Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing

Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3′ (10X 3′) and Chromium Next GEM Single Cell V(D)J (10X 5′) sequencing, we find that 10X 5′ with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

Proviral Turnover During Untreated HIV Infection Is Dynamic and Variable Between Hosts, Impacting Reservoir Composition on ART

Human immunodeficiency virus (HIV) can persist as an integrated provirus, in a transcriptionally repressed state, within infected cells. This small yet enduring pool of cellular reservoirs that harbor replication-competent HIV is the main barrier to cure. Entry of viral sequences into cellular reservoirs begins shortly after infection, and cells containing integrated proviral DNA are extremely stable once suppressive antiretroviral therapy (ART) is initiated. During untreated HIV infection however, reservoir turnover is likely to be more dynamic. Understanding these dynamics is important because the longevity of the persisting proviral pool during untreated infection dictates reservoir composition at ART initiation. If the persisting proviral pool turns over slowly pre-ART, then HIV sequences seeded into it during early infection would have a high likelihood of persisting for long periods. However, if pre-ART turnover was rapid, the persisting proviral pool would rapidly shift toward recently circulating HIV sequences. One-way to estimate this turnover rate is from the age distributions of proviruses sampled shortly after therapy initiation: this is because, at the time of sampling, the majority of proviral turnover would have already occurred prior to ART. Recently, methods to estimate a provirus’ age from its sequence have made this possible. Using data from 12 individuals with HIV subtype C for whom proviral ages had been determined phylogenetically, we estimated that the average proviral half-life during untreated infection was 0.78 (range 0.45–2.38) years, which is >15 times faster than that of proviral DNA during suppressive ART. We further show that proviral turnover during untreated infection correlates with both viral setpoint and rate of CD4+ T-cell decline during this period. Overall, our results support dynamic proviral turnover pre-ART in most individuals, which helps explain why many individuals’ reservoirs are skewed toward younger HIV sequences. Broadly, our findings are consistent with the notion that active viral replication creates an environment less favorable to proviral persistence, while viral suppression creates conditions more favorable to persistence, where ART stabilizes the proviral pool by dramatically slowing its rate of decay. Strategies to inhibit this stabilizing effect and/or to enhance reservoir turnover during ART could represent additional strategies to reduce the HIV reservoir.

Persistent viremia in an immunocompetent patient with inherited chromosomally integrated HHV-6B

Human herpesvirus-6 (HHV-6), the virus which causes roseola, has traditionally been associated with benign and self-limited childhood illness. However, HHV-6 establishes lifelong latency and can reactivate in immunocompromised adult patients. In about 1% of cases, it integrates into the human genome as inherited chromosomally integrated HHV-6 (iciHHV-6). We report the case of a 70-year-old man presenting with altered mental status and agitation. His infectious workup revealed a cerebrospinal fluid sample positive for HHV-6 with virus detectable in the blood as well. He was subsequently treated with ganciclovir. HHV-6 viremia (DNAemia) persisted, and the antiviral medications were switched to foscarnet under the assumption of treatment failure due to drug resistance. After several admissions to the hospital for the same complaint, and after noticing that DNAemia persisted despite adequate treatment for HHV-6, infectious disease specialists ordered testing for chromosomally integrated virus. Test results confirmed the presence of iciHHV-6, explaining his consistently elevated serum viral load. Primary HHV-6 infection in adults causes a transient increase in viral load with resolution and clearance after a few weeks while iciHHV-6 is characterized by persistent detection of viral DNA at a high copy number. Individuals with iciHHV-6 can develop HHV-6 disease and are at increased risk for active viral replication when treated with immunosuppressive medications, but only mRNA testing, which is not widely available can differentiate between latent and active infection. This makes the decision to treat challenging in this patient population. When faced with a positive HHV-6 DNA result in the setting of equivocal symptoms, clinicians should consider the possibility of chromosomally integrated virus rather than drug-resistant virus in order to reduce exposure to potentially toxic antiviral medications.

Timely Birth Dose Vaccine to Prevent Vertical Transmission of Hepatitis B: A Single Center Experience on the Road to the WHO Elimination Goals in Italy

Italy was one of the first industrialized countries to implement a program of routine vaccination against hepatitis B virus (HBV) infection. However, currently, no HBV vaccine is administered at birth if the screened mother is HBsAg negative, whilst babies born to HBsAg positive mothers are given vaccine and hepatitis B immunoglobulin, within 12–24 post-delivery hours. A single center retrospective analysis of policies and practices to prevent mother-to-child transmission of HBV was carried out, to evaluate their adherence to HBV care guidelines. Paired maternal-infant medical records for consecutive live births, between January 2015 and December 2019, were reviewed at the AOU Città della Salute e Scienza di Torino, where a total of 235/35,506 babies (0.7%) were born to HBsAg positive mothers. Markers of active viral replication, i.e., HBV DNA level and/or HBeAg, were reported in only 66/235 (28%) of the mothers’ medical records. All newborns had immunoprophylaxis at birth: 61% at <12 h, 31% between 12 and 24 h, 7% between 24 and 36 h and 1% at >36 h. In 2019, two cases of vertical HBV transmission occurred, despite timely immunoprophylaxis, as their mothers’ viral load was detected too late for antiviral prophylaxis. Missed early identification of pregnant women with high viremia levels or late vaccinations may contribute to perinatal HBV infection. Immunoprophylaxis should be given to babies born to HBsAg positive mothers at the latest within 12 h. In Italy, policies aimed at achieving the WHO 2030 goal of eliminating viral hepatitis should be further implemented.

Contribution to Herpesvirus Surveillance in Beaked Whales Stranded in the Canary Islands

Herpesviruses (HVs) (Alpha- and Gammaherpesvirinae subfamilies) have been detected in several species of cetaceans with different pathological implications. However, available information on their presence in beaked whales (BWs) is still scarce. In this study, a total of 55 BWs (35 Ziphius cavirostris and 20 animals belonging to the Mesoplodon genus) were analyzed. Samples (n = 294) were obtained from BWs stranded along the coasts of the Canary Islands (1990–2017). Molecular detection of HV was performed by means of a conventional nested PCR based on the DNA polymerase gene. Herpesvirus was detected in 14.45% (8/55) of the analyzed BWs, including 2 positive animals from a previous survey. A percentage positivity of 8.57% was found within the Cuvier’s BW group, while the percentage of positivity rose to 25% within the Mesoplodon genus group (three M. densirostris, one M. europaeus, and one M. bidens). All the obtained sequences from this study belonged to the Alphaherpesvirinae subfamily, from which three are considered novel sequences, all of them within the Mesoplodon genus group. In addition, to our knowledge, this is the first description of HV infection in Gervais’ and Sowerby’s BWs. Three out of eight HV-positive BWs displayed histopathological lesions indicative of active viral replication.

Massive Perivillous Fibrin Deposition and Chronic Histiocytic Intervillositis a Complication of SARS-CoV-2 Infection

An emerging complication of COVID-19 (SARS-CoV-2) infection is reported. A 23-year-old patient presented with high temperature and reduced fetal movements at 25 + 5/40 weeks of gestation. RT-PCR proved maternal COVID-19 infection. Ultrasound examination confirmed intrauterine death. Placenta histology showed necrosis of the villous trophoblast, associated with Chronic Histiocytic Intervillositis (CHI) and Massive Perivillous Fibrin Deposition (MPFD) with up to 90% - of the intervillous spaces being involved. Immunohistochemistry showed CD68 positive histiocytes in the intervillous spaces and the villous trophoblast was positive for the COVID-19 spike protein. RNA scope signal was indicative of the presence of the viral genome and active viral replication in the villous trophoblastic cells, respectively. MPFD is a gradually developing end-stage disease with various etiology, including autoimmune and alloimmune maternal response to antigens expressed at the feto-maternal interface and frequently accompanies chronic alloimmune villitis or histiocytic intervillositis. Covid-19 infection is associated with similar pattern of histological changes of the placenta leading to placental insufficiency and fetal death. This case report supports maternal- fetal vertical transmission of SARS-CoV-2 virus leading to placental insufficiency and fetal demise. MPFD and CHI appear to be the typical placental histology for SARS-CoV-2 virus infection associated fetal demise.

Frontline Screening for SARS-CoV-2 Infection at Emergency Department Admission by Third Generation Rapid Antigen Test: Can We Spare RT-qPCR?

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96–98), and a sensitivity of 85% (95% CI: 82–89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86–95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657–41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.

TLR4-Mediated Recognition of Mouse Polyomavirus Promotes Cancer-Associated Fibroblast-Like Phenotype and Cell Invasiveness

The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.

Central Nervous System (CNS) Viral Seeding by Mature Monocytes and Potential Therapies To Reduce CNS Viral Reservoirs in the cART Era

ABSTRACT The human immunodeficiency virus (HIV) enters the central nervous system (CNS) within a few days after primary infection, establishing viral reservoirs that persist even with combined antiretroviral therapy (cART). We show that monocytes from people living with HIV (PLWH) on suppressive cART harboring integrated HIV, viral mRNA, and/or viral proteins preferentially transmigrate across the blood-brain barrier (BBB) to CCL2 and are significantly enriched post-transmigration, and even more highly enriched posttransmigration than T cells with similar properties. Using HIV-infected ART-treated mature monocytes cultured in vitro, we recapitulate these findings and demonstrate that HIV+ CD14+ CD16+ ART-treated monocytes also preferentially transmigrate. Cenicriviroc and anti-JAM-A and anti-ALCAM antibodies significantly and preferentially reduce/block transmigration of HIV+ CD14+ CD16+ ART-treated monocytes. These findings highlight the importance of monocytes in CNS HIV reservoirs and suggest targets to eliminate their formation and reseeding. IMPORTANCE We characterized mechanisms of CNS viral reservoir establishment/replenishment using peripheral blood mononuclear cells (PBMC) of PLWH on cART and propose therapeutic targets to reduce/block selective entry of cells harboring HIV (HIV+) into the CNS. Using DNA/RNAscope, we show that CD14+ CD16+ monocytes with integrated HIV, transcriptionally active, and/or with active viral replication from PBMC of PLWH prescribed cART and virally suppressed, selectively transmigrate across a human BBB model. This is the first study to our knowledge demonstrating that monocytes from PLWH with HIV disease for approximately 22 years and with long-term documented suppression can still carry virus into the CNS that has potential to be reactivated and infectious. This selective entry into the CNS—and likely other tissues—indicates a mechanism of reservoir formation/reseeding in the cART era. Using blocking studies, we propose CCR2, JAM-A, and ALCAM as targets on HIV+ CD14+ CD16+ monocytes to reduce and/or prevent CNS reservoir replenishment and to treat HAND and other HIV-associated comorbidities.

Population Bottlenecks and Intra-host Evolution During Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.