Beneficial and detrimental effects of regulatory T cells in neurotropic virus infections

Neurotropic viruses infect the central nervous system (CNS) and cause acute or chronic neurologic disabilities. Regulatory T cells (Treg) play a critical role for immune homeostasis, but may inhibit pathogen-specific immunity in infectious disorders. The present review summarizes the current knowledge about Treg in human CNS infections and their animal models. Besides dampening pathogen-induced immunopathology, Treg have the ability to facilitate protective responses by supporting effector T cell trafficking to the infection site and the development of resident memory T cells. Moreover, Treg can reduce virus replication by inducing apoptosis of infected macrophages and attenuate neurotoxic astrogliosis and pro-inflammatory microglial responses. By contrast, detrimental effects of Treg are caused by suppression of antiviral immunity, allowing for virus persistence and latency. Opposing disease outcomes following Treg manipulation in different models might be attributed to differences in technique and timing of intervention, infection route, genetic background, and the host’s age. In addition, mouse models of virus-induced demyelination revealed that Treg are able to reduce autoimmunity and immune-mediated CNS damage in a disease phase-dependent manner. Understanding the unique properties of Treg and their complex interplay with effector cells represents a prerequisite for the development of new therapeutic approaches in neurotropic virus infections

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease

Human rhinovirus infection blocks SARS-CoV-2 replication within the respiratory epithelium: implications for COVID-19 epidemiology

Virus-virus interactions influence the epidemiology of respiratory infections. However, the impact of viruses causing upper respiratory infections on SARS-CoV-2 replication and transmission is currently unknown. Human rhinoviruses cause the common cold and are the most prevalent respiratory viruses of humans. Interactions between rhinoviruses and co-circulating respiratory viruses have been shown to shape virus epidemiology at the individual host and population level. Here, we examined the replication kinetics of SARS-CoV-2 in the human respiratory epithelium in the presence or absence of rhinovirus. We show that human rhinovirus triggers an interferon response that blocks SARS-CoV-2 replication. Mathematical simulations show that this virus-virus interaction is likely to have a population-wide effect as an increasing prevalence of rhinovirus will reduce the number of new COVID-19 cases

Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice

Background: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3+ CD4+ regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. Methods: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3+ Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). Results: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. Conclusion: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS

Mendelian Randomisation and Causal Inference in Observational Epidemiology

Nuala Sheehan and colleagues describe how Mendelian randomization provides an alternative way of dealing with the problems of observational studies, especially confounding

Detection of SARS‐CoV‐2 in respiratory samples from cats in the UK associated with human‐to‐cat transmission

Objectives: The aim of the study was to find evidence of SARS‐CoV‐2 infection in UK cats. Design: Tissue samples were tested for SARS‐CoV‐2 antigen using immunofluorescence and for viral RNA by in situ hybridisation. A set of 387 oropharyngeal swabs that had been submitted for routine respiratory pathogen testing was tested for SARS‐CoV‐2 RNA using reverse transcriptase quantitative PCR. Results: Lung tissue collected post‐mortem from cat 1 tested positive for both SARS‐CoV‐2 nucleocapsid antigen and RNA. SARS‐CoV‐2 RNA was detected in an oropharyngeal swab collected from cat 2 that presented with rhinitis and conjunctivitis. High throughput sequencing of the viral genome revealed five single nucleotide polymorphisms (SNPs) compared to the nearest UK human SARS‐CoV‐2 sequence, and this human virus contained eight SNPs compared to the original Wuhan‐Hu‐1 reference sequence. An analysis of the viral genome of cat 2 together with nine other feline‐derived SARS‐CoV‐2 sequences from around the world revealed no shared cat‐specific mutations. Conclusions: These findings indicate that human‐to‐cat transmission of SARS‐CoV‐2 occurred during the COVID‐19 pandemic in the UK, with the infected cats developing mild or severe respiratory disease. Given the ability of the new coronavirus to infect different species, it will be important to monitor for human‐to‐cat, cat‐to‐cat and cat‐to‐human transmission

SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication

COVID-19, caused by SARS-CoV-2, is a respiratory disease associated with inflammation and endotheliitis. Mechanisms underling inflammatory processes are unclear, but angiotensin converting enzyme 2 (ACE2), the receptor which binds the spike protein of SARS-CoV-2 may be important. Here we investigated whether spike protein binding to ACE2 induces inflammation in endothelial cells and determined the role of ACE2 in this process. Human endothelial cells were exposed to SARS-CoV-2 spike protein, S1 subunit (rS1p) and pro-inflammatory signaling and inflammatory mediators assessed. ACE2 was modulated pharmacologically and by siRNA. Endothelial cells were also exposed to SARS-CoV-2. rSP1 increased production of IL-6, MCP-1, ICAM-1 and PAI-1, and induced NFkB activation via ACE2 in endothelial cells. rS1p increased microparticle formation, a functional marker of endothelial injury. ACE2 interacting proteins involved in inflammation and RNA biology were identified in rS1p-treated cells. Neither ACE2 expression nor ACE2 enzymatic function were affected by rSP1. Endothelial cells exposed to SARS-CoV-2 virus did not exhibit viral replication. We demonstrate that rSP1 induces endothelial inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication. We define a novel role for ACE2 in COVID-19- associated endotheliitis

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning

The dataset contains the raw data underpinning some of the figures in the research article. In addition, files needed to use algorithm necessary to identify alveolar epithelial hyperproliferation using the HALO software

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science