Sylviane Bassot

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

A Severe Bite From a Nonhuman Primate Is a Major Risk Factor for HTLV-1 Infection in Hunters From Central Africa

BACKGROUND HTLV-1 infection is endemic to Central African populations. The risk factors for HTLV-1 acquisition in humans via the interspecies transmission of STLV-1 (its simian counterpart) remain largely unknown. METHODS We studied 269 individuals (254 men, 15 women) bitten by a nonhuman primate (NHP), mostly during hunting activities. These, Pygmies and Bantus, living in the southern Cameroonian rainforest, were matched for sex, age, and ethnicity with individuals from the same settlements reporting no NHP bites. HTLV-1 serology was performed by Western blot on plasma samples. PCR was carried out for HTLV-1 provirus on buffy-coat DNAs. The amplified products were sequenced and analyzed by phylogenetic analyses. RESULTS HTLV-1 prevalence was 8.6% (23/269) in individuals with bites, vs 1.5% (4/269) in matched controls (P < .001). Moreover, HTLV-1 infection was linked to bite severity. The 23 HTLV-1-positive bitten individuals reported being bitten by a gorilla (17), chimpanzee (3), or small monkey (3). Thirteen (56%) were coinfected with a simian foamy virus known to be acquired through severe bites. Mother-to-child infection was excluded in 6 HTLV-1-infected bitten individuals. All the HTLV-1-positive hunters bitten by a gorilla or chimpanzee were infected with a subtype B strain similar to that present in apes from the same area. Two hunters bitten by small monkeys (C. agilis in one case) were infected with a HTLV-1 subtype F strain very similar to the STLV-1 strains present in such monkeys. CONCLUSIONS These results strongly suggest ongoing direct zoonotic acquisition of STLV-1 in humans through severe NHP bites during hunting activities.

Modes of transmission and genetic diversity of foamy viruses in a Macaca tonkeana colony

BackgroundFoamy viruses are exogenous complex retroviruses that are highly endemic in several animal species, including monkeys and apes, where they cause persistent infection. Simian foamy viral (SFV) infection has been reported in few persons occupationally exposed to non-human primates (NHP) in zoos, primate centers and laboratories, and recently in few hunters from central Africa. Most of the epidemiological works performed among NHP populations concern cross-sectional studies without long-term follow-up. Therefore, the exact timing and the modes of transmission of SFVs remain not well known, although sexual and oral transmissions have been suspected. We have conducted a longitudinal study in a free-breeding colony of Macaca tonkeana in order (1) to determine the prevalence of the infection by foamy viruses, (2) to characterize molecularly the viruses infecting such animals, (3) to study their genetic variability overtime by long-term follow-up of several DNA samples in a series of specific animals, and (4) to get new insights concerning the timing and the modes of SFVs primary infection in these monkeys by combining serology and molecular means, as well as studies of familial structures and long-term behavioral observations.Results/conclusionWe first demonstrated that this colony was highly endemic for SFVs, with a clear increase of seroprevalence with age. Only 4.7% of immatures, and 43,7% of sub-adults were found seropositive, while 89.5% of adults exhibited antibodies directed against SFV. We further showed that 6 different strains of foamy viruses (exhibiting a very low intra-strain and overtime genetic variability in the integrase gene) are circulating within this group. This suggests a possible infection by different strains within an animal. Lastly, we provide strong evidence that foamy viruses are mostly acquired through severe bites, mainly in sub-adults or young adults. Most cases of seroconversion occur after 7 years of age; from this age individuals competed for access to sexual partners, thus increasing the likelihood of being wounded. Furthermore, all the serological and molecular data, obtained in this free-breeding colony, argue against a significant transmission of SFVs from mother or father to infants as well as between siblings.

Multiple retroviral infection by HTLV type 1, 2, 3 and simian foamy virus in a family of Pygmies from Cameroon.

To better understand the origins and modes of transmission of HTLV-3 and to search for other retroviral infections (HTLV-1, HTLV-2, foamy viruses), we studied the family of a HTLV-3-infected individual (Pyl43), from Cameroon. Thirty-five persons were included. All adult men were still actively hunting nonhuman primates (NHP). All women were also butchering and cutting-up animals. Five persons reported a bite by an NHP. While HTLV-3 infection was only found in Pyl43, HTLV-1 and HTLV-2 infections were found, respectively, in 5 and 9 persons with one being co-infected by both retroviruses. Phylogenetic analysis suggested intra-familial transmission of HTLV-1 subtypes B and D and HTLV-2. One man was infected by a chimpanzee foamy virus, acquired probably 45 years ago, through a bite. Acquisition of retroviral infections still occurs in central Africa involving to various extent not only intra-familial transmission for HTLV-1/HTLV-2 but also direct interspecies transmission from NHP for foamy virus and possibly for HTLV-1 and HTLV-3.

Human T Lymphotropic Virus Type 1 Subtype C Melanesian Genetic Variants of the Vanuatu Archipelago and Solomon Islands Share a Common Ancestor

BACKGROUND Melanesia is endemic for human T lymphotropic virus type 1 (HTLV-1) subtype C. In 2005, we identified 4 infected women from Ambae Island, Vanuatu. Subsequently, 4247 Ni-Vanuatu originating from 18 islands were enrolled to define HTLV-1 epidemiological determinants and to characterize the viral strains molecularly. METHODS Plasma from 1074 males and 3173 females were screened for HTLV-1/2 antibodies by particle agglutination (PA) and an immunofluorescence assay (IFA). Positive and/or borderline samples were then tested by a Western blot (WB) confirmatory assay. DNAs were amplified to obtain a 522-bp env gene fragment. Phylogenetic and molecular-clock analyses were performed. RESULTS Of 4247 samples, 762 were positive and/or borderline by IFA/PA, and 26 of them were confirmed to be HTLV-1 positive by WB. The overall HTLV-1 seroprevalence was 0.62%. Viral transmission was found within families of infected index case patients. A geographic heterogeneity of HTLV-1 seroprevalence was observed among the islands. All 41 of the new env sequences belonged to HTLV-1 subtype C. Phylogenetic and molecular-clock analyses suggested that Ni-Vanuatu and Solomon Islander strains emerged from a common ancestor ~10,000 years ago. CONCLUSION The Vanuatu archipelago is endemic for HTLV-1 with a diversity of subtype C variants. These strains were probably introduced into Vanuatu during ancient migration of the original settlers a few thousand years ago.

A New and Frequent Human T-Cell Leukemia Virus Indeterminate Western Blot Pattern: Epidemiological Determinants and PCR Results in Central African Inhabitants

ABSTRACT Human T-cell leukemia virus (HTLV) indeterminate Western blot (WB) serological patterns are frequently observed in plasma/serum from persons living in intertropical areas. In the framework of ongoing projects on HTLV-1/2 and related viruses in Central Africa, we systematically analyzed plasma from villagers living in South Cameroon by WB. The group included 1,968 individuals (mean age, 44 years; age range, 5 to 90 years; 978 women/990 men), both Bantus (1,165) and Pygmies (803). Plasma samples were tested by WB analysis (MPD HTLV Blot 2.4) and interpreted according to the manufacturers instructions. Only clear bands were considered in the analysis. Among the 1,968 plasma samples, 38 (1.93%) were HTLV-1, 13 (0.66%) were HTLV-2, and 6 (0.3%) were HTLV WB seropositive. Furthermore, 1,292 (65.65%) samples were WB sero-indeterminate, including 104 (5.28%) with an HTLV-1 Gag-indeterminate pattern (HGIP) and 68 (3.45%) with a peculiar yet unreported pattern exhibiting mostly a strong shifted GD21 and a p28. The other 619 (31.45%) samples were either WB negative or exhibited other patterns, mostly with unique p19 or p24 bands. DNA, extracted from peripheral blood buffy coat, was subjected to PCR using several primer pairs known to detect HTLV-1/2/3/4. Most DNAs from HTLV-1- and HTLV-seropositive individuals were PCR positive. In contrast, all the others, from persons with HTLV-2, HGIP, new WB, and other indeterminate patterns, were PCR negative. Epidemiological determinant analysis of the persons with this new peculiar WB pattern revealed that seroprevalence was independent from age, sex, or ethnicity, thus resembling the indeterminate profile HGIP rather than HTLV-1. Moreover, this new pattern persists over time.

Novel human herpesvirus 8 subtype D strains in Vanuatu, Melanesia.

We show human herpesvirus 8 with diverse molecular subtype D variants to be highly endemic among the Ni-Vanuatu population. Most K1 genes were nearly identical to Polynesian strains, although a few clustered with Australian or Taiwanese strains. These results suggest diverse origins of the Ni-Vanuatu population and raise questions about the ancient human population movements in Melanesia.

Divergent KSHV/HHV-8 subtype D strains in New Caledonia and Solomon Islands, Melanesia

BACKGROUND KSHV/HHV-8 is the etiological agent of Kaposis sarcoma, primary effusion lymphoma and most multicentric Castlemans disease cases. KSHV exhibits a high genetic variability comprising five genotypes (A-E). Few data are yet available concerning the situation of KSHV, its genetic variability and the associated diseases in Melanesia. OBJECTIVES We performed a study on 626 natives Melanesians from New Caledonia and Vanikoro Island to evaluate KSHV seroprevalence and characterize molecularly the viral strains. STUDY DESIGN Plasma from 343 males and 283 females (age range: 15-86 years, mean age: 60) were tested for KSHV latent antibodies by an immunofluorescence assay (IFA) using BC-3 cells. DNAs extracted from peripheral blood buffy-coat of KSHV seropositive individuals were amplified to obtain a 737-bp fragment of the ORF-K1 gene. Phylogenetic analyses were then performed. RESULTS Among 626 samples, 148 were IFA positive (dilution≥1:80). The overall seroprevalence was 23.6% (25.2% in New Caledonia, 17.5% in Vanikoro). Fifteen (8 men and 7 women, mean age 69 years) out of 148 DNA samples were found PCR positive. All ORF-K1 sequences belonged to KSHV genotype D. A geographic clustering according to the island of origin of KSHV infected persons was clearly observed with sequences from New Caledonia clustering with most Vanuatu strains. CONCLUSIONS New Caledonia and Vanikoro are endemic for KSHV with a high diversity of genotype D variants. These strains were probably introduced into New Caledonia during multiple waves of migrations of Melanesian and Polynesian individuals that have colonized this archipelago.

Human herpesvirus 8, Southern Siberia.

To the Editor: Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi sarcoma. Sequence analysis of the highly variable open reading frame (ORF)–K1 of HHV-8 has enabled the identification of 5 main molecular subtypes, A–E (1). A and C subtypes are prevalent in persons in Europe, Mediterranean countries, northwestern China, and the United States; subtype B, in persons in sub-Saharan Africa; subtype D, in persons in the Pacific Islands and Japan (2–6); and subtype E, in Native Americans in the United States. Considering that K1 gene polymorphisms of HHV-8–infected persons reflect the divergence accumulated during the early migrations of modern humans out of Africa (1), it is tempting to put the polymorphisms observed in the different subtypes into an evolutionary perspective with their geographic distribution. It is thought that Native Americans infected by subtype E and Pacific Islanders, including those infected by subtype D in the Japanese archipelago, originated from a common ancestral genetic stock in continental Asia. Because Siberia constitutes the geographic link between mainland Asia, North America, and the Pacific (Technical Appendix), it is likely that the Siberian region has served as a source or a corridor of human dispersals to these regions. Thus, we conducted a molecular epidemiology HHV-8 survey of the Buryat population, a major indigenous group in southern Siberia, to gain new insights into the origins, possibly common, of HHV-8 subtypes D and E. After consent of local authorities and participants, we collected 745 human blood samples in 1995 in 17 medicosocial structures (homes for elderly persons, veterans of the Russian army, hospitalized persons, blood donors) located near Lake Baikal and originating from Ulan Ude (344), Ust Orda (216), and Chita (185), Siberia, Russia (additional data can be obtained directly from the authors). The median age of those included was 52 years (range 25–98 years); 489 (66%) were women. Antibodies against HHV-8 latency–associated nuclear antigen were identified by immunofluorescent antibody assay by using the BC3 cell line (3). Punctuate nuclear staining of BC3 cells at a 1:160 dilution was observed for 187 (25.1%) patients with no difference according to investigated regions (p = 0.32 by χ2 test) or between men (25.8%) and women (24.7%) (p = 0.76 by χ2 test; Technical Appendix). However, HHV-8 seroprevalence increased with patient age, rising from 12.9% (25–43 years) to 46.4% (>61 years) (p = 1.8 × 10–13 by χ2 test for trend) (Figure; Technical Appendix). No significant difference was observed in antibody titers according to age (p = 0.45 by Fisher exact test). These results demonstrate that HHV-8 infection is highly prevalent in the Siberian adult population tested. Figure Age-dependent human herpesvirus 8 (HHV-8) seroprevalence rates for 745 persons in southern Siberia 25–98 years of age who lived in the Ust Orda, Ulan Ude, or Chita districts during 1995. Seropositivity was based on strict criteria; only samples ... HHV-8 infection was determined by nested PCR that amplified a 737-bp fragment of the ORFK1 in peripheral blood buffy coats of 85 HHV-8–seropositive and 10 HHV-8–seronegative persons (3). Amplification was positive in 19/85 (22.4%) samples; sequences were obtained for 18 of these samples (Technical Appendix). These sequences showed 0%–7.31% nucleotide divergence and 0%–3.55% amino acid divergence. Nevertheless, 17 strains were found to be closely related with <1.75% nucleotide differences for 684 nt, and only 1 sequence (1445 strain) displayed higher nucleotide divergence. A comparative sequence analysis, including 66 representatives of K1 gene sequences of the HHV-8 A/C subtypes/subgroups, and sequences obtained from persons originating from Russia, was performed (7–9). Seventeen of the 18 HHV-8 strains from Siberia belonged to the A subtype; 15 clustered in a newly identified specific subclade (Technical Appendix). Notably, the 1445-Siberian strain, which exhibits the typical 5 aa deletion at positions 201–205, belongs to subtype C and clustered with the 7848 strain previously described by Lacoste et al. (9). Furthermore, both strains originate from Chita. Our results indicate that HHV-8 infection is highly prevalent in the population tested in southern Siberia and extend current knowledge on the worldwide distribution of HHV-8 genotypes. The presence of a Siberian strain monophyletic subclade suggests the existence of HHV-8 strains preferentially spreading among this population in southern Siberia. To ascertain the maternal ancestry of these persons, we sequenced the hypervariable region I (HVS-I) of the maternally-inherited mitochondrial DNA (mtDNA) and assigned haplogroups on the basis of the HVS-I motifs. Our analyses showed that 17/18 persons analyzed showed a mtDNA motif of clear continental east Asian origin (e.g., A, D correspond to different mtDNA haplogroups). One person (1474-strain) had a lineage (i.e., HV1) that is thought to have a western Eurasian origin. Overall, these mtDNA analyses indicate that the maternal ancestry of the persons examined here can be unambiguously attributed to East Asia, and not to Western Eurasia. K1 subtype A sequences recently found in the Xinjiang Uygur region in China (10) do not correspond to the specific Siberian clade described in our study. Thus, we must now consider that the widely distributed HHV-8 A/C subtype, so far mainly observed in Europe and Mediterranean countries, is also largely predominant in continental Asia.

In vivo effects of ovalbumin-conjugated muramyl peptides on the anti-ovalbumin IgE and IgG responses in mice

The effects of pretreatments of BALB/c mice with several conjugates of MDP and MDP-Lys to ovalbumin before immunization with ovalbumin (OA) were tested on the anti-OA IgE responses. Pretreatment with MDP-Lys-OA, but not with MDP-OA, induced an inhibition of the anti-OA primary and secondary responses, as measured by passive cutaneous anaphylaxis (PCA) and also by mast cell degranulation. The inhibition by pretreatment with MDP-Lys-OA was obtained whether it was administered in Freunds incomplete adjuvant (FIA) or in saline. This IgE suppression was accompanied by an enhancement of IgG2a and IgG2b anti-OA antibodies, with no change in the specific IgG1 levels. Loss of antigenicity of OA, detected by the lack of degranulation of peritoneal mast cells sensitized by IgE anti-OA, was observed in the MDP-Lys-OA but not in the MDP-OA conjugates. This loss of antigenicity appears to correlate with the ability of the conjugate to induce suppression of the specific IgE response.