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.

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.

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data.

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

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.

Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic.

Monkeypox is an emerging and neglected zoonotic disease whose number of reported cases has been gradually increasing in Central Africa since 1980. This disease is caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae. Obtaining molecular data is particularly useful for establishing the relationships between the viral strains involved in outbreaks in countries affected by this disease. In this study, we evaluated the use of the MinION real-time sequencer as well as different polishing tools on MinION-sequenced genome for sequencing the MPXV genome originating from a pustular lesion in the context of an epidemic in a remote area of the Central African Republic. The reads corresponding to the MPXV genome were identified using two taxonomic classifiers, Kraken2 and Kaiju. Assembly of these reads led to a complete sequence of 196,956 bases, which is 6322 bases longer than the sequence previously obtained with Illumina sequencing from the same sample. The comparison of the two sequences showed mainly indels at the homopolymeric regions. However, the combined use of Canu with specific polishing tools such as Medaka and Homopolish was the best combination that reduced their numbers without adding mismatches. Although MinION sequencing is known to introduce a number of characteristic errors compared to Illumina sequencing, the new polishing tools allow a better-quality MinION-sequenced genome, thus to be used to help determine strain origin through phylogenetic analysis.

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 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.

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.