Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.     

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.     

Ebola and Marburg virus antibody prevalence in selected populations of the Central African Republic

With the natural history of the filovirus family seemingly unknown, filovirus ecology in its natural environment remains a rudimentary field of research. In order to investigate the maintenance cycle of filovirus in Central Africa, a study was conducted within the rain forest of the Central African Republic. The epidemiological study determines the frequency and distribution of filovirus seroprevalence in a selected human population. Using an ELISA, serum samples from Pygmy and non-Pygmy populations were tested for Ebola-Zaire virus and Marburg (MBG) virus antibody. Filovirus antibody reacting sera were found in all zones investigated, and in all populations studied (Ebola virus IgG 5.3%; Marburg virus IgG 2.4%). Pygmies appeared to have a significantly higher seroprevalence (P < 0.03) against Ebola-Zaire virus (7.02%) than non-Pygmies (4.2%). MBG virus or related unknown filovirus strains also seem to be present in the western part of Central Africa. MBG virus antibodies were present in different Pygmy groups (ranging from 0.7 to 5.6%, mean 2.05%) and in several non-Pygmy populations (ranging from 0.0 to 3.9%, mean 3.4%) without an overall significant difference between the two groups (P = 0.14). The potentialities of nonpathogenic filovirus strains circulating in the Central African Republic are discussed.     

Shifts in Mycobacterial Populations and Emerging Drug-Resistance in West and Central Africa

In this study, we retrospectively analysed a total of 605 clinical isolates from six West or Central African countries (Benin, Cameroon, Central African Republic, Guinea-Conakry, Niger and Senegal). Besides spoligotyping to assign isolates to ancient and modern mycobacterial lineages, we conducted phenotypic drug-susceptibility-testing for each isolate for the four first-line drugs. We showed that phylogenetically modern Mycobacterium tuberculosis strains are more likely associated with drug resistance than ancient strains and predict that the currently ongoing replacement of the endemic ancient by a modern mycobacterial population in West/Central Africa might result in increased drug resistance in the sub-region.     

一株鸡传染性贫血病毒野毒株致病性及其全基因组序列比较

从山东某商品代肉鸡场表现生长迟缓的14日龄病鸡群分离到一株鸡传染性贫血病毒(CAV)C14株。C14株感染1日龄SPF鸡能抑制对禽流感病毒(AIV)的抗体反应,还能与禽网状内皮增生病病毒(REV)在免疫抑制上起协同作用。用PCR方法分段扩增出C14基因组的三条部分重叠片段,分别克隆于T载体并进行测序,拼接后得到其全基因组序列。测序结果表明,CAV-C14株基因组全长2298bp,含有3个互相重叠的开放阅读框和1个调控区。将C14与国内外已发表的CAV参考株基因组比较,同源性为97.2%~99.2%。序列比较表明CAV非编码区中含有的多个与复制及转录调控相关已知基序的序列都非常保守。CAV的3个编码基因VP1、VP2和VP3均有一定程度变异,以VP1变异性最大,且不同毒株间的3个蛋白质氨基酸序列的变异是互不相关的。     

Comparative analysis of west nile virus strains isolated from human and animal hosts using monoclonal antibodies and cDNA restriction digest profiles

The West Nile (WN) virus strains isolated in Bangui, Central African Republic (CAR), from patients with hepatitis were analysed comparatively with the prototype WN virus strain and 7 WN strains previously isolated from birds (2 strains), mosquitoes (3 strains) and ticks (2 strains) in CAR.The comparison was based on two techniques: an epitopic analysis by indirect immunofluorescence assay using a panel of 9 monoclonal antibodies to WN virus, and an analysis of HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA.Similar results were obtained with both techniques: the 3 human strains were found to be identical to each other and identical or very close to mosquito and tick strains, whereas prototype WN virus and bird strains were significantly different from the human strains.As “classical” infections due to WN virus without hepatic involvement were also reported during the period of isolation of the arthropod strains, we concluded that the same virus subtype may have been the cause of different infection patterns. A new definition of the disease spectrum of WN virus, including the possibility of liver involvement, should be established.Clearly, the Egyptian prototype WN virus represents a different topotype. Bird strains also appear to be different from human and arthropod strains, raising the question of their transmissibility and pathogenicity for man, and of the role of birds in the natural cycle of WN virus.     

High influenza a virus infection rates in mallards bred for hunting in the camargue, South of france

During the last decade, the role of wildlife in emerging pathogen transmission to domestic animals has often been pointed out. Conversely, far less attention has been paid to pathogen transmission from domestic animals to wildlife. Here, we focus on the case of game restocking, which implies the release of millions of animals worldwide each year. We conducted a 2-year study in the Camargue (Southern France) to investigate the influence of hand-reared Mallard releases on avian influenza virus dynamics in surrounding wildlife. We sampled Mallards (cloacal swabs) from several game duck facilities in 2009 and 2010 before their release. A very high (99%) infection rate caused by an H10N7 strain was detected in the game bird facility we sampled in 2009. We did not detect this strain in shot ducks we sampled, neither during the 2008/2009 nor the 2009/2010 hunting seasons. In 2010 infection rates ranged from 0 to 24% in hand-reared ducks. The 2009 H10N7 strain was fully sequenced. It results from multiple reassortment events between Eurasian low pathogenic strains. Interestingly, H10N7 strains had previously caused human infections in Egypt and Australia. The H10 and N7 segments we sequenced were clearly distinct from the Australian ones but they belonged to the same large cluster as the Egyptian ones. We did not observe any mutation linked to increased virulence, transmission to mammals, or antiviral resistance in the H10N7 strain we identified. Our results indicate that the potential role of hand-reared Mallards in influenza virus epizootics must be taken into account given the likely risk of viral exchange between game bird facilities and wild habitats, owing to duck rearing conditions. Measures implemented to limit transmission from wildlife to domestic animals as well as measures to control transmission from domestic animals to wild ones need to be equally reinforced.     

A non-sense mutation in the putative anti-mutator gene <Emphasis Type="Italic">ada/alkA</Emphasis> of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and <Emphasis Type="Italic">M. bovis</Emphasis> isolates suggests convergent evolution

Background  

Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR).     

实时荧光定量PCR技术在SPF鸡四种垂直传播疾病监测中的应用

目的探讨荧光定量PCR检测技术对SPF鸡四种垂直传播病毒的检测应用。方法采集60份SPF鸡及70份普通鸡群蛋清、泄殖腔试子样品,提取样品核酸,分别进行ARV、REV、CAV、ALV四种病毒实时荧光定量PCR检测,根据标准曲线及溶解曲线分析判读样品病毒拷贝数。结果 SPF鸡ALV 2份阳性,检出率3.3%,其余病毒检测均为阴性;普通鸡样品REV检测2份阳性,检出率2.9%,ALV 10份阳性,检出率14.3%。结论荧光定量PCR检测方法最低可检测到100个拷贝核酸,检测灵敏度较高,有望应用于SPF鸡临床样品的病原检测。     

Rift Valley Fever Virus Circulating among Ruminants,Mosquitoes and Humans in the Central African Republic

BackgroundRift Valley fever virus (RVFV) causes a viral zoonosis, with discontinuous epizootics and sporadic epidemics, essentially in East Africa. Infection with this virus causes severe illness and abortion in sheep, goats, and cattle as well as other domestic animals. Humans can also be exposed through close contact with infectious tissues or by bites from infected mosquitoes, primarily of the Aedes and Culex genuses. Although the cycle of RVFV infection in savannah regions is well documented, its distribution in forest areas in central Africa has been poorly investigated.

Methodology/Principal Findings

To evaluate current circulation of RVFV among livestock and humans living in the Central African Republic (CAR), blood samples were collected from sheep, cattle, and goats and from people at risk, such as stock breeders and workers in slaughterhouses and livestock markets. The samples were tested for anti-RVFV immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. We also sequenced the complete genomes of two local strains, one isolated in 1969 from mosquitoes and one isolated in 1985 from humans living in forested areas. The 1271 animals sampled comprised 727 cattle, 325 sheep, and 219 goats at three sites. The overall seroprevalence of anti-RVFV IgM antibodies was 1.9% and that of IgG antibodies was 8.6%. IgM antibodies were found only during the rainy season, but the frequency of IgG antibodies did not differ significantly by season. No evidence of recent RVFV infection was found in 335 people considered at risk; however, 16.7% had evidence of past infection. Comparison of the nucleotide sequences of the strains isolated in the CAR with those isolated in other African countries showed that they belonged to the East/Central African cluster.

Conclusion and significance

This study confirms current circulation of RVFV in CAR. Further studies are needed to determine the potential vectors involved and the virus reservoirs.     

Phylogeography of Mus (Nannomys) minutoides (Rodentia,Muridae) in West Central African savannahs: singular vicariance in neighbouring populations

We studied the phylogeography of the strict savannah pygmy mice Mus (Nannomys) minutoides in West Central Africa. A total of 846 base pairs of the cytochrome b sequence were obtained for 66 individuals collected in Gabon, Cameroon, Republic of Congo and Central African Republic. These sequences were compared to those of M. minutoides from other African countries and to eight other species of the genus Mus. We performed maximum likelihood, Bayesian and nested clade analyses, as well as neutrality tests and time estimates. We show that M. minutoides is a well‐differentiated monophyletic species that separated from other pygmy mice 1.17 Myr ago. A distinct West Central African M. minutoides clade diverged early from the other African populations of the species, with a more recent common ancestor dating 0.14 Myr. West Central African populations are globally homogeneous, despite the present fragmentation of savannahs by the rain forest. However, our analyses show an unexpected vicariance between geographically close savannahs, embedded in the rain forest in Central Gabon. One of these populations is genetically more similar to very distant peripheral populations than to three closely neighbouring populations situated on both sides of the Ogooué River. A non‐river geographical barrier probably persisted in this area, durably isolating these local populations. This hypothesis about the history of the savannah landscape should be testable through the biogeographical analysis of other strict savannah small mammal species.     

Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi,India

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co‐circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT‐PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co‐infection with DENV and CHIKV in 10% of the samples by RT‐PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV‐1 strains, six DENV‐2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV‐1 strains grouped in the American African genotype, DENV‐2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV‐1 and DENV‐2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.     

Application of a Simplified Molecular Protocol to Reveal Mixed Infections with Begomoviruses in Cassava

Samples of cassava leaves exhibiting severe symptoms of cassava mosaic disease (CMD) were collected with the PhytoPASS kit in fields surrounding the city of Bujumbura (Burundi). These materials were then sent to Belgium for polymerase chain reaction determination of the CMD begomoviruses inducing the observed symptoms. Different pairs of specific primers were used to amplify DNA sequences specific to African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV), East African cassava mosaic Zanzibar virus (EACMZV), the Uganda variant of East African cassava mosaic virus (EACMV-UG) and South African cassava mosaic virus (SACMV). It was revealed that mixed infections were prevailing in the analyzed materials. Most of the samples submitted to this analysis were found to be co-infected by three different begomoviruses (ACMV + EACMV + EACMV-UG). The so revealed mixed infections could explain the high severity of CMD symptoms noticed on cassava in the region of Bujumbura while the diversity within the CMD causal agents illustrates the importance to take this parameter into consideration for a successful use of plant genetic resistance to control the disease.     

Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

The analysis of variation of mtDNA hypervariable region 1 suggests that Eastern and Western Pygmies diverged before the Bantu expansion

The Eastern Pygmies from Zaire and Western Pygmies from Cameroon, Congo, and the Central African Republic represent the two principal groups of African Pygmies. In the "recent divergence" hypothesis in which Western Pygmies are thought to be the result of hybridization between the ancestors of Eastern Pygmies and Bantu farmers who penetrated the equatorial belt and came into contact with Pygmies around 2-3 kiloyears ago. On the basis of recent archaeological research in the tropical rain forest, we propose a "pre-Bantu divergence" hypothesis, which posits the separation between the ancestors of Eastern and Western Pygmies earlier than 18 kiloyears ago. In order to test the two hypotheses, we analyzed the variation of the hypervariable region 1 of the mitochondrial DNA in the Mbenzele, Western Pygmies of the Central African Republic, and compared our results with those of previous mtDNA and Y chromosome studies. Distribution, sequence variation, and age of haplogroups along with genetic distances among populations, estimates of divergence times, and simulations based on the coalescent approach were found to be congruent with the pre-Bantu divergence but failed to support the recent divergence hypothesis.     

Molecular epidemiology of influenza A/H3N2 viruses circulating in Uganda

The increasing availability of complete influenza virus genomes is deepening our understanding of influenza evolutionary dynamics and facilitating the selection of vaccine strains. However, only one complete African influenza virus sequence is available in the public domain. Here we present a complete genome analysis of 59 influenza A/H3N2 viruses isolated from humans in Uganda during the 2008 and 2009 season. Isolates were recovered from hospital-based sentinel surveillance for influenza-like illnesses and their whole genome sequenced. The viruses circulating during these two seasons clearly differed from each other phylogenetically. They showed a slow evolution away from the 2009/10 recommended vaccine strain (A/Brisbane/10/07), instead clustering with the 2010/11 recommended vaccine strain (A/Perth/16/09) in the A/Victoria/208/09 clade, as observed in other global regions. All of the isolates carried the adamantane resistance marker S31N in the M2 gene and carried several markers of enhanced transmission; as expected, none carried any marker of neuraminidase inhibitor resistance. The hemagglutinin gene of the 2009 isolates differed from that of the 2008 isolates in antigenic sites A, B, D, and to a lesser extent, C and E indicating evidence of an early phylogenetic shift from the 2008 to 2009 viruses. The internal genes of the 2009 isolates were similar to those of one 2008 isolate, A/Uganda/MUWRP-050/2008. Another 2008 isolate had a truncated PB1-F2 protein. Whole genome sequencing can enhance surveillance of future seasonal changes in the viral genome which is crucial to ensure that selected vaccine strains are protective against the strains circulating in Eastern Africa. This data provides an important baseline for this surveillance. Overall the influenza virus activity in Uganda appears to mirror that observed in other regions of the southern hemisphere.     

Detection and molecular characterization of foamy viruses in Central African chimpanzees of the Pan troglodytes troglodytes and Pan troglodytes vellerosus subspecies

BACKGROUND: Foamy viruses are exogenous retroviruses that are highly endemic in non-human primates (NHPs). Recent studies, mainly performed in North America, indicated frequent simian foamy virus (SFV) infection in persons occupationally exposed to NHPs. This zoonotic infection was demonstrated mainly after bites by chimpanzees [Pan troglodytes (P. t.)] of the West African P. t. verus subspecies in primatology centers or zoos in the USA. METHODS: We studied 32 chimpanzees from the Central African subspecies P. t. troglodytes and P. t. vellerosus, originating from Cameroon (29 cases) or Gabon (3 cases). We screened first plasma or sera of the animals with a Western blot detecting the SFVs Gag doublet proteins. Then, we performed two nested polymerase chain reactions (PCRs) amplifying a fragment of the integrase and LTR regions and, finally, we made phylogenetical analyses on the sequences obtained from the integrase PCR products. RESULTS: By serological and/or molecular assays, we detected foamy viruses (FVs) infection in 14 chimpanzees. Sequence comparison and phylogenetic analyses of a 425 bp fragment of the integrase gene obtained for 10 of the 14 positive apes, demonstrated a wide diversity of new FVs strains that belong phylogenetically either to the P. t. troglodytes or P. t. vellerosus foamy viral clade. CONCLUSIONS: This study shows that chimpanzees living in these areas of Central Africa are infected by several specific foamy viruses. This raises, in such regions, the potential risk of a human retroviral infection of zoonotic origin linked to chimpanzees contacts, as already exemplified for STLV-1 and SIV infections.     

Simian immunodeficiency virus infection in wild-caught chimpanzees from cameroon

Simian immunodeficiency viruses (SIVcpz) infecting chimpanzees (Pan troglodytes) in west central Africa are the closest relatives to all major variants of human immunodeficiency virus type 1 ([HIV-1]; groups M, N and O), and have thus been implicated as the source of the human infections; however, information concerning the prevalence, geographic distribution, and subspecies association of SIVcpz still remains limited. In this study, we tested 71 wild-caught chimpanzees from Cameroon for evidence of SIVcpz infection. Thirty-nine of these were of the central subspecies (Pan troglodytes troglodytes), and 32 were of the Nigerian subspecies (Pan troglodytes vellerosus), as determined by mitochondrial DNA analysis. Serological analysis determined that one P. t. troglodytes ape (CAM13) harbored serum antibodies that cross-reacted strongly with HIV-1 antigens; all other apes were seronegative. To characterize the newly identified virus, 14 partially overlapping viral fragments were amplified from fecal virion RNA and concatenated to yield a complete SIVcpz genome (9,284 bp). Phylogenetic analyses revealed that SIVcpzCAM13 fell well within the radiation of the SIVcpzPtt group of viruses, as part of a clade including all other SIVcpzPtt strains as well as HIV-1 groups M and N. However, SIVcpzCAM13 clustered most closely with SIVcpzGAB1 from Gabon rather than with SIVcpzCAM3 and SIVcpzCAM5 from Cameroon, indicating the existence of divergent SIVcpzPtt lineages within the same geographic region. These data, together with evidence of recombination among ancestral SIVcpzPtt lineages, indicate long-standing endemic infection of central chimpanzees and reaffirm a west central African origin of HIV-1. Whether P. t. vellerosus apes are naturally infected with SIVcpz requires further study.