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While detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by diagnostic reverse-transcription polymerase chain reaction (RT-PCR) is highly sensitive for viral RNA, the nucleic acid amplification of subgenomic RNAs (sgRNAs) that are the product of viral replication may more accurately identify replication. We characterized the diagnostic RNA and sgRNA detection by RT-PCR from nasal swab samples collected daily by participants in postexposure prophylaxis or treatment studies for SARS-CoV-2. Among 1932 RT-PCR–positive swab samples with sgRNA tests, 40% (767) had detectable sgRNA. Above a diagnostic RNA viral load (VL) threshold of 5.1 log10 copies/mL, 96% of samples had detectable sgRNA with VLs that followed a linear trend. The trajectories of diagnostic RNA and sgRNA VLs differed, with 80% peaking on the same day but duration of sgRNA detection being shorter (8 vs 14 days). With a large sample of daily swab samples we provide comparative sgRNA kinetics and a diagnostic RNA threshold that correlates with replicating virus independent of symptoms or duration of illness.

Selection of a pre-clinical non-human primate (NHP) model is essential when evaluating therapeutic vaccine and treatment strategies for HIV. SIV and SHIV-infected NHPs exhibit a range of viral burdens, pathologies, and responses to combinatorial antiretroviral therapy (cART) regimens and the choice of the NHP model for AIDS could influence outcomes in studies investigating interventions. Previously, in rhesus macaques (RMs) we showed that maintenance of mucosal Th17/Treg homeostasis during SIV infection correlated with a better virological response to cART. Here, in RMs we compared viral kinetics and dysregulation of gut homeostasis, defined by T cell subset disruption, during highly pathogenic SIVΔB670 compared to SHIV-1157ipd3N4 infection. SHIV infection resulted in lower acute viremia and less disruption to gut CD4 T-cell homeostasis. Additionally, 24/24 SHIV-infected versus 10/19 SIV-infected animals had sustained viral suppression &lt, 100 copies/mL of plasma after 5 months of cART. Significantly, the more profound viral suppression during cART in a subset of SIV and all SHIV-infected RMs corresponded with less gut immune dysregulation during acute SIV/SHIV infection, defined by maintenance of the Th17/Treg ratio. These results highlight significant differences in viral control during cART and gut dysregulation in NHP AIDS models and suggest that selection of a model may impact the evaluation of candidate therapeutic interventions for HIV treatment and cure strategies.

Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-based “typing” assays. Here we describe a case of HSV mistyping caused by a previously undescribed HSV-1 × HSV-2 recombination event in UL27, the HSV gene that encodes glycoprotein B. This is the first documented case of HSV mistyping caused by an HSV-1 × HSV-2 recombination event and the first description of an HSV interspecies recombination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and probe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that about 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.

The recent outbreak of the novel coronavirus SARS-CoV-2, which causes COVID-19, can be diagnosed using RT-qPCR, but inadequate access to reagents and equipment has slowed disease detection and impeded efforts to mitigate viral spread. Alternative approaches based on combinations of isothermal amplification and CRISPR-mediated detection, such as the SHERLOCK (SpecificHighSensitivityEnzymaticReporter UnLOCKing) technique, offer reduced dependence on RT-qPCR equipment, but previously reported methods required multiple fluid handling steps, complicating their deployment outside clinical labs. Here we developed a simple test chemistry called STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use. This simplified test, STOPCovid, provides sensitivity comparable to RT-qPCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns result in 70 minutes, and using fluorescence readout, the test returns result in 40 minutes. Moreover, we validated STOPCovid using nasopharyngeal swabs from COVID-19 patients and were able to correctly diagnose 12 positive and 5 negative patients out of 3 replicates. We envision that implementation of STOPCovid will significantly aid “test-trace-isolate” efforts, especially in low-resource settings, which will be critical for long-term public health safety and effective reopening of the society.

Background NGU is common with no known etiology in ∼50% of cases. We evaluated the association of urethral bacteria with NGU among men who have sex with men (MSM) and men who have sex with women (MSW). Methods Urine samples were collected from MSM and MSW attending a Seattle STD Clinic and enrolled in a cross-sectional study. Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) were detected by TMA (Aptima), and adenovirus, HSV-1 and HSV-2 by PCR. NGU was defined as having urethral symptoms or visible discharge and ≥5 PMNs/high powered field (HPF). Absence of CT, MG, adenovirus, and HSV was considered as idiopathic NGU. Men without NGU had no urethral symptoms, no discharge, and Results Of 434 (199 MSM, 235 MSW) urine samples, 330 yielded sequence data. NGU+ men were less likely to yield sequence data (70% vs 84%, Fisher’s p=0.001). Of 328 men with ≥1000 sequence reads/sample, 95 MSM (44 NGU+) and 143 MSW (46 NGU+) were negative for CT, MG, adenovirus, and HSV. Higher relative abundances of Haemophilus influenzae (β=0.0139) and Mycoplasma penetrans (β=0.0095) were positively associated with idiopathic NGU in MSM, while H. influenzae was positively associated with idiopathic NGU in MSW (β=0.0184). The model also identified bacterial species that were negatively associated with NGU in MSM and MSW. Notably, Lactobacillus iners was negatively associated with idiopathic NGU in MSW (β=−0.0006) but not MSM. Conclusion Different bacterial species are associated with NGU in MSM and MSW. We identified two bacterial species infrequently detected in the male urethra as positively associated with NGU. Disclosure No significant relationships.

Background Genital HSV-1 has surpassed HSV-2 as a cause of first episode genital herpes in high-income settings. To inform counseling messages regarding prevention of genital HSV-1 transmission, we assessed oral and genital shedding patterns among persons with laboratory documented first episode genital HSV-1 infection. Methods Participants with virologic evidence of first episode genital HSV-1 infection self-collected oral and genital swabs for HSV PCR and completed symptom diaries for 30 days at 2 and 11 months after the first episode. Questionnaires about sexual practices were completed. Blood samples were collected at serial timepoints to assess antibody and cellular immune responses to HSV-1. HSV serostatus was determined using the HSV Western Blot, and those who were HSV seronegative at the time of enrollment had primary infection. The per-participant risk of oral and genital HSV-1 shedding during the first and second collection periods was determined. Results Of 62 participants who completed both swabbing sessions, 42 (68%) were women and 36 (58%) had primary HSV-1 infection. Of 54 who responded, 44 (81%) had a sex partner of the opposite gender and 43 (80%) had a single partner within 4 weeks prior to symptom onset. Genital HSV was detected on 205 (12.2%) of 1684 days at 2 months and declined significantly to 92 (5.5%) of 1668 days at 11 months (RR=0.45, 95% CI=0.24–0.85). On days when genital HSV was detected, the median quantity was higher at 11 months (4.2 log10 copies/ml) as compared to 2 months (3.2 log10 copies/ml), p Conclusion HSV-1 genital shedding is rapidly contained after the first year of genital HSV-1 infection. Genital HSV-1 shedding is relatively infrequent, but does persist, one year after first episode infection. Disclosure No significant relationships.

Among the most ubiquitous of human pathogens, HSV-1 and HSV-2 are distinct viral species that diverged about six million years ago. At least four ancient HSV-1 x HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and non-recombinants at each locus circulating today. Though interspecies recombination has occurred in the past, its importance in HSV evolution remains incompletely defined. Using 255 newly-sequenced and 219 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV. The novel recombinants we identify demonstrate that the sizes and locations of interspecies recombination events in HSV-2 are more variable than previously appreciated. One novel recombinant arose in its current host, showing for the first time that interspecies recombination occurs in contemporary HSV populations. We also demonstrate that interspecies recombination affects T-cell recognition of HSV. Our findings indicate that interspecies recombination can significantly influence genetic variation in and host immunologic response to HSV-2.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD., Allogeneic hematopoietic cell transplantation (allo-HCT) has led to the cure of HIV in one individual, but the underlying mechanisms are unclear. Here, the authors present a model of allo-HCT in SHIV-infected nonhuman primates and show that the SHIV reservoir persists in multiple tissues early after transplantation.

Background Human rhinovirus (HRV) is the most common virus detected in the respiratory tract following hematopoietic cell transplantation (HCT). Mortality following virologically proven lower respiratory tract infection (LRTI) may be up to 40%. Specific immune processes activated in upper respiratory tract infection (URTI) vs LRTI are not well understood. Methods Whole blood was collected in PAXgene tubes at the time of URTI or proven (HRV detected in the lower tract with radiographic changes) or possible (HRV detected in the upper tract with radiographic changes) LRTI. RNA was extracted and globin-reduced (PAXgene RNA kit; GlobinClear). Libraries were prepared with the TruSeq RNA Access kit (Illumina), and were pooled and sequenced (2 × 50bp) on an Illumina HiSeq 2500. Low quality reads were filtered prior to alignment (TopHat), and associated with genes (HTSeq). Normalization and significance testing were done using the Bioconductor package, "edgeR". Differential expression was defined as |log2 (ratio)| ≥ 1 (± 2-fold) with the FDR set to 5%. Gene ontology (GO) enrichment analysis of a concise set of biological process terms (DAVID GO Direct) was performed using the Bioconductor package, "goseq". Results Seventeen subjects with HRV infection following HCT were included (URTI, N = 7; proven LRTI without copathogens, N = 4; possible LRTI, N = 6). RNA was of sufficient quantity and quality to perform RNA-seq. We demonstrated differential gene expression between HRV URTI and proven LRTI (Figure 1A). Differential gene expression between HRV URTI and possible LRTI was not as robust (Figure 1B). Principle component analysis of the top 500 most variable genes demonstrated separation between subjects with HRV URTI and HRV proven LRTI (Figure 2). GO enrichment analyses demonstrated enrichment of terms associated with adaptive immune responses in the URTI group (Table). Conclusions HRV URTI and proven LRTI have highly differential gene expression patterns, whereas possible LRTI gene expression appears to more closely resemble that of URTI. Gene enrichment analysis demonstrates potential increased immune activation in the URTI subjects, which may lead to better control of infection. Larger studies are needed to verify these findings. Whole blood RNA-seq is feasible and may be a useful tool for demonstrating unique transcriptomic signatures for clinical phenotypes in HCT recipients and for identifying specific pathways in pathogenesis.

The United States Food and Drug Administration recently issued guidance that CMV DNAemia (quantitative CMV viral load as measured by PCR) could be used in a composite endpoint along with tissue-invasive CMV disease as the primary endpoint in allogeneic HCT CMV prophylaxis trials. However, the specific time points after antiviral treatment initiation at which to measure viral load and the viral load thresholds that most accurately predict clinical CMV disease have not been identified. In order to estimate the treatment effect of an antiviral agent on CMV clinical outcomes, placebo-controlled, randomized trial data with accompanying viral load measurements are needed. Unfortunately, in the modern era of PCR and early preemptive treatment, ethically, placebo-controlled, randomized trials that allow for the development of CMV disease after HCT can no longer be performed. To overcome this limitation, we performed CMV DNA PCR testing on frozen plasma samples collected during the first 100 days post-HCT in the only placebo-controlled, double-blind RCT of ganciclovir for the early treatment of CMV infection after HCT (Goodrich et al. NEJM 1991). We used three analytic methods (Cox proportional hazards models, machine learning, and a novel non-parametric method) to determine which of 14 viral load markers, measured in the first four weeks of treatment, are the most useful surrogates of antiviral treatment for the prevention of CMV disease. All methods identified peak viral load and percentage of positive samples by week 4 as highly associated with or predictive of tissue-invasive CMV disease by week 8 post-randomization. Cox proportional hazards models (Figure 1) yielded significant associations for peak viral load measured by week 4 (HR 1.4, 95% CI 1.1-1.9) and percentage of positive samples by week 4 (HR 1.6, CI 1.1-2.3). The machine-learning ensemble algorithm SuperLearner implemented in R (Figure 2) demonstrated cross-validated area under the receiver-operator curves of at least 75% for peak viral load (75%, CI 62-87%) and percentage of positive plasma samples (77%, CI 63-90%) measured by week 4. Using a novel statistical technique that allows direct estimation of biomarker surrogacy (Parast et al. Stat Med 2017), we estimated that the percentage of ganciclovir treatment effect explained by peak viral load and percentage of positive samples measured by week 4 was greater than 85%—peak viral load 89%, CI 56-100%; percent positive samples 91%, CI 60-100% (Figure 3). Peak viral load and percentage of positive samples by week 4 are highly predictive of CMV disease and the treatment effect of ganciclovir. Our analysis suggests that these may be the best surrogate markers of ganciclovir treatment and introduces novel methods for identifying optimal candidate biomarkers as surrogates for clinical endpoints.

Background Human herpesvirus 6 (HHV-6) is a recently described herpesvirus that is epidemiologically and biologically similar to cytomegalovirus. It is the cause of exanthem subitum (roseola) in children. Methods To evaluate the possible role of HHV-6 infection in pneumonitis in immunocompromised patients, we used quantitative HHV-6 polymerase chain reactions to study lung-biopsy specimens from 15 patients with pneumonitis after bone marrow transplantation and lung tissue from 15 immunocompetent subjects without pneumonitis and 6 fetuses. Results HHV-6 DNA was detected in lung tissue from all 15 patients, from 14 seropositive control subjects, and from none of the 7 seronegative control subjects. Six patients had levels of HHV-6 DNA in lung tissue that were 10 to 500 times higher than those in any of the other patients or control subjects. Increased levels of HHV-6 DNA correlated with a decreased risk of death from pneumonitis (P = 0.015), an increased severity of graft-versus-host disease (P = 0.023), an...

Background The association between prior seropositivity to human adenovirus (HAd) type 5 and increased HIV acquisition in the Step Vaccine Study has raised questions concerning the frequency of endemic HAd infection in adults. Few studies have documented the frequency and types of HAd infections in HIV-infected populations using sensitive molecular methods. In this study we retrospectively tested rectal swabs collected from a prospective cohort of Peruvian HIV-positive MSM for the presence of HAds.

Human herpesvirus 6 (HHV-6) DNA levels in peripheral blood mononuclear cells were prospectively evaluated in 20 cytomegalovirus-seronegative allogeneic marrow transplant patients and in 10 healthy control subjects. Blood and saliva specimens obtained weekly for 3 months after transplant were evaluated by quantitative HHV-6 polymerase chain reaction. One of 20 patients experienced primary HHV-6 infection after marrow transplant (seroconversion, HHV-6 viremia, skin rash); 18 of 20 had increased peripheral blood mononuclear cell HHV-6 DNA levels consistent with asymptomatic reactivations, and 1 patient experienced a reactivation-associated skin rash. Genotyping revealed HHV-6 variant B DNA in all cases. Therapy with acyclovir or intravenous immunoglobulin was not correlated with lower HHV-6 DNA levels. Thus, asymptomatic HHV-6 reactivations appear to be common following allogeneic marrow transplantation. Among HHV-6-seronegative and viral DNA-negative patients, primary HHV-6 infection can ensue in association with self-limited clinical symptoms, including diffuse maculopapular rash.

Human herpesvirus 6 (HHV-6) variant B is frequently identified in peripheral blood, but identification of HHV-6 variant A is relatively rare. We devised a PCR-based method for sensitive, simultaneous detection of both HHV-6 variants. The method was applied to 34 lung tissue specimens that were previously shown to contain HHV-6 DNA. A total of 22 lung tissue samples showed coinfections with HHV-6 variants A and B, 2 had only HHV-6 variant A DNA, and 10 had only HHV-6 variant B DNA. The prevalences of coinfections in lung tissues from healthy controls (54% coinfected) and in those from bone marrow transplant patients with pneumonia (67% coinfected) were similar. These data indicate that coinfections of HHV-6 variants A and B commonly occur in lung tissues of healthy and diseased individuals.

Human herpesvirus 6 (HHV-6) genome equivalents were quantitated in peripheral blood mononuclear cells (PBMCs) and saliva from 20 healthy individuals by the polymerase chain reaction (PCR). Nineteen of 20 subjects (95%) harbored HHV-6 DNA: 18 (90%) had HHV-6 in their PBMCs and 18 had HHV-6 in their saliva. Quantitative PCR revealed HHV-6 DNA levels ranging from negative to 4,000 HHV-6 genome equivalents per 10(6) PBMCs and from negative to 200,000 HHV-6 genome equivalents per ml of saliva. Longitudinal saliva samples from 15 HHV-6-seropositive subjects revealed salivary HHV-6 DNA persistence in 13 subjects. HHV-6 antibodies were detected in 17 of 19 subjects, with titers ranging from 1:400 to 1:51,200 (geometric mean titer, 1:2,500). Antibody titers did not correlate with HHV-6 DNA levels in PBMCs or saliva (P = 0.27 and P = 0.44, respectively). One subject with persistent HHV-6 DNA lacked detectable HHV-6 antibodies. The high prevalence of HHV-6 DNA in PBMCs and saliva supports the concept that HHV-6 exists at these sites in normal individuals.