Histopathology and SARS-CoV-2 Cellular Localization in Eye Tissues of COVID-19 Autopsies.

Ophthalmic manifestations and tissue tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported in association with coronavirus disease 2019 (COVID-19), but the pathology and cellular localization of SARS-CoV-2 are not well characterized. The objective of this study was to evaluate macroscopic and microscopic changes and investigate cellular localization of SARS-CoV-2 across ocular tissues at autopsy. Ocular tissues were obtained from 25 patients with COVID-19 at autopsy. SARS-CoV-2 nucleocapsid gene RNA was previously quantified by droplet digital PCR from one eye. Herein, contralateral eyes from 21 patients were fixed in formalin and subject to histopathologic examination. Sections of the droplet digital PCR-positive eyes from four other patients were evaluated by in situ hybridization to determine the cellular localization of SARS-CoV-2 spike gene RNA. Histopathologic abnormalities, including cytoid bodies, vascular changes, and retinal edema, with minimal or no inflammation in ocular tissues were observed in all 21 cases evaluated. In situ hybridization localized SARS-CoV-2 RNA to neuronal cells of the retinal inner and outer layers, ganglion cells, corneal epithelia, scleral fibroblasts, and oligodendrocytes of the optic nerve. In conclusion, a range of common histopathologic alterations were identified within ocular tissue, and SARS-CoV-2 RNA was localized to multiple cell types. Further studies will be required to determine whether the alterations observed were caused by SARS-CoV-2 infection, the host immune response, and/or preexisting comorbidities.

UV-C Light Completely Blocks Aerosol Transmission of Highly Contagious SARS-CoV-2 Variants WA1 and Delta in Hamsters.

Behavioral and medical control measures have not been effective in containing the spread of SARS-CoV-2 in large part due to the unwillingness of populations to adhere to "best practices". Ultraviolet light with wavelengths of between 200 and 280 nm (UV-C) and, in particular, germicidal ultraviolet light, which refers to wavelengths around 254 nm, have the potential to unobtrusively reduce the risk of SARS-CoV-2 transmission in enclosed spaces. We investigated the effectiveness of a strategy using UV-C light to prevent airborne transmission of the virus in a hamster model. Treatment of environmental air with 254 nm UV-C light prevented transmission of SARS-CoV-2 between individuals in a model using highly susceptible Syrian golden hamsters. The prevention of transmission of SARS-CoV-2 in a natural system by treating elements of the surrounding environment is one more weapon in the arsenal to combat COVID. The results presented indicate that coupling mitigation strategies utilizing UV-C light, along with current methods to reduce transmission risk, have the potential to allow a return to normal indoor activities.

UV-C light completely blocks highly contagious Delta SARS-CoV-2 aerosol transmission in hamsters.

Behavioral and medical control measures are not effective in containing the spread of SARS-CoV-2. Here we report on the effectiveness of a preemptive environmental strategy using UV-C light to prevent airborne transmission of the virus in a hamster model and show that UV-C exposure completely prevents airborne transmission between individuals.

Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses.

Ambient temperature and humidity strongly affect inactivation rates of enveloped viruses, but a mechanistic, quantitative theory of these effects has been elusive. We measure the stability of SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities (RH); median estimated virus half-life is >24 hr at 10°C and 40% RH, but ∼1.5 hr at 27°C and 65% RH. Our mechanistic model uses fundamental chemistry to explain why inactivation rate increases with increased temperature and shows a U-shaped dependence on RH. The model accurately predicts existing measurements of five different human coronaviruses, suggesting that shared mechanisms may affect stability for many viruses. The results indicate scenarios of high transmission risk, point to mitigation strategies, and advance the mechanistic study of virus transmission.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

Heat-treated virus inactivation rate depends strongly on treatment procedure: illustration with SARS-CoV-2.

Decontamination helps limit environmental transmission of infectious agents. It is required for the safe re-use of contaminated medical, laboratory and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.00 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature temperature-dependent coronavirus stability and found that specimen containers, and whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines.

Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses.

Environmental conditions affect virus inactivation rate and transmission potential. Understanding those effects is critical for anticipating and mitigating epidemic spread. Ambient temperature and humidity strongly affect the inactivation rate of enveloped viruses, but a mechanistic, quantitative theory of those effects has been elusive. We measure the stability of the enveloped respiratory virus SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities; median estimated virus half-life is over 24 hours at 10 °C and 40 % RH, but approximately 1.5 hours at 27 °C and 65 % RH. Our mechanistic model uses simple chemistry to explain the increase in virus inactivation rate with increased temperature and the U-shaped dependence of inactivation rate on relative humidity. The model accurately predicts quantitative measurements from existing studies of five different human coronaviruses (including SARS-CoV-2), suggesting that shared mechanisms may determine environmental stability for many enveloped viruses. Our results indicate scenarios of particular transmission risk, point to pandemic mitigation strategies, and open new frontiers in the mechanistic study of virus transmission.

Clinical relevance of plasma virome dynamics in liver transplant recipients.

BACKGROUND: The role of the microbiome in liver transplantation (LT) outcome has received a growing interest in the past decades. In contrast to bacteria, the role of endogenous viral communities, known as the virome, is poorly described. Here, we applied a viral metagenomic approach to study the dynamic evolution of circulating viruses in the plasma of LT recipients and its effect on the clinical course of patients. METHODS: Patients chronically infected with hepatitis B virus (HBV) that received a LT due to endstage liver disease were included in this study. Longitudinal plasma samples were collected pre- and post-LT. Intact viral particles were isolated and sequenced on an Illumina HiSeq 2500 platform. Short read libraries were analysed with an in-house bioinformatics pipeline. Key endpoints were the dynamics of viral families and post-LT complications. FINDINGS: The initiation of immunosuppression induced a bloom of the Anelloviridae that dominated the post-LT plasma virome. A variety of post-LT complication were observed. Nephrotoxicity was reported in 38% of the patients and was associated with a high abundance of anelloviruses. Besides nephrotoxicity, 16 (67%) patients experienced flares of viral or bacterial infections in post-transplant follow-up. These flares were recognized by an increased burden of anelloviruses (p < 0.05). Interestingly, no mortality was observed in patients infected with human pegivirus. INTERPRETATION: These findings suggest a diagnostic potential for the Anelloviridae family in post-LT complications. Furthermore, the impact of human pegivirus infection on post-transplant survival should be further investigated. FUNDING: This trial was supported by Gilead Sciences grant number BE-2017-000133.

Persistence of SARS-CoV-2 in Water and Wastewater.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is frequently detected in the feces of infected individuals. While infectious SARS-CoV-2 has not previously been identified in wastewater, infectious SARS-CoV-2 has been isolated from the feces of at least one patient, raising concerns about the presence of infectious SARS-CoV-2 in wastewater. The fate and inactivation characteristics of SARS-CoV-2 in water and wastewater are unknown, with current inactivation estimates based on surrogate models. In this study, the persistence of SARS-CoV-2 infectivity and RNA signal was determined in water and wastewater. The times for 90% reduction (T90) of viable SARS-CoV-2 in wastewater and tap water at room temperature were 1.5 and 1.7 days, respectively. In high-starting titer (105 TCID50 mL-1) experiments, infectious virus persisted for the entire 7-day sampling time course. In wastewater at 50 and 70 °C, the observed T90 values for infectious SARS-CoV-2 were decreased to 15 and 2 min, respectively. SARS-CoV-2 RNA was found to be significantly more persistent than infectious SARS-CoV-2, indicating that the environmental detection of RNA alone does not substantiate risk of infection.

Whole Genome Analysis of Selected Human Group A Rotavirus Strains Revealed Evolution of DS-1-Like Single- and Double-Gene Reassortant Rotavirus Strains in Pakistan During 2015-2016.

Acute gastroenteritis due to group A rotaviruses (RVAs) is the leading cause of infant and childhood morbidity and mortality particularly in developing countries including Pakistan. In this study we have characterized the whole genomes of five RVA strains (PAK56, PAK419, PAK585, PAK622, and PAK663) using the Illumina HiSeq platform. The strains PAK56 and PAK622 exhibited a typical Wa-like genotype constellation (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, respectively), whereas PAK419, PAK585, and PAK663 exhibited distinct DS-1-like genotype constellations (G3P[4]-I2-R2-C2-M2-A2-N2-T1-E2-H2, G1P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and G3P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, respectively). Despite their DS-1-like genotype constellation, strain PAK585 possessed the typical Wa-like G1P[8] genotypes, whereas both PAK419 and PAK663 possessed the G3 genotype. In addition, PAK419 also possessed the Wa-like NSP3 genotype T1, suggesting that multiple reassortments have occurred. On Phylogenetic analysis, all of the gene segments of the five strains examined in this study were genetically related to globally circulating human G1, G2, G3, G6, G8, G9, and G12 strains. Interestingly, the NSP2 gene of strain PAK419 showed closest relationship with Indian bovine strain (India/HR/B91), suggesting the occurrence of reassortment between human and bovine RVA strains. Furthermore, strains PAK419, PAK585, and PAK663 were closely related to one another in most of their gene segments, indicating that these strains might have been derived from a common ancestor. To our knowledge this is the first whole genome-based molecular characterization of human rotavirus strains in Pakistan. The results of our study will enhance our existing knowledge on the diversity and evolutionary dynamics of novel RVA strains including DS-1-like intergenogroup reassortant strains spreading in Asian countries including Pakistan, in the pre-vaccine era. Therefore, continuous surveillance is recommended to monitor the evolution, spread and genetic stability of novel reassortant rotavirus strains derived from such events.

Stable distinct core eukaryotic viromes in different mosquito species from Guadeloupe, using single mosquito viral metagenomics.

BACKGROUND: Mosquitoes are the most important invertebrate viral vectors in humans and harbor a high diversity of understudied viruses, which has been shown in many mosquito virome studies in recent years. These studies generally performed metagenomics sequencing on pools of mosquitoes, without assessment of the viral diversity in individual mosquitoes. To address this issue, we applied our optimized viral metagenomics protocol (NetoVIR) to compare the virome of single and pooled Aedes aegypti and Culex quinquefasciatus mosquitoes collected from different locations in Guadeloupe, in 2016 and 2017. RESULTS: The total read number and viral reads proportion of samples containing a single mosquito have no significant difference compared with those of pools containing five mosquitoes, which proved the feasibility of using single mosquito for viral metagenomics. A comparative analysis of the virome revealed a higher abundance and more diverse eukaryotic virome in Aedes aegypti, whereas Culex quinquefasciatus harbors a richer and more diverse phageome. The majority of the identified eukaryotic viruses were mosquito-species specific. We further characterized the genomes of 11 novel eukaryotic viruses. Furthermore, qRT-PCR analyses of the six most abundant eukaryotic viruses indicated that the majority of individual mosquitoes were infected by several of the selected viruses with viral genome copies per mosquito ranging from 267 to 1.01 × 108 (median 7.5 × 106) for Ae. aegypti and 192 to 8.69 × 106 (median 4.87 × 104) for Cx. quinquefasciatus. Additionally, in Cx. quinquefasciatus, a number of phage contigs co-occurred with several marker genes of Wolbachia sp. strain wPip. CONCLUSIONS: We firstly demonstrate the feasibility to use single mosquito for viral metagenomics, which can provide much more precise virome profiles of mosquito populations. Interspecific comparisons show striking differences in abundance and diversity between the viromes of Ae. aegypti and Cx. quinquefasciatus. Those two mosquito species seem to have their own relatively stable "core eukaryotic virome", which might have important implications for the competence to transmit important medically relevant arboviruses. The presence of Wolbachia in Cx. quinquefasciatus might explain (1) the lower overall viral load compared to Ae. aegypti, (2) the identification of multiple unknown phage contigs, and (3) the difference in competence for important human pathogens. How these viruses, phages, and bacteria influence the physiology and vector competence of mosquito hosts warrants further research.

Molecular characterization of human group A rotavirus genotypes circulating in Rawalpindi, Islamabad, Pakistan during 2015-2016.

Group A rotaviruses (RVA) are one of the major causes of acute gastroenteritis (AGE) in young children worldwide. Owing to lack of proper surveillance programs and health facilities, developing countries of Asia and Africa carry a disproportionately heavy share of the RVA disease burden. The aim of this hospital-based study was to investigate the circulation of RVA genotypes in Rawalpindi and Islamabad, Pakistan in 2015 and 2016, prior to the implementation of RVA vaccine. 639 faecal samples collected from children under 10 years of age hospitalized with AGE were tested for RVA antigen by ELISA. Among 171 ELISA positive samples, 143 were successfully screened for RT-PCR and sequencing. The prevalence of RVA was found to be 26.8% with the highest frequency (34.9%) found among children of age group 6-11 months. The most predominant circulating genotypes were G3P[8] (22.4%) followed by G12P[6] (20.3%), G2P[4] (12.6%), G1P[8] (11.9%), G9P[6] (11.9%), G3P[4] (9.1%), G1P[6] (4.2%), G9P[8] (4.2%), and G3P[6] (0.7%). A single mixed genotype G1G3P[8] was also detected. The findings of this study provide baseline data, that will help to assess if future vaccination campaigns using currently available RVA vaccine will reduce RVA disease burden and instigate evolutionary changes in the overall RVA biology. The high prevalence of RVA infections in Pakistan require to improve and strengthen the surveillance and monitoring system for RVA. This will provide useful information for health authorities in planning public health care strategies to mitigate the disease burden caused by RVA.

Rotavirus: Genetics, pathogenesis and vaccine advances.

Since its discovery 40 years ago, rotavirus (RV) is considered to be a major cause of infant and childhood morbidity and mortality particularly in developing countries. Nearly every child in the world under 5 years of age is at the risk of RV infection. It is estimated that 90% of RV-associated mortalities occur in developing countries of Africa and Asia. Two live oral vaccines, RotaTeq (RV5, Merck) and Rotarix (RV1, GlaxoSmithKline) have been successfully deployed to scale down the disease burden in Europe and America, but they are less effective in Africa and Asia. In April 2009, the World Health Organization recommended the inclusion of RV vaccination in national immunization programs of all countries with great emphasis in developing countries. To date, 86 countries have included RV vaccines into their national immunization programs including 41 Global Alliance for Vaccines and Immunization eligible countries. The predominant RV genotypes circulating all over the world are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8], while G12[P6] and G12[P8] are emerging genotypes. On account of the segmented genome, RV shows an enormous genetic diversity that leads to the evolution of new genotypes that can influence the efficacy of current vaccines. The current need is for a global RV surveillance program to monitor the prevalence and antigenic variability of new genotypes to formulate future vaccine development planning. In this review, we will summarize the previous and recent insights into RV structure, classification, and epidemiology and current status of RV vaccination around the globe and will also cover the status of RV research and vaccine policy in Pakistan.

NetoVIR: Modular Approach to Customize Sample Preparation Procedures for Viral Metagenomics.

The democratization of next-generation sequencing (NGS) technologies has enabled scientists to explore the diversity of microbial life in various ecological niches in an unpreceded depth. The role of viruses as a key player in health and disease is becoming increasingly clear. To address the need for an up scalable, reproducible protocol to purify RNA and DNA viruses from a sample, we describe our optimized method. The Novel Enrichment Technique Of Viromes (NetoVIR) allows researchers to attain a fast, reproducible, and high-throughput sample preparation protocol for NGS gut viromics studies. With appropriate prior homogenization steps, this protocol can be extended to any biological samples.