Viral Exploration of Negative Acute Febrile Cases Observed during Chikungunya Outbreaks in Gabon.

Non-malarial febrile illness outbreaks were documented in 2007 and 2010 in Gabon. After investigation, these outbreaks were attributed to the chikungunya and dengue viruses (CHIKV and DENV). However, for more than half of the samples analyzed, the causative agent was not identified. Given the geographical and ecological position of Gabon, where there is a great animal and microbial diversity, the circulation of other emerging viruses was suspected in these samples lacking aetiology. A total of 436 undiagnosed samples, collected between 2007 and 2013, and originating from 14 urban, suburban, and rural Gabonese locations were selected. These samples were used for viral isolation on newborn mice and VERO cells. In samples with signs of viral replication, cell supernatants and brain suspensions were used to extract nucleic acids and perform real-time RT-PCR targeting specific arboviruses, i.e., CHIKV, DENV, yellow fever, Rift Valley fever, and West Nile and Zika viruses. Virus isolation was conclusive for 43 samples either on newborn mice or by cell culture. Virus identification by RT-PCR led to the identification of CHIKV in 37 isolates. A total of 18 complete genomes and 19 partial sequences containing the E2 and E1 genes of CHIKV were sequenced using next-generation sequencing technology or the Sanger method. Phylogenetic analysis of the complete genomes showed that all the sequences belong to the East Central South Africa lineage. Furthermore, we identified 2 distinct clusters. The first cluster was made up of sequences from the western part of Gabon, whereas the second cluster was made up of sequences from the southern regions, reflecting the way CHIKV spread across the country following its initial introduction in 2007. Similar results were obtained when analyzing the CHIKV genes of the E2 and E1 structural proteins. Moreover, study of the mutations found in the E2 and E1 structural proteins revealed the presence of several mutations that facilitate the adaptation to the Aedes albopictus mosquito, such as E2 I211T and E1 A226V, in all the Gabonese CHIKV strains. Finally, sequencing of 6 additional viral isolates failed to lead to any conclusive identification.

Ebola virus disease in the Democratic Republic of Congo.

BACKGROUND: The seventh reported outbreak of Ebola virus disease (EVD) in the equatorial African country of the Democratic Republic of Congo (DRC) began on July 26, 2014, as another large EVD epidemic continued to spread in West Africa. Simultaneous reports of EVD in equatorial and West Africa raised the question of whether the two outbreaks were linked. METHODS: We obtained data from patients in the DRC, using the standard World Health Organization clinical-investigation form for viral hemorrhagic fevers. Patients were classified as having suspected, probable, or confirmed EVD or a non-EVD illness. Blood samples were obtained for polymerase-chain-reaction-based diagnosis, viral isolation, sequencing, and phylogenetic analysis. RESULTS: The outbreak began in Inkanamongo village in the vicinity of Boende town in Équateur province and has been confined to that province. A total of 69 suspected, probable, or confirmed cases were reported between July 26 and October 7, 2014, including 8 cases among health care workers, with 49 deaths. As of October 7, there have been approximately six generations of cases of EVD since the outbreak began. The reported weekly case incidence peaked in the weeks of August 17 and 24 and has since fallen sharply. Genome sequencing revealed Ebola virus (EBOV, Zaire species) as the cause of this outbreak. A coding-complete genome sequence of EBOV that was isolated during this outbreak showed 99.2% identity with the most closely related variant from the 1995 outbreak in Kikwit in the DRC and 96.8% identity to EBOV variants that are currently circulating in West Africa. CONCLUSIONS: The current EVD outbreak in the DRC has clinical and epidemiologic characteristics that are similar to those of previous EVD outbreaks in equatorial Africa. The causal agent is a local EBOV variant, and this outbreak has a zoonotic origin different from that in the 2014 epidemic in West Africa. (Funded by the Centre International de Recherches Médicales de Franceville and others.).

Human papillomavirus detection using the Abbott RealTime high-risk HPV tests compared with conventional nested PCR coupled to high-throughput sequencing of amplification products in cervical smear specimens from a Gabonese female population.

BACKGROUND: Cervical cancer is the fourth most common malignancy in women worldwide. However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTime High-Risk HPV test (AbRT) was evaluated in 83 cervical smear specimens and compared with a conventional nested PCR coupled to high-throughput sequencing (HTS) to identify the amplicons. RESULTS: The AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR. CONCLUSIONS: The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test.

Contribution of Next-Generation Sequencing to Aquatic and Fish Virology.

The recent technological advances in nucleic acid sequencing, called next-generation sequencing (NGS), have revolutionized the field of genomics and have also influenced viral research. Aquatic viruses, and especially those infecting fish, have also greatly benefited from NGS technologies, which provide a huge amount of molecular information at a low cost in a relatively short period of time. Here, we review the use of the current high-throughput sequencing platforms with a special focus on the associated challenges (regarding sample preparation and bioinformatics) in their applications to the field of aquatic virology, especially for: (i) discovering novel viruses that may be associated with fish mortalities, (ii) elucidating the mechanisms of pathogenesis, and finally (iii) studying the molecular epidemiology of these pathogens.

The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo.

BACKGROUND: The involvement of NF-κB signaling in prostate cancer (PCa) has largely been established through the study of the classical p65 subunit. Nuclear localization of p65 in PCa patient tissues has been shown to correlate with biochemical recurrence, while in vitro studies have demonstrated that the classical NF-κB signaling pathway promotes PCa progression and metastatic potential. More recently, the nuclear location of RelB, a member of the alternative NF-κB signaling, has also been shown to correlate with the Gleason score. The current study aims to clarify the role of alternative NF-κB in PCa cells by exploring, in vitro and in vivo, the effects of RelB overexpression on PCa biology. METHODS: Using a lentivirus-expression system, we constitutively overexpressed RelB or control GFP into 22Rv1 cells and monitored alternative transcriptional NF-κB activity. In vivo, tumor growth was assessed after the injection of 22Rv1-derived cells into SCID mice. In vitro, the impact of RelB on 22Rv1 cell proliferation was evaluated in monolayer culture. The anchorage-independent cell growth of derived-22Rv1 cells was assessed by soft agar assay. Apoptosis and autophagy were evaluated by Western blot analysis in 22Rv1-derived cells cultured in suspension using poly-HEMA pre-coated dishes. RESULTS: The overexpression of RelB in 22Rv1 cells induced the constitutive activation of the alternative NF-κB pathway. In vivo, RelB expression caused a lag in the initiation of 22Rv1-induced tumors in SCID mice. In vitro, RelB stimulated the proliferation of 22Rv1 cells and reduced their ability to grow in soft agar. These observations may be reconciled by our findings that, when cultured in suspension on poly-HEMA pre-coated dishes, 22Rv1 cells expressing RelB were more susceptible to cell death, and more specifically to autophagy controlled death. CONCLUSIONS: This study highlights a role of the alternative NF-κB pathway in proliferation and the controlled autophagy. Thus, the interplay of these properties may contribute to tumor survival in stress conditions while promoting PCa cells growth contributing to the overall tumorigenicity of these cells.

Molecular Characterization of Canine Parvovirus Variants CPV-2a and CPV-2c, Associated with Vaccinated Dogs at Libreville, Gabon.

The first detection of canine parvovirus type-2 (CPV-2) was in the early 1970s, when it was known to cause severe gastroenteritis in dogs. However, it has evolved over the years into CPV-2a within 2 years, into CPV-2b after 14 years, into CPV-2c after 16 years and more recently CPV-2a-, 2b- and 2c-like variants reported in 2019, with a global distribution. Reports on the molecular epidemiology of this virus are missing in most African countries. The report of clinical cases among vaccinated dogs in Libreville in Gabon triggered the execution of this study. The objective of this study was to characterize circulating variants from dogs showing clinical signs suggestive of CPV that were examined by a veterinarian. A total of eight (8) fecal swab samples were collected, and all had positive PCR results. Sequencing, Blast analysis and assembly of two whole genomes and eight partial VP2 sequences were performed, and the sequences submitted to GenBank. Genetic characterization revealed the presence of CPV-2a and CPV-2c variants with predominance of the former. Phylogenetically, the Gabonese CPVs formed distinct groups similar to Zambian CPV-2c and Australian CPV-2a sequences. The antigenic variants CPV-2a and CPV-2c have not yet been reported in Central Africa. However, these CPV-2 variants circulate in young, vaccinated dogs in Gabon. These results suggest additional epidemiological and genomic studies are required in order to evaluate the occurrence of different CPV variants in Gabon and effectiveness of the commercial vaccines used against protoparvovirus in the country.

Genomic history of human monkey pox infections in the Central African Republic between 2001 and 2018.

Monkeypox is an emerging infectious disease, which has a clinical presentation similar to smallpox. In the two past decades, Central Africa has seen an increase in the frequency of cases, with many monkeypox virus (MPXV) isolates detected in the Democratic Republic of Congo (DRC) and the Central African Republic (CAR). To date, no complete MPXV viral genome has been published from the human cases identified in the CAR. The objective of this study was to sequence the full genome of 10 MPXV isolates collected during the CAR epidemics between 2001 and 2018 in order to determine their phylogenetic relationships among MPXV lineages previously described in Central Africa and West Africa. Our phylogenetic results indicate that the 10 CAR isolates belong to three lineages closely related to those found in DRC. The phylogenetic pattern shows that all of them emerged in the rainforest block of the Congo Basin. Since most human index cases in CAR occurred at the northern edge of western and eastern rainforests, transmissions from wild animals living in the rainforest is the most probable hypothesis. In addition, molecular dating estimates suggest that periods of intense political instability resulting in population movements within the country often associated also with increased poverty may have led to more frequent contact with host wild animals. The CAR socio-economic situation, armed conflicts and ecological disturbances will likely incite populations to interact more and more with wild animals and thus increase the risk of zoonotic spillover.

Genome-wide profiling of human papillomavirus DNA integration in liquid-based cytology specimens from a Gabonese female population using HPV capture technology.

Human papillomavirus (HPV) is recognised as the cause of precancerous and cancerous cervical lesions. Furthermore, in high-grade lesions, HPV is frequently integrated in the host cell genome and associated with the partial or complete loss of the E1 and E2 genes, which regulate the activity of viral oncoproteins E6 and E7. In this study, using a double-capture system followed by high-throughput sequencing, we determined the HPV integration status present in liquid-based cervical smears in an urban Gabonese population. The main inclusion criteria were based on cytological grade and the detection of the HPV16 genotype using molecular assays. The rate of HPV integration in the host genome varied with cytological grade: 85.7% (6/7), 71.4% (5/7), 66.7% (2/3) 60% (3/5) and 30.8% (4/13) for carcinomas, HSIL, ASCH, LSIL and ASCUS, respectively. For high cytological grades (carcinomas and HSIL), genotypes HPV16 and 18 represented 92.9% of the samples (13/14). The integrated form of HPV16 genotype was mainly found in high-grade lesions in 71.4% of samples regardless of cytological grade. Minority genotypes (HPV33, 51, 58 and 59) were found in LSIL samples, except HPV59, which was identified in one HSIL sample. Among all the HPV genotypes identified after double capture, 10 genotypes (HPV30, 35, 39, 44, 45, 53, 56, 59, 74 and 82) were detected only in episomal form. Our study revealed that the degree of HPV integration varies with cervical cytological grade. The integration event might be a potential clinical prognostic biomarker for the prediction of the progression of neoplastic lesions.

Molecular characterization of complete genome of a canine distemper virus associated with fatal infection in dogs in Gabon, Central Africa.

Canine distemper (CD) is the most deadly disease in dogs with mortality rates reaching 50%. The pathological agent, the CD virus (CDV), generally causes a severe systemic disease, although the nervous form can coexist with the acute catarrhal form in the same individual. In this study, we describe an outbreak of 18 cases of CD that occurred in 2015 in a German Shepherd dog population in northwestern Gabon. In addition, we determined the sequence of the CDV genotype associated with this fatal distemper infection in Gabon and compared it with other published CDV sequences. The CDV was detected using RT-PCR on cDNA from RNA of harvested brains and other organs. The identification was confirmed by sequencing amplicons. Moreover, we obtained the whole genome sequence using high-throughput sequencing. Phylogenetic analysis revealed that Gabonese CDV strain clustered with European strains belonging to the Europe genotype. This study provided the first molecular detection of the CDV strain associated with this fatal distemper infection in Central Africa region.

Erratum to: Impact of human papillomavirus on head and neck squamous cell cancers in Gabon.

[This corrects the article DOI: 10.1186/s13027-015-0036-7.].

Molecular analysis of human Papillomavirus detected among women positive for cervical lesions by visual inspection with acetic acid/Lugol's iodine (VIA/VILI) in Libreville, Gabon.

BACKGROUND: The human papillomavirus (HPV) is the causative agent of cervical cancer, which is the leading cancer-related cause of death for women in Sub-Saharan Africa. In 2013, the Gabonese Ministry of Health and the Sylvia Bongo Ondimba Foundation implemented cervical cancer screening programs based on the detection of cancerous lesions by visual inspection with acetic acid and/or Lugol's iodine (VIA/VILI). This pilot study was set up to determine the HPV profile and analyze the nucleotide sequence variation of HPV16 circulating in patients with cervical abnormalities detected by VIA/VILI testing. METHODS: The cervical abnormalities observed upon VIA/VILI were confirmed by liquid-based cytology for all tested women. Nested PCR using the MY09/11 and GP5+/6+ primer sets was used to detect HPVs present in the extracted DNA. HPV genotypes were determined after sequencing of amplicons based on a high-throughput sequencing approach. For isolates of the HPV16 genotype, the E6 gene and the long control region (LCR) were directly sequenced using Sanger method. RESULTS: The study included 87 women who showed a positive VIA/VILI result. Cervical abnormalities were found in 40.23 % of women and 40 % were classified as high-grade lesions. The HPV detection rate was 82.9 % among women with abnormal cytology. Among all the identified high-risk HPV genotypes, HPV16, 18 and 33 were the most frequent. Multiple HPV infections were observed in 42.31 % of HPV-infected women. Analysis of the HPV16 sequence variation in the E6 gene and in the LCR showed that 85.3 and 14.7 % belonged to the African and European lineages, respectively. Among the African branch variants, Af2 was the most frequently identified in this study. CONCLUSION: This study offers the first report of the HPV detection rate and molecular epidemiology among Gabonese women with a positive result in a VIA/VILI screening test. Moreover, data on the HPV16 sequence variation confirm the predominance of African variants in high-grade lesions.

Potential Cross-Talk between Alternative and Classical NF-κB Pathways in Prostate Cancer Tissues as Measured by a Multi-Staining Immunofluorescence Co-Localization Assay.

BACKGROUND: While the classical NF-κB/p65 pathway is known to be involved in prostate cancer progression and is associated with poor patient outcome, the role of the NF-κB /RelB alternative protein is not well defined. Here we analyzed the activation of both NF-κB pathways in prostate cancer tissues and correlate this activation with clinical features of the disease. METHODS: A multiple immunofluorescence technique was employed to concomitantly and quantitatively visualize the nuclear localization of p65 and RelB in 200 paraffin embedded samples. Epithelia were defined using appropriate fluorochrome markers and the resulting immunofluorescent signals were quantified with an automated scoring system. RESULTS: The nuclear frequency of p65 was found to be significantly increased in tumor tissues as compared with normal adjacent tissue, whereas the frequency for RelB was decreased (p < 0.001, Wilcoxon test). As previously reported, p65 nuclear frequency was associated with a risk of biochemical recurrence. Although, RelB nuclear frequency alone did not predict recurrence, the presence of activated RelB reduced the risk of recurrence associated with the activation of p65. CONCLUSION: For the first time p65/RelB co-distribution was assessed in prostate cancer tissues and suggested a negative crosstalk between the two NF-κB pathways in prostate cancer progression.

Positional mapping and candidate gene analysis of the mouse Ccs3 locus that regulates differential susceptibility to carcinogen-induced colorectal cancer.

The Ccs3 locus on mouse chromosome 3 regulates differential susceptibility of A/J (A, susceptible) and C57BL/6J (B6, resistant) mouse strains to chemically-induced colorectal cancer (CRC). Here, we report the high-resolution positional mapping of the gene underlying the Ccs3 effect. Using phenotype/genotype correlation in a series of 33 AcB/BcA recombinant congenic mouse strains, as well as in groups of backcross populations bearing unique recombinant chromosomes for the interval, and in subcongenic strains, we have delineated the maximum size of the Ccs3 physical interval to a ∼2.15 Mb segment. This interval contains 12 annotated transcripts. Sequencing of positional candidates in A and B6 identified many either low-priority coding changes or non-protein coding variants. We found a unique copy number variant (CNV) in intron 15 of the Nfkb1 gene. The CNV consists of two copies of a 54 bp sequence immediately adjacent to the exon 15 splice site, while only one copy is found in CRC-susceptible A. The Nfkb1 protein (p105/p50) expression is much reduced in A tumors compared to normal A colonic epithelium as analyzed by immunohistochemistry. Studies in primary macrophages from A and B6 mice demonstrate a marked differential activation of the NfκB pathway by lipopolysaccharide (kinetics of stimulation and maximum levels of phosphorylated IκBα), with a more robust activation being associated with resistance to CRC. NfκB has been previously implicated in regulating homeostasis and inflammatory response in the intestinal mucosa. The interval contains another positional candidate Slc39a8 that is differentially expressed in A vs B6 colons, and that has recently been associated in CRC tumor aggressiveness in humans.

BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis.

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR=0.651, p=0.006 and HR=0.642, p=0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR=1.355 p=0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.