Viral Culture in Hospitalized Congregate Care Patients With Prolonged SARS-CoV-2 Viral RNA Detection

Prolonged detection of SARS-CoV-2 viral RNA has been observed in hospitalized congregate care patients following resolution of clinical symptoms. It is unknown whether patients with persistent PCR positivity pose a risk for COVID-19 transmission. The purpose of this study was to examine the results of serial PCR testing, viral load, and viral culture in patients awaiting discharge prior to a negative PCR test. We sampled 14 patients who were admitted from skilled nursing and/or rehabilitation facilities to a large academic medical center, had clinical signs and symptoms of COVID-19, and had multiple PCR-positive tests separated by at least 14 days. PCR-positive nasopharyngeal swabs were obtained from each patient for viral load quantification and viral culture. The mean age of patients was 72.5 years (55 – 92), with a mean peak SOFA score of 5.6 (1 – 11). Patients were hospitalized for a mean of 37.0 days (25 – 60). RNA was detected by PCR for a mean of 32.9 days (19 – 47). Mean viral load for the first PCR-positive nasopharyngeal swab collected at our hospital was 5.81 genomic copies/mL (2.12 – 9.72). Viral load decreased significantly with days from clinical symptom onset (R = -0.69, 95% CI, -0.80 – -0.55). Four out of 28 samples grew active virus via culture, with no active virus isolates after 2 days of symptom onset. Our viral culture data suggests that persistent PCR positivity may not correlate with infectivity, which has important implications for COVID-19 infection control precautions among older congregate care patients.

Integration of SARS-CoV-2 RNA in infected human cells by retrotransposons: an unlikely hypothesis and old viral relationships

Zhang et al. (Proc Natl Acad Sci 118:e2105968118, 2021) recently reported that SARS-CoV-2 RNA can be retrotranscribed and integrated into the DNA of human cells by the L1 retrotransposon machinery. This phenomenon could cause persistence of viral sequences in patients and may explain the prolonged PCR-positivity of SARS-CoV-2 infected patients, even long after the phase of active virus replication has ended. This commentary does critically review the available data on this topic and discusses them in the context of findings made for other exogenous viruses and ancestral endogenous retroviral elements.

Developing a Biomimetic Evaluation Method for Antiviral Coatings Using Artificial Saliva Droplets.

Respiratory infections pose a serious threat worldwide, and many new antiviral agents and coatings have been developed to reduce the overall risk of viral infection. Here, we evaluate the methodology used to test these antiviral coatings and developed a novel system that is more similar to real-world conditions. Contact infection is largely mediated via contact with saliva containing the active virus released as droplets by coughing or sneezing, with these droplets adhering to objects and surfaces and subsequently entering the human body via indirect contact with the mucous membranes. Here, we evaluated the antiviral effect of a known antiviral coating agent using an artificial saliva based system, where artificial saliva containing phages were sprayed onto the antiviral coating under various conditions associated with viral replication and infectious spread. We used a commercially available antiviral coating in this evaluation, and M13 bacteriophages as model viruses. This method enables simple biomimetic evaluations of any product antiviral effects.

Active virus-host interactions at sub-freezing temperatures in Arctic peat soil

Background Winter carbon loss in northern ecosystems is estimated to be greater than the average growing season carbon uptake and is primarily driven by microbial decomposers. Viruses modulate microbial carbon cycling via induced mortality and metabolic controls, but it is unknown whether viruses are active under winter conditions (anoxic and sub-freezing temperatures). Results We used stable isotope probing (SIP) targeted metagenomics to reveal the genomic potential of active soil microbial populations under simulated winter conditions, with an emphasis on viruses and virus-host dynamics. Arctic peat soils from the Bonanza Creek Long-Term Ecological Research site in Alaska were incubated under sub-freezing anoxic conditions with H218O or natural abundance water for 184 and 370 days. We sequenced 23 SIP-metagenomes and measured carbon dioxide (CO2) efflux throughout the experiment. We identified 46 bacterial populations (spanning 9 phyla) and 243 viral populations that actively took up 18O in soil and respired CO2 throughout the incubation. Active bacterial populations represented only a small portion of the detected microbial community and were capable of fermentation and organic matter degradation. In contrast, active viral populations represented a large portion of the detected viral community and one third were linked to active bacterial populations. We identified 86 auxiliary metabolic genes and other environmentally relevant genes. The majority of these genes were carried by active viral populations and had diverse functions such as carbon utilization and scavenging that could provide their host with a fitness advantage for utilizing much-needed carbon sources or acquiring essential nutrients. Conclusions Overall, there was a stark difference in the identity and function of the active bacterial and viral community compared to the unlabeled community that would have been overlooked with a non-targeted standard metagenomic analysis. Our results illustrate that substantial active virus-host interactions occur in sub-freezing anoxic conditions and highlight viruses as a major community-structuring agent that likely modulates carbon loss in peat soils during winter, which may be pivotal for understanding the future fate of arctic soils' vast carbon stocks.

Natural Attenuation as a Decontamination Approach for SARS-CoV-2 on Library Materials

Based on the current scientific understanding regarding COVID-19, this article presents the results of new studies (Tests 4, 5, 6), conducted by the Institute of Museum and Library Services (IMLS) and the Online Computer Library Center (OCLC), in partnership with Battelle. The purpose of the experiments is to determine how long frequently circulated library, museum and archival materials should be quarantined before being put back into public circulation.Each test has been conducted by applying the virulent SARS-CoV-2 virus on various materials held at standard room temperature and relative humidity conditions, and then examining the rate of natural attenuation of the virus. The items were not sterilized before testing. Battelle propagated the clinical isolate of the SARS-CoV-2 virus in-house, followed by characterization and testing to establish a certified titer. The materials in Test 4 included binding and cover items placed in a stacked configuration to imitate the common practice that libraries employ when handling book/DVD returns, as well as expanded polyethylene foam, which is typically used in museums for storage and shipping. Test 5 focused on textiles, while Test 6 included building materials (glass, marble, brass, laminate, and powder-coated steel). Test coupons cut from the materials were inoculated with active virus, and then allowed to dry. The test coupons were then examined at the following timepoints: on the second, third, fourth and sixth day (Test 4); on the second, fourth, sixth and eighth day (Tests 5 and 6). The results of the tests showed how much virus was detectable at the selected timepoints. In Test 4, the virus was still detected on all the five materials after six days. In Test 5, the virus was still detected on leather and synthetic leather publication after eight days. In Test 6, the virus was not detected on the tested materials after six days.This publication is provided for informational purposes only, and readers are encouraged to review federal, regional, and local guidance. The authors, sponsors and researchers are not liable for any damages resulting from use, misuse, or reliance upon this information, or any errors or omissions herein.

Natural Attenuation as a Decontamination Approach for SARS-CoV-2 on Library Materials

Based on the current scientific understanding regarding COVID-19, this article presents the results of new studies (Tests 4, 5, 6), conducted by the Institute of Museum and Library Services (IMLS) and the Online Computer Library Center (OCLC), in partnership with Battelle. The purpose of the experiments is to determine how long frequently circulated library, museum and archival materials should be quarantined before being put back into public circulation.Each test has been conducted by applying the virulent SARS-CoV-2 virus on various materials held at standard room temperature and relative humidity conditions, and then examining the rate of natural attenuation of the virus. The items were not sterilized before testing. Battelle propagated the clinical isolate of the SARS-CoV-2 virus in-house, followed by characterization and testing to establish a certified titer. The materials in Test 4 included binding and cover items placed in a stacked configuration to imitate the common practice that libraries employ when handling book/DVD returns, as well as expanded polyethylene foam, which is typically used in museums for storage and shipping. Test 5 focused on textiles, while Test 6 included building materials (glass, marble, brass, laminate, and powder-coated steel). Test coupons cut from the materials were inoculated with active virus, and then allowed to dry. The test coupons were then examined at the following timepoints: on the second, third, fourth and sixth day (Test 4); on the second, fourth, sixth and eighth day (Tests 5 and 6). The results of the tests showed how much virus was detectable at the selected timepoints. In Test 4, the virus was still detected on all the five materials after six days. In Test 5, the virus was still detected on leather and synthetic leather publication after eight days. In Test 6, the virus was not detected on the tested materials after six days.This publication is provided for informational purposes only, and readers are encouraged to review federal, regional, and local guidance. The authors, sponsors and researchers are not liable for any damages resulting from use, misuse, or reliance upon this information, or any errors or omissions herein.

Eastern Equine Encephalitis Virus Rapidly Infects and Disseminates in the Brain and Spinal Cord of Infected Cynomolgus Macaques Following Aerosol Challenge

ABSTRACTEastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state of the art telemetry to measure critical physiological parameters. Following challenge, all parameters were rapidly and profoundly altered, and all nonhuman primates (NHPs) met the euthanasia criteria. In this study, we performed the first comprehensive pathology investigation of tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation of EEEV clinical disease course and pathogenesis in a NHP model and will provide significant insights into the evaluation of countermeasure.Author SummaryEEEV is an arbovirus endemic in parts of North America and is able to produce fatal encephalitis in humans and domesticated animals. Despite multiple human outbreaks during the last 80 years, there are still no therapeutic or vaccines to treat or prevent human disease. One critical obstacle in the development of effective countermeasure is the lack of insights into EEEV pathogenesis in a susceptible animal host. We recently conducted a study in cynomolgus macaques to investigate the disease course by measuring clinical parameters relevant to humans. Following infection, these parameters were rapidly and profoundly altered leading to severe disease. In this study, we examined the potential mechanisms that underlie pathogenesis to cause severe disease. The virus was present in many parts of the brain and spinal cord, however, little or no pathological lesions as well as active virus replication were observed. Additionally, neurons were the predominant target of EEEV and virus transport was facilitated by axonal transport system to spread neuron-to-neuron throughout the brain and spinal cord. These data show that EEEV likely hijacks host cell transport system to rapidly spread in the brain and local/global neuronal dysfunction rather than death is the principal cause of severe disease.

On the study of the dynamic model of COVID-19 in Wuhan

The current outbreak of coronavirus disease 2019 (COVID-19) has quickly spread across countries and become a global crisis. Understanding the transmission mechanism and effects of interventions is critical to the prevention and control of the COVID-19 pandemic. A recent study by Hao et al (2020) provided an interesting perspective on the transmission dynamics of COVID-19 in Wuhan and inferred that 87% of the infections before 8 March 2020 were not laboratory-confirmed. However we believe that there are a few major issues due to the vagueness in the definitions of compartments and inconsistence in the settings of parameters. In this paper, we clarify the definitions of the model compartments and raise questions in regard to the underlying homogenous assumption within compartments and settings of the parameters in the dynamic model by Hao et al (2020), and furthermore offer a modified model to resolve these potential limitations. Compared with the model in Hao et al (2020), the active virus carriers were predicted to persist for a longer period in our model which is well consistent with the active virus carriers detected in Wuhan in mid-May. Our model suggests that control measures cannot be easily lifted while continuous efforts are needed to contain the spread of the pandemic; a universal PT-PCR screening is essential to detect hidden cases before lifting control measure. In addition, we also provide a possible solution to solve the problem of heterogeneity transmission rate in disease courses.

On the study of full transmission dynamics of COVID-19 in Wuhan

The current outbreak of coronavirus disease 2019 (COVID-19) has quickly spread across countries and become a global crisis. Understanding the transmission mechanism and effects of interventions is critical to the prevention and control of the COVID-19 pandemic. A recent study by Hao et al (2020) provided an interesting perspective on the transmission dynamics of COVID-19 in Wuhan and inferred that 87% of the infections before 8 March 2020 were not laboratory-confirmed. However we believe that there are a few major issues due to the vagueness in the definitions of compartments and inconsistence in the settings of parameters. In this paper, we clarify the definitions of the model compartments and raise questions in regard to the underlying homogenous assumption within compartments and settings of the parameters in the dynamic model by Hao et al (2020), and furthermore offer a modified model to resolve these potential limitations. Compared with the model in Hao et al (2020), the active virus carriers were predicted to persist for a longer period in our model which is well consistent with the active virus carriers detected in Wuhan in mid-May. Our model suggests that control measures cannot be easily lifted while continuous efforts are needed to contain the spread of the pandemic; a universal PT-PCR screening is essential to detect hidden cases before lifting control measure. In addition, we also provide a possible solution to solve the problem of heterogeneity transmission rate in disease courses.

Rotavirus Infection and Cytopathogenesis in Human Biliary Organoids Potentially Recapitulate Biliary Atresia Development

ABSTRACT Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease. IMPORTANCE There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients.