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ObjectivePrior genomewide association studies have identified variation in major histocompatibility complex (MHC) class I alleles and C-C chemokine receptor type 5 gene (CCR5Δ32) as genetic predictors of viral control, especially in 'elite' controllers, individuals who remain virally suppressed in the absence of therapy.DesignCross-sectional genomewide association study.MethodsWe analyzed custom whole exome sequencing and direct human leukocyte antigen (HLA) typing from 202 antiretroviral therapy (ART)-suppressed HIV+ noncontrollers in relation to four measures of the peripheral CD4+ T-cell reservoir: HIV intact DNA, total (t)DNA, unspliced (us)RNA, and RNA/DNA. Linear mixed models were adjusted for potential covariates including age, sex, nadir CD4+ T-cell count, pre-ART HIV RNA, timing of ART initiation, and duration of ART suppression.ResultsPreviously reported 'protective' host genetic mutations related to viral setpoint (e.g. among elite controllers) were found to predict smaller HIV reservoir size. The HLA 'protective' B∗57:01 was associated with significantly lower HIV usRNA (q = 3.3 × 10-3), and among the largest subgroup, European ancestry individuals, the CCR5Δ32 deletion was associated with smaller HIV tDNA (P = 4.3 × 10-3) and usRNA (P = 8.7 × 10-3). In addition, genomewide analysis identified several single nucleotide polymorphisms in MX1 (an interferon stimulated gene) that were significantly associated with HIV tDNA (q = 0.02), and the direction of these associations paralleled MX1 gene eQTL expression.ConclusionsWe observed a significant association between previously reported 'protective' MHC class I alleles and CCR5Δ32 with the HIV reservoir size in noncontrollers. We also found a novel association between MX1 and HIV total DNA (in addition to other interferon signaling relevant genes, PPP1CB, DDX3X). These findings warrant further investigation in future validation studies.

Real‐time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real‐time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT‐ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS‐CoV‐2 detected in Washington State by using a combination of reverse‐transcription polymerase chain reaction (RT‐PCR), RT‐ddPCR, and next‐generation sequencing. We initially screened 1035 samples positive for SARS‐CoV‐2 by our CDC‐based laboratory‐developed assay using ThermoFisher's multiplex RT‐PCR COVID‐19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT‐ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69‐70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow‐up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT‐ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT‐ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS‐CoV‐2., Highlights ‐B.1.1.7 (alpha) lineage of SARS‐CoV‐2 was detected in Washington State using RT‐ddPCR.‐Specific allelic discrimination can be used to distinguish SARS‐CoV‐2 variants of concern.‐Allelic discrimination can identify variants of concern more rapidly than conventional sequencing techniques.‐S‐gene target failures can be used to screen for certain SARS‐CoV‐2 variants.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOCs) and had a median Ct of 20.2 (IQR, 17.1–23.3). When compared with 5174 contemporaneous samples sequenced in our laboratory, VOCs were significantly enriched among breakthrough infections (P < .05).

The global spread of the SARS-CoV-2 virus has resulted in emergence of lineages which impact the effectiveness of immunotherapies and vaccines that are based on the early Wuhan isolate. All currently approved vaccines employ the spike protein S, as it is the target for neutralizing antibodies. Here we describe two SARS-CoV-2 isolates with unusually large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis shows that the deletions result in complete reshaping of the NTD supersite, an antigenically important region of the NTD. For both spike variants the remodeling of the NTD negatively affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite. For one of the variants, we observed a P9L mediated shift of the signal peptide cleavage site resulting in the loss of a disulfide-bridge; a unique escape mechanism with high antigenic impact. Although the observed deletions and disulfide mutations are rare, similar modifications have become independently established in several other lineages, indicating a possibility to become more dominant in the future. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

The COVID-19 epidemic of 2019–20 is due to the novel coronavirus SARS-CoV-2. Following first case description in December, 2019 this virus has infected over 10 million individuals and resulted in at least 500,000 deaths world-wide. The virus is undergoing rapid mutation, with two major clades of sequence variants emerging. This study sought to determine whether SARS-CoV-2 sequence variants are associated with differing outcomes among COVID-19 patients in a single medical system. Whole genome SARS-CoV-2 RNA sequence was obtained from isolates collected from patients registered in the University of Washington Medicine health system between March 1 and April 15, 2020. Demographic and baseline clinical characteristics of patients and their outcome data including their hospitalization and death were collected. Statistical and machine learning models were applied to determine if viral genetic variants were associated with specific outcomes of hospitalization or death. Full length SARS-CoV-2 sequence was obtained 190 subjects with clinical outcome data. 35 (18.4%) were hospitalized and 14 (7.4%) died from complications of infection. A total of 289 single nucleotide variants were identified. Clustering methods demonstrated two major viral clades, which could be readily distinguished by 12 polymorphisms in 5 genes. A trend toward higher rates of hospitalization of patients with Clade 2 infections was observed (p = 0.06, Fisher’s exact). Machine learning models utilizing patient demographics and co-morbidities achieved area-under-the-curve (AUC) values of 0.93 for predicting hospitalization. Addition of viral clade or sequence information did not significantly improve models for outcome prediction. In summary, SARS-CoV-2 shows substantial sequence diversity in a community-based sample. Two dominant clades of virus are in circulation. Among patients sufficiently ill to warrant testing for virus, no significant difference in outcomes of hospitalization or death could be discerned between clades in this sample. Major risk factors for hospitalization and death for either major clade of virus include patient age and comorbid conditions.

The sequencing of SARS-CoV-2 provides essential information on viral evolution, transmission, and epidemiology. In this paper, we performed the whole-genome sequencing of SARS-CoV-2 using nanopore and Illumina sequencing to describe the circulation of the virus lineages in Armenia. The analysis of 145 full genomes identified six clades (19A, 20A, 20B, 20I, 21J, and 21K) and considerable intra-clade PANGO lineage diversity. Phylodynamic and transmission analysis allowed to attribute specific clades as well as infer their importation routes. Thus, the first two waves of positive case increase were caused by the 20B clade, the third peak caused by the 20I (Alpha), while the last two peaks were caused by the 21J (Delta) and 21K (Omicron) variants. The functional analyses of mutations in sequences largely affected epitopes associated with protective HLA loci and did not cause the loss of the signal in PCR tests targeting ORF1ab and N genes as confirmed by RT-PCR. We also compared the performance of nanopore and Illumina short-read sequencing and showed the utility of nanopore sequencing as an efficient and affordable alternative for large-scale molecular epidemiology research. Thus, our paper describes new data on the genomic diversity of SARS-CoV-2 variants in Armenia in the global context of the virus molecular genomic surveillance.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections., Graphical Abstract, This study analyzes CRISPR-Cas9-based antiviral therapy in a model of chronic hepatitis B virus (HBV) infection. The study demonstrates that therapy is safe, and that gene editing of HBV can occur in vivo, although low levels of CRISPR-Cas9 delivery limit the current efficacy of the approach.

Background More than 2 months separated the initial description of SARS-CoV-2 and discovery of its widespread dissemination in the United States. Despite this lengthy interval, implementation of specific quantitative reverse transcription (qRT)-PCR-based SARS-CoV-2 tests in the US has been slow, and testing is still not widely available. Metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence. Methods To evaluate metagenomic approaches in the context of the current SARS-CoV-2 epidemic, laboratory-confirmed positive and negative samples from Seattle, WA were evaluated by metagenomic sequencing, with comparison to a 2019 reference genomic database created before the emergence of SARS-CoV-2. Results Within 36 h our results showed clear identification of a novel human Betacoronavirus, closely related to known Betacoronaviruses of bats, in laboratory-proven cases of SARS-CoV-2. A subset of samples also showed superinfection or colonization with human parainfluenza virus 3 or Moraxella species, highlighting the need to test directly for SARS-CoV-2 as opposed to ruling out an infection using a viral respiratory panel. Samples negative for SARS-CoV-2 by RT-PCR were also negative by metagenomic analysis, and positive for Rhinovirus A and C. Unlike targeted SARS-CoV-2 qRT-PCR testing, metagenomic analysis of these SARS-CoV-2 negative samples identified candidate etiological agents for the patients’ respiratory symptoms. Conclusion Taken together, these results demonstrate the value of metagenomic analysis in the monitoring and response to this and future viral pandemics.

The mechanisms underlying rapid elimination of herpes simplex virus-2 (HSV-2) in the human genital tract despite low CD8(+) and CD4(+) tissue-resident T cell (Trm cell) density are unknown. We analyzed shedding episodes during chronic HSV-2 infection; viral clearance always predominated within 24 hours of detection even when viral load exceeded 1 × 10(7) HSV DNA copies, and surges in granzyme B and IFN-γ occurred within the early hours after reactivation and correlated with local viral load. We next developed an agent-based mathematical model of an HSV-2 genital ulcer to integrate mechanistic observations of Trm cells in in situ proliferation, trafficking, cytolytic effects, and cytokine alarm signaling from murine studies with viral kinetics, histopathology, and lesion size data from humans. A sufficiently high density of HSV-2–specific Trm cells predicted rapid elimination of infected cells, but our data suggest that such Trm cell densities are relatively uncommon in infected tissues. At lower, more commonly observed Trm cell densities, Trm cells must initiate a rapidly diffusing, polyfunctional cytokine response with activation of bystander T cells in order to eliminate a majority of infected cells and eradicate briskly spreading HSV-2 infection.

The long-term evolution of viruses is ultimately due to viral mutants that arise within infected individuals and transmit to other individuals. Here, we use deep sequencing to investigate the transmission of viral genetic variation among individuals during a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that infected the vast majority of crew members on a fishing boat. We deep-sequenced nasal swabs to characterize the within-host viral population of infected crew members, using experimental duplicates and strict computational filters to ensure accurate variant calling. We find that within-host viral diversity is low in infected crew members. The mutations that did fix in some crew members during the outbreak are not observed at detectable frequencies in any of the sampled crew members in which they are not fixed, suggesting that viral evolution involves occasional fixation of low-frequency mutations during transmission rather than persistent maintenance of within-host viral diversity. Overall, our results show that strong transmission bottlenecks dominate viral evolution even during a superspreading event with a very high attack rate.

With the COVID‐19 pandemic caused by SARS‐CoV‐2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT–PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g., subgenomic RNA [sgRNA]) and replicative intermediates (e.g., negative‐strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS‐CoV‐2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3 to 37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative‐strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative‐strand, and sgE) detected with the WHO/Charité primer‐probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS‐CoV‐2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cutoff of > 31 or specifically testing for sgRNA can serve as an effective rule‐out test for the presence of culturable virus.

ObjectivePrior genomewide association studies have identified variation in MHC Class I alleles andCCR5Δ32as genetic predictors of viral control, especially in “elite” controllers, individuals who remain virally suppressed in the absence of therapy.DesignCross-sectional genomewide association study.MethodsWe analyzed custom whole exome sequencing and direct HLA typing from 202 ART-suppressed HIV+ non-controllers in relation to four measures of the peripheral CD4+ T cell reservoir: HIV intact DNA, total (t)DNA, unspliced (us)RNA, and RNA/DNA. Linear mixed models were adjusted for potential covariates including age, sex, nadir CD4+ T cell count, pre-ART HIV RNA, timing of ART initiation, and duration of ART suppression.ResultsPreviously reported “protective” host genetic mutations related to viral setpoint (e.g., among elite controllers) were found to predict smaller HIV reservoir size. The HLA “protective” B*57:01 was associated with significantly lower HIV usRNA (q=3.3×10−3), and among the largest subgroup, European ancestry individuals, theCCR5Δ32deletion was associated with smaller HIV tDNA (p=4.3×10−3) and usRNA (p=8.7×10−3). In addition, genomewide analysis identified several SNPs inMX1(an interferon stimulated gene) that were significantly associated with HIV tDNA (q=0.02), and the direction of these associations paralleledMX1gene eQTL expression.ConclusionsWe observed a significant association between previously reported “protective” MHC class I alleles andCCR5Δ32with the HIV reservoir size in non-controllers. We also found a novel association betweenMX1and HIV total DNA (in addition to other interferon signaling relevant genes,PPP1CB,DDX3X). These findings warrant further investigation in future validation studies.

SARS-CoV-2 is spreading worldwide with continuously evolving variants, some of which occur in the Spike protein and appear to increase the viral transmissibility. However, variants that cause severe COVID-19 or lead to other breakthroughs have not been well characterized. To discover such viral variants, we assembled a cohort of 683 COVID-19 patients; 388 inpatients (“cases”) and 295 outpatients (“controls”) from April to August 2020 using electronically captured COVID test request forms and sequenced their viral genomes. To improve the analytic power, we accessed 7,137 viral sequences in Washington State to filter out viral single nucleotide variants (SNVs) that did not have significant expansions over the collection period. Applying this filter led to the identification of 53 SNVs that were statistically significant, of which 13 SNVs each had 3 or more variant copies in the discovery cohort. Correlating these selected SNVs with case/control status, eight SNVs were found to significantly associate with inpatient status (q-values

Monkeypox virus, the causative agent of the 2022 monkeypox outbreak, is a double-stranded DNA virus in the Orthopoxvirus genus of the Poxviridae family. Genes in terminal regions of Orthopoxvirus genomes mostly code for host-pathogen interaction proteins and are prone to selective pressure and modification events. Using viral whole genome sequencing, we identified twenty-five total clinical samples with ORF-disrupting mutations, including twenty samples encoding nonsense mutations in MPXVgp001/191 (OPG001), MPXVgp004/188 (OPG015), MPXVgp010 (OPG023), MPXVgp030 (OPG042), MPXVgp159 (OPG0178), or MPXVgp161 (OPG181). Additional mutations include a frameshift leading to an alternative C-terminus in MPXVgp010 (OPG023) and an insertion in an adenine homopolymer at the beginning of the annotated ORF for MPXVgp153 (OPG151), encoding a subunit of the RNA polymerase, suggesting the virus may instead use the start codon that encodes Met9 as annotated. Finally, we detected three samples with large (>900 bp) deletions. These included a 913 bp deletion that truncates the C-terminus of MPXVgp010 (OPG023); a 4205 bp deletion that eliminates MPXVgp012 (OPG025), MPXVgp013 (OPG027), and MPXVgp014 (OPG029) and truncates MPXVgp011 (OPG024; D8L) and MPXVgp015 (OPG030); and a 6881 bp deletion that truncates MPXVgp182 (OPG210) and eliminates putative ORFs MPXVgp184, MPXVgp185 (OPG005), and MPXVgp186, as well as MPXVgp187 (OPG016), and MPXVgp188 (OPG015) from the 3’ ITR only. MPXVgp182 encodes the monkeypox-specific, highly immunogenic surface glycoprotein B21R which has been proposed as a serological target. Overall, we find greater than one-tenth of our sequenced MPXV isolates have at least one gene inactivating mutation and these genes together comprised greater than one-tenth of annotated MPXV genes. Our findings highlight non-essential genes in monkeypox virus that may be evolving as a result of selective pressure in humans, as well as the limitations of targeting them for therapeutics and diagnostic testing.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOC) and had a median Ct of 20.2 (IQR=17.1-23.3). When compared to 5174 contemporaneous samples sequenced in our laboratory, VOC were significantly enriched among breakthrough infections (p < .05).

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus., RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection.

Whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a better understanding of the virus’s origin, transmission, and evolution ([1][1][–][2][6][3]). Multiplex amplicon sequencing for viral WGS is a preferred approach to library preparation

Summary Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells., Graphical abstract, Highlights HIV cure studies rely on precise HIV reservoir assays that can be done at scale Multiplexed droplet digital PCR can probe multiple HIV genome targets per sample HIV-1 proviruses containing 5 probed genomic regions are likely intact PCR-based counting of T cells improves reservoir quantification in tissues, Evaluation of HIV cure interventions requires accurate and scalable measurement of the replication-competent HIV reservoir. Levy at al. describe a highly multiplexed droplet digital PCR assay that simultaneously quantifies likely intact HIV-1 proviruses and T lymphocytes. They validate the assay in cell and tissue specimens from several patient cohorts.

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have been performed only in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral reverse transcription-PCR (RT-PCR) testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32., The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have been performed only in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral reverse transcription-PCR (RT-PCR) testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range, 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR-positive viral test with a cycle threshold (CT) of

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell–specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell–specific and NK cell–specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity., Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations; the mechanisms behind these host differences are still unclear. This study reveals that SARS-CoV-2 infection induces inflammation by activation of interferon signaling, with differences in host response observed due to sex and age.

PURPOSE: This is a SARS-CoV-2 specific protocol that covers the steps needed to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal. For new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission. We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. If you need a pipeline for frequent or large volume submissions, follow Step 1 in the SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject to get your NCBI submission environment established, then contact gb-admin@ncbi.nlm.nih.gov to set up an account for submitting through the API. These protocols cover submission using NCBI's Submission Portal web-interface. Complete in order (1 then 2): 1. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort. SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample. 2. SARS-CoV-2 NCBI consensus submission protocol: GenBank (included protocol) Required: established BioProject and BioSamples Submit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data. Metadata templates, explained! SOP for populating NCBI submission templates

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, resulting in >300,000 reported cases worldwide as of March 21st, 2020. Here we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 in Northern California using samples from returning travelers, cruise ship passengers, and cases of community transmission with unclear infection sources. Virus genomes were sampled from 29 patients diagnosed with COVID-19 infection from Feb 3rd through Mar 15th. Phylogenetic analyses revealed at least 8 different SARS-CoV-2 lineages, suggesting multiple independent introductions of the virus into the state. Virus genomes from passengers on two consecutive excursions of the Grand Princess cruise ship clustered with those from an established epidemic in Washington State, including the WA1 genome representing the first reported case in the United States on January 19th. We also detected evidence for presumptive transmission of SARS-CoV-2 lineages from one community to another. These findings suggest that cryptic transmission of SARS-CoV-2 in Northern California to date is characterized by multiple transmission chains that originate via distinct introductions from international and interstate travel, rather than widespread community transmission of a single predominant lineage. Rapid testing and contact tracing, social distancing, and travel restrictions are measures that will help to slow SARS-CoV-2 spread in California and other regions of the USA.

A series of unfortunate events The history of how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread around the planet has been far from clear. Several narratives have been propagated by social media and, in some cases, national policies were forged in response. Now that many thousands of virus sequences are available, two studies analyzed some of the key early events in the spread of SARS-CoV-2. Bedford et al. found that the virus arrived in Washington state in late January or early February. The viral genome from the first case detected had mutations similar to those found in Chinese samples and rapidly spread and dominated subsequent undetected community transmission. The other viruses detected had origins in Europe. Worobey et al. found that early introductions into Germany and the west coast of the United States were extinguished by vigorous public health efforts, but these successes were largely unrecognized. Unfortunately, several major travel events occurred in February, including repatriations from China, with lax public health follow-up. Serial, independent introductions triggered the major outbreaks in the United States and Europe that still hold us in the grip of control measures. Science, this issue p. 571, p. 564, The first case of SARS-CoV-2 in Washington state occurred at the end of January and, by going undetected, sparked community transmission., After its emergence in Wuhan, China, in late November or early December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus rapidly spread globally. Genome sequencing of SARS-CoV-2 allows the reconstruction of its transmission history, although this is contingent on sampling. We analyzed 453 SARS-CoV-2 genomes collected between 20 February and 15 March 2020 from infected patients in Washington state in the United States. We find that most SARS-CoV-2 infections sampled during this time derive from a single introduction in late January or early February 2020, which subsequently spread locally before active community surveillance was implemented.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection., Herpes simplex virus establishes lifelong latency in ganglionic neurons, which are the source for recurrent infection. Here Aubert et al. report a promising antiviral therapy based on gene editing with adeno-associated virus-delivered meganucleases, which leads to a significant reduction in ganglionic HSV loads and HSV reactivation.

BACKGROUND: Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins. METHODS: A total of 29 MZ and 22 DZ HSV-1–seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4(+) T-cell responses to HSV-1 proteins were studied. RESULTS: The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4(+) T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002). CONCLUSION: Viral strain characteristics likely contribute to oral HSV-1 shedding rates.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

Introduction Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. Methods Genital lesion swabs containing ≥ 107 log10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (UL_US) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. Results Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated UL_US segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in Conclusions Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens.

We describe a case of acute liver failure and myopericarditis due to herpes simplex virus–1 (HSV-1) in an immunocompetent adult. We estimate that, at the height of viremia, the patient contained a quantity of HSV-1 virions approaching that of human cells. The patient recovered with acyclovir that was dose-adjusted for neurotoxicity and developed a vigorous anti-HSV-1 T-cell response.

Peculiar among human RNA viruses, coronaviruses have large genomes containing accessory genes that are not required for replication. Numerous mutations within the SARS-CoV-2 genome have been described but few deletions in the accessory genes of SARS-CoV-2 have been reported. Here, we report two large deletions in ORF7a, both of which produce new open reading frames (ORFs) through the fusion of the N-terminus of ORF7a and a downstream ORF.

Tissue-resident CD8+ T cells (Trm) can rapidly eliminate virally infected cells, but their heterogeneous spatial distribution may leave gaps in protection within tissues. Although Trm patrol prior sites of viral replication, murine studies suggest they do not redistribute to adjacent uninfected sites to provide wider protection. We perform mathematical modeling of HSV-2 shedding in Homo sapiens and predict that infection does not induce enough Trm in many genital tract regions to eliminate shedding; a strict spatial distribution pattern of mucosal CD8+ T cell density is maintained throughout chronic infection, and trafficking of Trm across wide genital tract areas is unlikely. These predictions are confirmed with spatial analysis of CD8+ T cell distribution in histopathologic specimens from human genital biopsies. Further simulations predict that the key mechanistic correlate of protection following therapeutic HSV-2 vaccination would be an increase in total Trm rather than spatial reassortment of these cells. The fixed spatial structure of Trm induced by HSV-2 is sufficient for rapid elimination of infected cells but only in a portion of genital tract microregions.

Herpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding the global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen., Herpes simplex viruses (HSVs) are difficult to sequence due to their large DNA genome, high GC content, and the presence of repeats. To date, most HSV genomes have been recovered from culture isolates, raising concern that these genomes may not accurately represent circulating clinical strains. We report the development and validation of a DNA oligonucleotide hybridization panel to recover nearly complete HSV genomes at abundances up to 50,000-fold lower than previously reported. Using copy number information on herpesvirus and host DNA background via quantitative PCR, we developed a protocol for pooling for cost-effective recovery of more than 50 HSV-1 or HSV-2 genomes per MiSeq run. We demonstrate the ability to recover >99% of the HSV genome at >100× coverage in 72 h at viral loads that allow whole-genome recovery from latently infected ganglia. We also report a new computational pipeline for rapid HSV genome assembly and annotation. Using the above tools and a series of 17 HSV-1-positive clinical swabs sent to our laboratory for viral isolation, we show limited evolution of HSV-1 during viral isolation in human fibroblast cells compared to the original clinical samples. Our data indicate that previous studies using low-passage-number clinical isolates of herpes simplex viruses are reflective of the viral sequences present in the lesion and thus can be used in phylogenetic analyses. We also detect superinfection within a single sample with unrelated HSV-1 strains recovered from separate oral lesions in an immunosuppressed patient during a 2.5-week period, illustrating the power of direct-from-specimen sequencing of HSV. IMPORTANCE Herpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding the global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen.

Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae. IMPORTANCE Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.

Chronic viral infections remain a major public health issue affecting millions of people worldwide. Highly active antiviral treatments have significantly improved prognosis and infection-related morbidity and mortality, but have failed to eliminate persistent viral forms. Therefore, new strategies to either eradicate or control these viral reservoirs are paramount to allow patients to stop antiretroviral therapy and realize a cure. Viral genome disruption based on gene editing by programmable endonucleases is one promising curative gene therapy approach. Recent findings on RNA-guided Human immunodeficiency virus (HIV)-1 genome cleavage by Cas9 and other gene-editing enzymes in latently infected cells have shown high levels of site-specific genome disruption and potent inhibition of virus replication. However, HIV-1 can readily develop resistance to genome editing at a single antiviral target site. Current data suggests that cellular repair associated with DNA double stranded breaks (DSBs) can accelerate emergence of resistance. On the other hand, a combination antiviral target strategy can exploit the same repair mechanism to functionally cure HIV-1 infection in vitro while avoiding the development of resistance. This perspective summarizes recent findings on the biology of resistance to genome editing, and discusses the significance of viral genetic diversity on application of gene-editing strategies towards cure.

A large portion of the global population carries latent herpes simplex virus (HSV), which can periodically reactivate, resulting in asymptomatic shedding or formation of ulcerative lesions. Current anti-HSV drugs do not eliminate latent virus from sensory neurons where HSV resides, and therefore do not eliminate the risk of transmission or recurrent disease. Here, we report the ability of HSV-specific endonucleases to induce mutations of essential HSV genes both in cultured neurons and in latently infected mice. In neurons, viral genomes are susceptible to endonuclease-mediated mutagenesis, regardless of the time of treatment after HSV infection, suggesting that both HSV lytic and latent forms can be targeted. Mutagenesis frequency after endonuclease exposure can be increased nearly 2-fold by treatment with a histone deacetylase (HDAC) inhibitor. Using a mouse model of latent HSV infection, we demonstrate that a targeted endonuclease can be delivered to viral latency sites via an adeno-associated virus (AAV) vector, where it is able to induce mutation of latent HSV genomes. These data provide the first proof-of-principle to our knowledge for the use of a targeted endonuclease as an antiviral agent to treat an established latent viral infection in vivo.

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

Background Quantitative estimation of the extent to which the immune system’s protective effect against one herpes simplex virus type 2 (HSV-2) infection protects against infection with additional HSV-2 strains is important for understanding the potential for HSV-2 vaccine development. Using viral genotyping, we estimated the prevalence of HSV-2 dual-strain infection and identified risk factors. Methods and findings People with and without HIV infection participating in HSV-2 natural history studies (University of Washington Virology Research Clinic) and HIV prevention trials (HIV Prevention Trials Network 039 and Partners in Prevention HSV/HIV Transmission Study) in the US, Africa, and Peru with 2 genital specimens each containing ≥105 copies herpes simplex virus DNA/ml collected a median of 5 months apart (IQR: 2–11 months) were included. It is unlikely that 2 strains would be detected in the same sample simultaneously; therefore, 2 samples were required to detect dual-strain infection. We identified 85 HSV-2 SNPs that, in aggregate, could determine whether paired HSV-2 strains were the same or different with >90% probability. These SNPs were then used to create a customized high-throughput array-based genotyping assay. Participants were considered to be infected with more than 1 strain of HSV-2 if their samples differed by ≥5 SNPs between the paired samples, and dual-strain infection was confirmed using high-throughput sequencing (HTS). We genotyped pairs of genital specimens from 459 people; 213 (46%) were men, the median age was 34 years (IQR: 27–44), and 130 (28%) were HIV seropositive. Overall, 272 (59%) people were from the US, 59 (13%) were from Peru, and 128 (28%) were from 8 countries in Africa. Of the 459 people, 18 (3.9%) met the criteria for dual-strain infection. HTS and phylogenetic analysis of paired specimens confirmed shedding of 2 distinct HSV-2 strains collected at different times in 17 pairs, giving an estimated dual-strain infection prevalence of 3.7% (95% CI = 2.0%–5.4%). Paired samples with dual-strain infection differed by a median of 274 SNPs in the UL_US region (range 129–413). Matching our observed dual-strain infection frequency to simulated data of varying prevalences and allowing only 2 samples per person, we inferred the true prevalence of dual-strain infection to be 7%. In multivariable analysis, controlling for HIV status and continent of origin, people from Africa had a higher risk for dual-strain infection (risk ratio [RR] = 9.20, 95% CI = 2.05–41.32), as did people who were HIV seropositive (RR = 4.06, 95% CI = 1.42–11.56). Conclusions HSV-2 dual-strain infection was detected in 3.7% of paired samples from individual participants, and was more frequent among people with HIV infection. Simulations suggest that the true prevalence of dual-strain infection is 7%. Our data indicate that naturally occurring immunity to HSV-2 may be protective against infection with a second strain. This study is limited by the inability to determine the timing of acquisition of the second strain., In a nested cross-sectional study, Christine Johnston and colleagues examine the prevalence of dual-strain HSV-2 infection as a measure of immune system efficacy and potential for vaccine development., Author summary Why was this study done? There is a global need for an effective vaccine that prevents herpes simplex virus type 2 (HSV-2) infection. The authors asked: does naturally occurring immunity to HSV-2 infection prevent against infection with another HSV-2 strain (“dual-strain infection”)? Understanding how frequently HSV-2 dual-strain infection occurs and the risk factors for HSV-2 dual-strain infection is important for HSV-2 vaccine design. What did the researchers do and find? Two genital swabs containing HSV-2 were collected at 2 different time points from 459 participants enrolled in natural history studies and HIV prevention studies in the US, Peru, and Africa. These paired samples underwent a genotyping assay to determine if different HSV-2 strains were present in single individuals. For paired samples that appeared different at the 2 time points, high-throughput sequencing of the HSV-2 genome was performed to confirm the presence of HSV-2 dual-strain infection. HSV-2 dual-strain infection was found in 17 (3.7%) of 459 people. People with HIV infection had a 4-fold increased risk of dual-strain infection, and people from Africa had a 9-fold increased risk of dual-strain infection. What do these findings mean? In this study, HSV-2 dual-strain infection occurred infrequently, suggesting that naturally occurring HSV-2 immunity may be effective at preventing infection with a second strain. This study is limited by the inability to determine the timing of acquisition of the second strain and the inability to determine the clinical consequences of HSV-2 dual-strain infection. The genotyping assay could only be performed on samples with moderate levels of HSV-2 present, which may have led to underestimation of the frequency of dual-strain infection.

Background Most clinical and natural microbial communities live and evolve in spatially structured environments. When changes in environmental conditions trigger evolutionary responses, spatial structure can impact the types of adaptive response and the extent to which they spread. In particular, localized competition in a spatial landscape can lead to the emergence of a larger number of different adaptive trajectories than would be found in well-mixed populations. Our goal was to determine how two levels of spatial structure affect genomic diversity in a population and how this diversity is manifested spatially. Methodology/Principal Findings We serially transferred bacteriophage populations growing at high temperatures (40°C) on agar plates for 550 generations at two levels of spatial structure. The level of spatial structure was determined by whether the physical locations of the phage subsamples were preserved or disrupted at each passage to fresh bacterial host populations. When spatial structure of the phage populations was preserved, there was significantly greater diversity on a global scale with restricted and patchy distribution. When spatial structure was disrupted with passaging to fresh hosts, beneficial mutants were spread across the entire plate. This resulted in reduced diversity, possibly due to clonal interference as the most fit mutants entered into competition on a global scale. Almost all substitutions present at the end of the adaptation in the populations with disrupted spatial structure were also present in the populations with structure preserved. Conclusions/Significance Our results are consistent with the patchy nature of the spread of adaptive mutants in a spatial landscape. Spatial structure enhances diversity and slows fixation of beneficial mutants. This added diversity could be beneficial in fluctuating environments. We also connect observed substitutions and their effects on fitness to aspects of phage biology, and we provide evidence that some substitutions exclude each other.

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One newly emerging approach to cure persistent viral infections is via the use of targeted endonucleases. To date, endonucleases have been used as therapeutic agents targeting the genomes of viruses including EBV, JCV, HBV, HCV, HIV, HPV, HSV and HTLV, and have been shown to inhibit viral replication and subsequently persistence. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of a panel of four HIV pol-specific zinc finger nucleases (ZFNs) we identified a provirus encoding a treatment-resistant and infectious mutant virus that was derived from a ZFN2-mediated disruption of reverse transcriptase (RT). Although ZFN-mediated disruption of HIV protease (ZFN1), RT (ZFN2 & ZFN3) and integrase (ZFN4) coding sequences could inhibit viral replication, a RT mutant provirus was detected that produced a replication competent and ZFN2 cleavage resistant HIV. Mutant virus ZFN2(+3) contained a single amino acid insertion that introduced a Leucine-Leucine (LL) motif in the thumb domain of RT. The RT of mutant virus ZFN2(+3) likely remained functional since a LL motif is also found in the RT of several hominid, old world monkey or equine spumaviruses. In ZFN2-treated HEK293 and CD4+ SupT1 T cells, we found that up to 25% and 6% of all ZFN2 HIV pol target site mutations detected by Illumina sequencing encoded the ZFN2(+3) mutant respectively. We found that ZFN2(+3) mutant virus could replicate at levels comparable to wild type HIV in primary CD4+ T cells, but importantly remained susceptible to treatment with both NRTi and NNRTi antiretroviral inhibitors. When secondary ZFN-derived mutations were introduced into other RT or integrase domains of mutant virus ZFN2(+3), replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.