 to 2004. These results suggest that the most recent Madagascar outbreak was caused by a virus likely arriving in the country some time between 2003 and 2008 and that this outbreak may be an extension of the 2006-2007 East African outbreak. Clustering of the Malagasy sequences into subclades indicates that the viruses have continued to evolve during their short-term circulation within the country. These data are consistent with the notion that RVFV outbreaks in Madagascar result not from emergence from enzootic cycles within the country but from recurrent virus introductions from the East African mainland.     

First laboratory confirmation and sequencing of Zaire ebolavirus in Uganda following two independent introductions of cases from the 10th Ebola Outbreak in the Democratic Republic of the Congo,June 2019

Uganda established a domestic Viral Hemorrhagic Fever (VHF) testing capacity in 2010 in response to the increasing occurrence of filovirus outbreaks. In July 2018, the neighboring Democratic Republic of Congo (DRC) experienced its 10^th Ebola Virus Disease (EVD) outbreak and for the duration of the outbreak, the Ugandan Ministry of Health (MOH) initiated a national EVD preparedness stance. Almost one year later, on 10^th June 2019, three family members who had contracted EVD in the DRC crossed into Uganda to seek medical treatment.Samples were collected from all the suspected cases using internationally established biosafety protocols and submitted for VHF diagnostic testing at Uganda Virus Research Institute. All samples were initially tested by RT-PCR for ebolaviruses, marburgviruses, Rift Valley fever (RVF) virus and Crimean-Congo hemorrhagic fever (CCHF) virus. Four people were identified as being positive for Zaire ebolavirus, marking the first report of Zaire ebolavirus in Uganda. In-country Next Generation Sequencing (NGS) and phylogenetic analysis was performed for the first time in Uganda, confirming the outbreak as imported from DRC at two different time point from different clades. This rapid response by the MoH, UVRI and partners led to the control of the outbreak and prevention of secondary virus transmission.     

Cross-protection against Marburg virus strains by using a live, attenuated recombinant vaccine

Marburg virus (MARV) has been associated with sporadic episodes of hemorrhagic fever, including a recent highly publicized outbreak in Angola that produced severe disease and significant mortality in infected patients. MARV is also considered to have potential as a biological weapon. Recently, we reported the development of a promising attenuated, replication-competent vaccine against MARV based on recombinant vesicular stomatitis virus (VSV) expressing the glycoprotein of the Musoke strain of MARV (VSVDeltaG/MARVGP-Musoke). We used this vaccine to demonstrate complete protection of cynomolgus monkeys against a homologous MARV challenge. While these results are highly encouraging, an effective vaccine would need to confer protection against all relevant strains of MARV. Here, we evaluated the protective efficacy of the VSVDeltaG/MARVGP-Musoke vaccine against two heterologous MARV strains, the seemingly more pathogenic Angola strain and the more distantly related Ravn strain. In this study, seven cynomolgus monkeys were vaccinated with the VSVDeltaG/MARVGP-Musoke vector. Three of these animals were challenged with the Angola strain, three with the Ravn strain, and a single animal with the Musoke strain of MARV. Two animals served as controls and were each injected with a nonspecific VSV vector; these controls were challenged with the Angola and Ravn strains, respectively. Both controls succumbed to challenge by day 8. However, none of the specifically vaccinated animals showed any evidence of illness either from the vaccination or from the MARV challenges and all of these animals survived. These data suggest that the VSVDeltaG/MARVGP-Musoke vaccine should be sufficient to protect against all known MARV strains.     

Molecular Variability and Distribution of Cassava Mosaic Begomoviruses in Nigeria

Several begomovirus species and strains causing Cassava mosaic disease (CMD) have been reported from cassava in Africa. In Nigeria, African cassava mosaic virus (ACMV) was the predominant virus in this important crop, and East African cassava mosaic virus (EACMV), first reported from eastern Nigeria in 1999, was also found occasionally. A survey was conducted in 2002 to resolve the diversity of the virus types present in cassava in Nigeria and to further understand the increasing complexity of the viruses contributing to CMD. A total of 234 leaf samples from cassava with conspicuous CMD symptoms were collected in farmers’ fields across different agroecological zones of Nigeria and subjected to polymerase chain reaction (PCR) with type‐specific primers. In addition and, to provide a full characterization of the viruses present, DNA‐A genome components of several viruses and informative genome fragments were sequenced. In Nigeria, ACMV proved to be the dominant virus with 80% of all samples being positive for ACMV. The East African cassava mosaic Cameroon virus (EACMCV) prevalent in Cameroon and Ivory Coast was detected in single infections (2%) and in mixed infections (18%) with ACMV. There was no indication for other virus strains of EACMV present in the country. The EACMCV samples collected showed a high nucleotide sequence identity >98% and resembled the described sequence of a Cameroon isolate (EACMCV‐CM) more than an Ivory Coast isolate, EACMCV‐CM[CI]. Evidence is provided that the EACMCV has reached epidemiological significance in Nigeria.     

Batai and Ngari viruses: M segment reassortment and association with severe febrile disease outbreaks in East Africa

Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.     

Evidence for X-linked introgression between molecular forms of Anopheles gambiae from Angola

The M and S molecular forms of the African malaria vector Anopheles gambiae (Diptera: Culicidae) are morphologically identical incipient species in which reproductive isolation is incomplete, enabling low-level gene flow between forms. In an attempt to find differences between the M and S forms, sequence variation was studied at loci along the X chromosome in adult female An. gambiae from Angola. A high proportion of M form specimens from Angola (79% of the 456 X chromosomes sampled) were found to contain a 16-bp insertion in intron 4 of the X-linked GPRCCK1 locus, relative to the AgamP3 release of the An. gambiae PEST genome sequence. The insertion was in Hardy-Weinberg equilibrium in Angolan M form populations. The same insertion was found in all S form specimens examined, regardless of where in Africa they were sampled, but was absent from a sample of M form specimens collected in Ghana, Bioko and Mali. In M form specimens from Angola, there was an association between alleles at the GPRCCK1 locus and those at a microsatellite locus, AGXH678, close to the centromere of the X chromosome, with significant linkage disequilibrium between loci separated by 0.472 Mbp (P < 0.033). We show that the insertion results from introgression from the S form into the M form, rather than from the retention of an ancestral character. Gene flow from the S to M form could allow genes of adaptive value to be transferred, including those conferring insecticide resistance and others influencing ecology and behaviour, and thus malaria transmission and control. We discuss factors that may have led to this introgression event.     

Viruses in sanctuary chimpanzees across Africa

Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild-born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long-term conservation strategies. We used metagenomic next-generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other “atypical” or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas-Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector-borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood-borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.     

Chikungunya: an overview

Chikungunya (CHIK), a mosquito borne debilitating disease, is caused by CHIK virus, an alphavirus belonging to the family Togaviridae. The sudden onset of very high fever along with rash, and severe arthralgia especially in the small joints of hands and toes are the characteristics of the disease. It was first reported from Tanzania in 1952–53 and spread subsequently to sub-Saharan Africa, South East Asia and Pacific causing large epidemics. The virus exists in three genotypes, the Asian, West African and East Central South African that are responsible for outbreaks in the respective areas. The first outbreak in Asia was in Bangkok in 1958 followed by other Asian countries. India experienced massive outbreaks of CHIK in the 1960s and early 70s mainly in cities. After a gap of 32 years an explosive outbreak of CHIK devastated the country affecting more than 1.4 million people in 13 states. The epidemic also witnessed many unusual clinico-pathological complications including CHIK associated deaths and mother to child transmission. High morbidity with severe arthralgia persisted for several months made the people mentally and physically weak. This review describes CHIK in general and highlights the various clinico-pathological aspects observed during the recent outbreak.     

Bactrocera invadens (Diptera: Tephritidae), a new invasive fruit fly pest for the Afrotropical region: host plant range and distribution in West and Central Africa

In 2003, the invasive fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) (Drew et al. 2005), of possible Sri Lankan origin, has been detected in the East and about 1 yr later in West Africa. In regular surveys in Benin and Cameroon covering 4 yr, samples from 117 plant species across 43 families have been obtained. Incubation of field-collected fruits demonstrate that in West and Central Africa (WCA) B. invadens is highly polyphagous, infesting wild and cultivated fruits of at least 46 species from 23 plant families with guava (Psidium spp.), mango (Mangifera spp.), and citrus (spp.), and the wild hosts tropical almond (Terminalia catappa L.), African wild mango (Irvingia gabonensis (Aubry-Lecomte) Baill.), and sheanut (Vitellaria paradoxa C.F.Gaertn.) showing the highest infestation index. B. invadens occurs in 22 countries of WCA with new records for Angola, Central African Republic, the Congo, DR Congo, Equatorial Guinea, Gabon, Gambia, Guinea Bissau, Mali, Mauritania, Niger, and Sierra Leone. Overall, the pest has spread across a North-South distance of ?5,000 km representing a contiguous area of >8.3 million km(2) within WCA. B. invadens has adapted to a wide range of ecological and climatic conditions extending from low land rainforest to dry savanna. Because of its highly destructive and invasive potential, B. invadens poses a serious threat to horticulture in Africa if left uncontrolled. Moreover, the presence of this quarantine pest causes considerable restrictions on international trade of affected crops.     

Genetic characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop.

It has been proposed that human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that are natural infections of sooty mangabeys (Cercocebus torquatus atys). To test this hypothesis, SIVs from eight sooty mangabeys, including six new viruses from West Africa, were genetically characterized. gag and env sequences showed that while the viruses of all eight sooty mangabeys belonged to the SIVsm/HIV-2 family, each was widely divergent from SIVs found earlier in captive monkeys at American primate centers. In two SIVs from sooty mangabeys discovered about 100 miles (ca. 161 Km) from each other in rural West Africa, the amino acids of a conserved gag p17-p26 region differed by 19.3%, a divergence greater than that in four of five clades of HIV-2 and in SIVs found in other African monkey species. Analysis of gag region sequences showed that feral mangabeys in one small troop harbored four distinct SIVs. Three of the newly found viruses were genetically divergent, showing as much genetic distance from each other as from the entire SIVsm/HIV-2 family. Sequencing and heteroduplex analysis of one feral animal-derived SIV showed a mosaic genome containing an env gene that was homologous with other feral SIVsm env genes in the troop but having a gag gene from another, distinct SIV. Surprisingly a gag phylogenetic tree based on nucleotide sequences showed that the African relatives closest to all three household-derived SIVs were HIV-2 subtypes D and E from humans in the same West African areas. In one case, the SIV/HIV-2 cluster was from the same village. The findings support the hypothesis that each HIV-2 subtype in West Africans originated from widely divergent SIVsm strains, transmitted by independent cross-species events in the same geographic locations.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Phylogeographic Reconstruction of African Yellow Fever Virus Isolates Indicates Recent Simultaneous Dispersal into East and West Africa

Yellow fever virus (YFV) is a mosquito-borne flavivirus that is a major public health problem in tropical areas of Africa and South America. There have been detailed studies on YFV ecology in West Africa and South America, but current understanding of YFV circulation on the African continent is incomplete. This inadequacy is especially notable for East and Central Africa, for which the unpredictability of human outbreaks is compounded by limitations in both historical and present surveillance efforts. Sparse availability of nucleotide sequence data makes it difficult to investigate the dispersal of YFV in these regions of the continent. To remedy this, we constructed Bayesian phylogenetic and geographic analyses utilizing 49 partial genomic sequences to infer the structure of YFV divergence across the known range of the virus on the African continent. Relaxed clock analysis demonstrated evidence for simultaneous divergence of YFV into east and west lineages, a finding that differs from previous hypotheses of YFV dispersal from reservoirs located on edges of the endemic range. Using discrete and continuous geographic diffusion models, we provide detailed structure of YFV lineage diversity. Significant transition links between extant East and West African lineages are presented, implying connection between areas of known sylvatic cycling. The results of demographic modeling reinforce the existence of a stably maintained population of YFV with spillover events into human populations occurring periodically. Geographically distinct foci of circulation are reconstructed, which have significant implications for studies of YFV ecology and emergence of human disease. We propose further incorporation of Bayesian phylogeography into formal GIS analyses to augment studies of arboviral disease.