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Infectious disease modeling has played a prominent role in recent outbreaks, yet integrating these analyses into public health decision-making has been challenging. We recommend establishing ‘outbreak science’ as an inter-disciplinary field to improve applied epidemic modeling.

The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in the non-ORF1 region of the genome containing structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.

A fundamental mystery for dengue and other infectious pathogens is how observed patterns of cases relate to actual chains of individual transmission events. These pathways are intimately tied to the mechanisms by which strains interact and compete across spatial scales. Phylogeographic methods have been used to characterize pathogen dispersal at global and regional scales but have yielded few insights into the local spatiotemporal structure of endemic transmission. Using geolocated genotype (800 cases) and serotype (17,291 cases) data, we show that in Bangkok, Thailand, 60% of dengue cases living <200 meters apart come from the same transmission chain, as opposed to 3% of cases separated by 1 to 5 kilometers. At distances <200 meters from a case (encompassing an average of 1300 people in Bangkok), the effective number of chains is 1.7. This number rises by a factor of 7 for each 10-fold increase in the population of the ÒenclosedÓ region. This trend is observed regardless of whether population density or area increases, though increases in density over 7000 people per square kilometer do not lead to additional chains. Within Thailand these chains quickly mix, and by the next dengue season viral lineages are no longer highly spatially structured within the country. In contrast, viral flow to neighboring countries is limited. These findings are consistent with local, density-dependent transmission and implicate densely populated communities as key sources of viral diversity, with home location the focal point of transmission. These findings have important implications for targeted vector control and active surveillance.

Handling cultured isolates and clinical, environmental, or wildlife surveillance samples containing Risk Group 3 and 4 pathogens presents considerable biosafety challenges in minimizing human exposure during processing and transport. Safe handling typically requires high- or maximum-containment facilities, demanding substantial logistical planning and resources. We evaluated PrimeStore Molecular Transport Medium (PS-MTM), a guanidine-based solution created to kill pathogens and preserve nucleic acids at ambient temperatures, for inactivating Crimean-Congo hemorrhagic fever, eastern equine encephalitis, Ebola, Hendra, Japanese encephalitis, Lassa, Marburg, Nipah, Rift Valley fever, and West Nile viruses. To mimic diagnostic conditions, human whole blood spiked with any of these viruses was incubated with PS-MTM for 20-, 30-, or 60-min. Samples with titers up to 107 PFU/mL exposed to PS-MTM at all time points resulted in complete loss of infectivity judged by plaque assays. A 30-min incubation provided a 50% safety margin over the minimum inactivation time and was used for quantification with the tissue culture infectious dose (TCID50) assay, enabling evaluation of PS-MTM’s activity for viruses that do or do not produce well-defined plaques. Results confirmed that PS-MTM inactivated all tested viruses at titers up to 107 TCID50/mL, underscoring its reliability for enhancing biosafety in diagnostics, outbreak management, and surveillance.

Background With the emergence and spread of SARS-CoV-2 variants, genomic epidemiology and surveillance have proven invaluable tools for variant tracking. Here, we analyzed SARS-CoV-2 samples collected from personnel located at the US/NATO bases across Afghanistan. Results Sequencing and phylogenetic analyses revealed at least 16 independent introductions of SARS-CoV-2 into four of these relatively isolated compounds during April and May 2021, including multiple introductions of Alpha and Delta variants. Four of the introductions resulted in sustained spread of the virus within, and in two cases between, the compounds. Three of these outbreaks, one Delta and two Alpha, occurred simultaneously. Conclusions Even in rigorously controlled and segregated environments, SARS-CoV-2 introduction and spread may occur frequently.

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5’ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5’ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5’ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5’ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5’ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

Despite unprecedented global sequencing and surveillance of SARS-CoV-2, timely identification of the emergence and spread of novel variants of concern (VoCs) remains a challenge. Several million raw genome sequencing runs are now publicly available. We sought to survey these datasets for intrahost variation to study emerging mutations of concern. We developed iSKIM (“intrahost SARS-CoV-2 k-mer identification method”) to relatively quickly and efficiently screen the many SARS-CoV-2 datasets to identify intrahost mutations belonging to lineages of concern. Certain mutations surged in frequency as intrahost minor variants just prior to, or while lineages of concern arose. The Spike N501Y change common to several VoCs was found as a minor variant in 834 samples as early as October 2020. This coincides with the timing of the first detected samples with this mutation in the Alpha/B.1.1.7 and Beta/B.1.351 lineages. Using iSKIM, we also found that Spike L452R was detected as an intrahost minor variant as early as September 2020, prior to the observed rise of the Epsilon/B.1.429/B.1.427 lineages in late 2020. iSKIM rapidly screens for mutations of interest in raw data, prior to genome assembly, and can be used to detect increases in intrahost variants, potentially providing an early indication of novel variant spread.

Background Increasing evidence suggests that influenza reassortment not only contributes to the emergence of new human pandemics but also plays an important role in seasonal influenza epidemics, disease severity, evolution, and vaccine efficacy. We studied this process within 2091 H3N2 full genomes utilizing a combination of the latest reassortment detection tools and more conventional phylogenetic analyses. Results We found that the amount of H3N2 intra-subtype reassortment depended on the number of sampled genomes, occurred with a steady frequency of 3.35%, and was not affected by the geographical origins, evolutionary patterns, or previous reassortment history of the virus. We identified both single reassortant genomes and reassortant clades, each clade representing one reassortment event followed by successful spread of the reassorted variant in the human population. It was this spread that was mainly responsible for the observed high presence of H3N2 intra-subtype reassortant genomes. The successfully spread variants were generally sampled within one year of their formation, highlighting the risk of their rapid spread but also presenting an opportunity for their rapid detection. Simultaneous spread of several different reassortant lineages was observed, and despite their limited average lifetime, second and third generation reassortment was detected, as well as reassortment between viruses belonging to different vaccine-associated clades, likely displaying differing antigenic properties. Some of the spreading reassortants remained confined to certain geographical regions, while others, sharing common properties in amino acid positions of the HA, NA, and PB2 segments, were found throughout the world. Conclusions Detailed surveillance of seasonal influenza reassortment patterns and variant properties may provide unique information needed for prediction of spread and construction of future influenza vaccines.

Tecovirimat is available for the treatment of mpox (formerly known as monkeypox) in Europe and the United States, on the basis of findings from efficacy studies in animals and safety evaluations in healthy humans. Evidence from randomized, controlled trials of safety and efficacy in patients with mpox is lacking. We conducted a double-blind, randomized, placebo-controlled trial of tecovirimat in patients with mpox in the Democratic Republic of Congo (DRC). Patients with at least one mpox skin lesion and positive polymerase-chain-reaction results for clade I MPXV were assigned in a 1:1 ratio to receive tecovirimat or placebo. All patients received supportive care. The primary end point was resolution of mpox lesions, measured in number of days after randomization. Safety was also assessed. From October 7, 2022, through July 9, 2024, a total of 597 patients underwent randomization - 295 to receive tecovirimat and 302 to receive placebo. The median time from randomization to lesion resolution was 7 days with tecovirimat and 8 days with placebo; the competing-risks hazard ratio for lesion resolution was 1.13 (95% confidence interval [CI], 0.97 to 1.31; P = 0.14). Results were similar whether patients began the trial regimen within 7 days after the reported onset of symptoms (competing-risks hazard ratio, 1.16; 95% CI, 0.98 to 1.37) or more than 7 days after onset (competing-risks hazard ratio, 1.00; 95% CI, 0.71 to 1.40). Overall mortality was 1.7%, which was lower than the case fatality rate of 4.6% reported in the DRC in 2023. At 14 days, the percentages of patients who had blood, lesion, and oropharyngeal samples negative for MPXV by PCR were similar in the two groups. Adverse events occurred in 72.9% of the patients in the tecovirimat group and 70.5% of those in the placebo group, and serious adverse events were reported in 5.1% and 5.0%, respectively. Tecovirimat did not reduce the number of days to lesion resolution in patients with mpox caused by clade I MPXV. No safety concerns were identified. (Funded by the National Institute of Allergy and Infectious Diseases and others; PALM007 ClinicalTrials.gov number, NCT05559099.).