Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93-94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our PLOS Pathogens |

Surveillance of emerging viral variants is critical to ensuring that blood screening and diagnostic tests detect all infections regardless of strain or geographic location. In this study, we conducted serological and molecular surveillance to monitor the prevalence and diversity of HIV, HBV, and HTLV in South Cameroon. The prevalence of HIV was 8.53%, HBV was 10.45%, and HTLV was 1.04% amongst study participants. Molecular characterization of 555 HIV-1 specimens identified incredible diversity, including 7 subtypes, 12 CRFs, 6 unclassified, 24 Group O and 2 Group N infections. Amongst 401 HBV sequences were found a rare HBV AE recombinant and two emerging sub-genotype A strains. In addition to HTLV-1 and HTLV-2 strains, sequencing confirmed the fifth known HTLV-3 infection to date. Continued HIV/HBV/HTLV surveillance and vigilance for newly emerging strains in South Cameroon will be essential to ensure diagnostic tests and research stay a step ahead of these rapidly evolving viruses.

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens.Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software.Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads 90% for 35/40 samples with higher viral loads.Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

While diagnosis of COVID-19 relies on qualitative molecular testing for the absence or presence of SARS-CoV-2 RNA, quantitative viral load determination for SARS-CoV-2 has many potential applications in antiviral therapy and vaccine trials as well as implications for public health and quarantine guidance. To date, no quantitative SARS-CoV-2 viral load tests have been authorized for clinical use by the FDA. In this study, we modified the FDA emergency use authorized qualitative RealTi m e SARS-CoV-2 assay into a quantitative SARS-CoV-2 Laboratory Developed Test (LDT) using newly developed Abbott SARS-CoV-2 calibration standards. Both analytical and clinical performance of this SARS-CoV-2 quantitative LDT was evaluated using nasopharyngeal swabs (NPS). We further assessed the correlation between Ct and the ability to culture virus on Vero CCL81 cells. The SARS-CoV-2 quantitative LDT demonstrated high linearity with R 2 value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ±0.08-0.14 log 10 copies/mL for inter-assay reproducibility and ±0.09 to 0.19 log 10 copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log 10 copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log 10 copies/mL. Quantitative tests, validated against viral culture capacity, may allow more accurate identification of individuals with and without infectious viral shedding from the respiratory tract.