Gregory S. Orf

HIV-1 diversified for decades within the Democratic Republic of the Congo (DRC) before spreading globally in the early 1980s. Thus, the DRC is home to some of the most ancestral and diverse HIV-1 strains. Recent serosurveys conducted from 2017 to 2019 in Kinshasa, DRC, indicated high prevalence of HIV-1, yet sequence data is lacking from this period. Given the history of circulating rare HIV-1 subtypes in the DRC, a viral whole-genome sequencing study was conducted to determine current diversity in the greater Kinshasa area. Next-generation sequencing (NGS) through metagenomic and target enrichment methods was conducted on 197 specimens collected from 2017 to 2019. A large array of HIV subtypes (A, B, C, D, F1, G, H, J, and K), circulating recombinant forms (CRF01_AE, CRF02_AG, CRF05_DF, CRF11_cpx, CRF13_cpx, CRF25_cpx, CRF 45_cpx, and CRF92_C2U), unique recombinant forms, and unclassifiable sequences were observed, with many branching in basal positions within, or outside of, many subtypes on phylogenetic trees. Incorporating these new sequences into Bayesian inference of phylogeny pushes back the dates of the most recent common ancestors of HIV-1 group M and the rare subtypes G, H, and J by between 3 and 7 years each. The DRC continues to harbor diverse and rare HIV-1 subtypes that could challenge diagnostic tests, treatments, and vaccines. In addition to shifting subtype emergence dates, the sequences from our study are evidence that rare strains continue to circulate and should be regularly monitored.

Probe-based capture represents a highly sensitive and cost-effective approach for overcoming host background and enriching viruses in metagenomic NGS (mNGS) libraries. Using clinical specimens collected globally from patients with fever or respiratory illness, we generated mNGS libraries by random priming and Nextera XT tagmentation, followed by target enrichment (teNGS) with Comprehensive Viral Research Panel (CVRP) probes. Capture pool sizes and total reads were optimized, and libraries were initially sequenced separately. Using only 3–4% of reads required for standard mNGS, teNGS achieved increased sensitivity, 100–10,000× increases in depth, and >50% genome coverage for pathogens with titers ≥ 1000 cp/mL. Application to >2000 clinical specimens from various matrices and to contrived samples containing viruses absent from the CVRP probe set enabled detection of diverse viral families and established a minimum 65% nucleotide identity for hybridization, respectively. To save time and resources, teNGS and mNGS libraries were then combined into one sequencing run: teamNGS. In addition to streamlining the workflow, teamNGS also improved genome recovery. Coupling methods maintain the sensitivity and coverage for viruses achieved by enrichment alone while also ensuring comprehensive recovery of non-viral microbes. teamNGS has the potential to improve patient management and lower the rates of unnecessary testing and antibiotic use.

Dengue virus (DENV) infection has not been previously reported from Bayelsa State, Nigeria. We aimed to determine the prevalence of dengue virus (DENV) infection, malaria, and coinfection, and other pathogens among febrile patients in the capital city, Yenagoa.We conducted a cross-sectional study among persons aged ≥1 year who presented with acute febrile illnesses (AFI) at four hospitals in Bayelsa State during 20 May – 15 June 2022. Blood samples from 443 participants were tested for DENV seromarkers (NS1, IgM, IgG), using serology and RT-PCR, and malaria was diagnosed by thick smear microscopy. Sociodemographic and risk factor data were collected using electronic questionnaires administered via smart phones/tablets and analyzed using univariate and multivariate methods. Metagenomic libraries were prepared and enriched by viral target capture and sequenced by NGS. The seroprevalence of acute DENV infection was 14.5% (n = 64) while the prevalence of malaria was 42.4% (n = 188); 6.5% (n = 29) of participants were coinfected with acute DENV infection and malaria. An additional 17.6% (n = 78) of participants had markers for past DENV infection. Rural/suburban residence and age ≥ 31 years were significantly correlated with having any dengue seromarker. Residence in a larger household (≥5 persons), and borehole water-use were predictors for malaria fever. RT-PCR results revealed multiple DENV serotypes, with serotype 3 dominant. Sequencing of unknown AFI cases revealed numerous viral causes such as adenovirus, EBV, and hepatitis A, as well as additional dengue and malarial infections missed by conventional testing. Of interest were Coxsackievirus A5 (hand, foot and mouth disease; HFMD) which has been diversifying locally for years in Nigeria and an mPox clade IIb (lineage A.2.3) strain that emerged in Nigeria during the 2022 global outbreak. The results of this study provide the first documentation of human DENV infection in Bayelsa State, Nigeria and suggests that dengue is an emerging and important cause of febrile illness in this area. Our findings support the need for routine testing to identify DENV among patients who present with acute febrile disease. Metagenomic NGS results highlight the benefits of unbiased surveillance to identify circulating and emerging pathogens.

Objectives: Utilizing post-mortem examination for routine monitoring of infectious diseases and pandemic preparedness is a common-sense, yet uncommon, public health measure. Here, we established a novel mortuary surveillance program in Uganda that leverages the unbiased nature of metagenomic next-generation sequencing (mNGS) to detect pathogens in recently deceased individuals.Methods: Between October 2022 and December 2023, specimens and patient metadata were collected from 2,607 individuals across five mortuary sites around Kampala. Specimens were pre-screened for hemorrhagic fever viruses by RT-qPCR and a subset (n = 134) of RT-qPCR negatives were sequenced by mNGS.Results: A variety of DNA (herpes, parvovirus, bufavirus) and RNA (Saffold, Salivirus, HAV) viruses, vectored (Bartonella, Rickettsia) and nosocomial (Enterobacter, Klebsiella) bacterial infections, and potentially lethal respiratory pathogens (e.g., Cryptococcus neoformans, Corynebacterium diphtheria) were detected. A localized outbreak of Enterovirus B (EV-B), specifically a recombinant Echovirus E7, was observed in Kampala. An epidemiologic assessment indicated that most identified pathogens were acquired via direct and/or indirect contact (e.g., fecal-oral, fomites) and that other modes of transmission (e.g., food-borne, insect-vectored) played a less significant role.Conclusion: Integration of mortuary surveillance, coupled with mNGS, into public health systems represents a powerful strategy for identifying unrecognized outbreaks and monitoring the (re-) emergence of infectious diseases.

Rocahepevirus ratti (hepatitis E virus [HEV]‐C), originally discovered in rats in 2010, has been recently linked to hepatitis in humans. Although rare and typically detected in the immunocompromised, much like Paslahepevirus balayani (HEV‐A), it can manifest as acute or persistent hepatitis. In a next‐generation sequencing (NGS)‐based screen for causes of acute febrile illness (AFI) in Thailand, we assembled a complete Rocahepevirus (rat HEV [rHEV]) genome from a patient admitted to the hospital who developed abnormal liver functions 2–3 months after a heart transplant. Despite withdrawal of medications suspected of inducing hepatitis, he progressed from parenchymal liver disease to cirrhosis. The absence of other viral etiologies suggested rHEV may have been the cause of chronic hepatitis. Thailand strain Ma617‐09869 is the sole human representative in a clade of genogroup C1 composed of sequences found in rats from Thailand and neighboring Southeast Asian countries, including Laos, Cambodia, Vietnam, and Indonesia. Principal component analysis (PCA) of viral sequences indicates humans are incidental hosts and suggests that white bellied rats ( Niviventer spp.) are the putative original host, with black and common rats ( Rattus spp.) serving as the natural reservoir. While Rocahepevirus adaptation may not currently facilitate human‐to‐human transmission, specific diagnostics are needed to identify additional sequences and cases, not only to gain a better understanding of the biology of this virus, but also to assess the risk for continued evolution, virulence, and increased zoonotic events.

Introduction: Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non-Plasmodium pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI.Methods: In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020–2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens.Results and Discussion: Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): Borrelia crocidurae (N = 7), West Nile virus (N = 3), Rickettsia felis (N = 2), Bartonella quintana (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to Plasmodium falciparum were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.

Before the appearance of the Omicron variant in late 2021, numerous SARS-CoV-2 variants emerged, were established, and declined, often with different outcomes in different geographic areas. In this study, we considered the trajectory of the Mu variant, which only successfully dominated the epidemic landscape of a single country: Colombia.