SARS-CoV2 RNA Research Articles

SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

Theoretically estimated risk of severe acute respiratory syndrome transmission through blood transfusion during an epidemic in Shenzhen, Guangdong, China in 2003

Background Severe acute respiratory syndrome (SARS) is a newly recognized infectious disease that caused an outbreak in south China in 2003. The cause of SARS was identified as a novel coronavirus (CoV). The existence of asymptomatic seroconvertors and the detection of the SARS-CoV RNA in plasma during the course of infection all suggest that SARS could, as least theoretically, be transmitted by transfusion. An estimate of the risk of SARS transmission through blood transfusion will contribute to decisions concerning blood safety monitoring and may be useful in the design of strategies to decrease the risk of transfusion-transmitted infections. Study design and methods Case onset dates from the 2003 Shenzhen SARS epidemic and investigational results from Taiwan on viremia in humans are used to estimate the number of cases that were viremic throughout the epidemic. Estimates of the asymptomatic-to-clinically confirmed SARS-CoV infection ratio, the proportion of asymptomatic infections reported in a seroprevalence survey in Hongkong, and the population size of Shenzhen are used to infer the SARS-CoV transfusion–transmission risk. Statistical resampling methods are used. Results Based on data from Shenzhen, Hongkong and Taiwan, the maximum and mean risk (per million) of SARS-CoV transmission from donors in Shenzhen were estimated as 23.57 (95% CI: 6.83–47.69) and 14.11 (95% CI: 11.00–17.22), respectively. The estimated risk peaked on April 02, 2003. Conclusions Although there are currently no confirmed reports of the transmission of SARS-CoV from asymptomatic individuals, recent research data indicate that transfusion-transmitted SARS-CoV is at least theoretically possible. Although the risk is low, with its rapid spread of the disease, appearance of alarmingly high infectivity and high fatality rate, public health authorities need to consider strategies for blood donor recruitment and virus inactivation during an epidemic to further ensure blood safety.

Pathology and Pathogenesis of Severe Acute Respiratory Syndrome

Severe acute respiratory syndrome (SARS) is an emerging infectious viral disease characterized by severe clinical manifestations of the lower respiratory tract. The pathogenesis of SARS is highly complex, with multiple factors leading to severe injury in the lungs and dissemination of the virus to several other organs. The SARS coronavirus targets the epithelial cells of the respiratory tract, resulting in diffuse alveolar damage. Several organs/cell types may be infected in the course of the illness, including mucosal cells of the intestines, tubular epithelial cells of the kidneys, neurons of the brain, and several types of immune cells, and certain organs may suffer from indirect injury. Extensive studies have provided a basic understanding of the pathogenesis of this disease. In this review we describe the most significant pathological features of SARS, explore the etiological factors causing these pathological changes, and discuss the major pathogenetic mechanisms. The latter include dysregulation of cytokines/chemokines, deficiencies in the innate immune response, direct infection of immune cells, direct viral cytopathic effects, down-regulation of lung protective angiotensin converting enzyme 2, autoimmunity, and genetic factors. It seems that both abnormal immune responses and injury to immune cells may be key factors in the pathogenesis of this new disease.

Follow up study on viruses associated with SARS among the SARS patients

To study the existence status of the SARS-CoV, retrovirus, and the poliovirus in the bodies of the patients with SARS and the possible relationship between the three viruses and SARS. The clinical specimens of the nasopharyngeal swabs, sputum (or saliva), urine, fecal specimens were collected on three consecutive days from 8 patients with SARS 2 years after the recovery from SARS. SARS-CoV, reovirus and poliovirus RNA was detected by using reverse transcription (RT)-PCR; IgG antibody to the poliovirus type 1 and 3 and the antibody to SARS-CoV were determined using enzyme linked immunosorbent assay (ELISA). All the specimens were negative for SARS-CoV and reovirus by RT-PCR, but the fecal specimens from 4 persons were positive for poliovirus. The sequences of these poliovirus were highly homologous to that of human poliovirus type 1 strain sabin 1 genome at nucleotide level, but back mutations have occurred in the primary attenuating mutation sites at nucleotide position 480 (G --> A) in the 5' UTR and the nucleotide position 2795 (A --> G). No SARS-CoV, reovirus, and poliovirus were found in the normal controls. Three serum specimens were positive for the antibody to SARS-CoV. The IgG antibody to poliovirus were detected in 4 SARS patients and 23 healthy persons. No positive results for antibody to SARS-CoV were detected in the 25 healthy persons. The positive rate of the poliovirus antibody in the serum of SARS patients 2 years after recovery was significantly different from that of the normal controls, and the positive rate of poliovirus in the fecal specimens was still very high, and more importantly back mutations have occurred in the attenuating mutation sites at nucleotide position which plays an important role in the poliomyelitis.

Pulmonary Tuberculosis and SARS, China

To the Editor: As part of a cohort study of 83 patients with severe acute respiratory syndrome (SARS) in Beijing, China, we conducted a follow-up study of all the patients by routine medical examination. During the process, 3 patients with chest radiographs consistent with active disease were identified as having pulmonary tuberculosis (TB). Here we describe the 1-year clinical outcome and immune response in these patients. Demographic details and coexisting conditions are shown in the Table. Patient 1 was a healthcare worker who became infected with SARS-associated coronavirus (CoV) while on duty with SARS patients. After he was transferred to a hospital dedicated to SARS management, pulmonary TB was diagnosed (positive acid-fast bacilli smear on sputum samples). Patients 2 and 3 were known to have cases of pulmonary TB and became infected with SARS-CoV after contact with other patients hospitalized for SARS. These 2 patients were sputum smear–negative for acid-fast bacilli, and diagnosis was made on the basis of previous exposure to TB, relevant symptoms of typical pulmonary TB, chest radiographs consistent with active disease, a positive tuberculin skin test result, and the finding of cavity regression on chest radiographs after anti-TB treatment was initiated. No cultures were obtained for isolation and comparison of Mycobacterium tuberculosis strains (1). All 3 patients had confirmed SARS based on amplification of SARS-CoV RNA by reverse transcriptase–polymerase chain reaction (RT-PCR) from sputum and stool specimens (2). Patients 2 and 3 recovered without complications; patient 1 had the most severe disease and required mechanical ventilation in an intensive care unit before recovering. Table Demographic and clinical information on severe acute respiratory syndrome (SARS) patients with pulmonary tuberculosis (TB), Beijing, China, 2003 Both cellular and humoral immunity were evaluated during the follow-up of these patients. T-lymphocyte subsets were measured 6 months after disease onset by flow cytometry using fluorescein isothiocyanate–labeled specific monoclonal antibodies. Compared to other SARS patients (n = 47), the 3 patients with TB had lower mean CD4+ T cells (368.4/μL vs. 656.6/μL, respectively; p = 0.05) and lower mean CD8+ T cells (371.0/μL vs. 490.1/μL, respectively; p = 0.39). SARS-CoV immunoglobulin G (IgG) antibody titers were measured by enzyme-linked immunosorbent assay kit (Huada Company, Beijing, China) at months 1, 2, 3, 4, 7, 10, and 16 after disease onset. (Titers were not measured for the 3 TB patients at month l.) Compared to most (26 [78.8%] of 33) other SARS patients whose antibodies remained detectable throughout follow-up, 2 of the 3 TB patients (patients 1 and 3) had undetectable antibody titers as of months 7 and 16, respectively. In patient 1, antibody titers, when detectable, were unusually low (40). Both patients 1 and 3 had prolonged viral excretion in stools, sputum, or both. While the median (range) duration of virus excretion in stools and sputa for the entire measurable cohort (n = 56) was 27 (16–127) and 21 (14–52) days, respectively (3), it was 125 and 16 days for patient 1, and 109 and 52 days for patient 3 (viral excretion data could not be obtained from patient 2 because sequential specimens for detection were unavailable). TB in SARS patients has been reported on rare occasions (4,5). In a cohort of 236 patients in Singapore, it was diagnosed in 2 patients after recovery from SARS (4). As with patient 1 in this study, TB had developed after the patient acquired SARS, most likely as the result of reactivation of past infection or new infection with M. tuberculosis, while temporarily immunosuppressed because of SARS (6) and corticoid therapy. Such phenomena have been described with other viral infections such as measles and HIV (7,8). By contrast, patients 2 and 3 were known TB patients who acquired SARS through exposure to SARS patients in the same hospital wards. Both diseases are known to be transiently immunosuppressive (6,9), and their combined effect resulted in more pronounced CD4+ cell decreases in coinfected SARS patients than others. Such immunosuppression also resulted in poorer IgG antibody response in coinfected SARS patients than in others and delayed viral clearance, as shown by longer viral excretion in sputum and stools. While viral excretion could be prolonged in coinfected patients, no virus could be isolated from any RT-PCR–positive specimen collected after 6 weeks of illness, which suggests that excreted viruses were no longer infectious (3). These case reports remind us of the importance of strict isolation of SARS patients, careful use of steroids for their case management, and the possibility of coinfection with TB in SARS patients with incomplete recovery.

Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

BackgroundWe have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay.MethodsAn automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision.ResultsThe newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method.ConclusionAs the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Laboratory detection on severe acute respiratory syndrome

目的 为严重急性呼吸综合征(SARS)病例的诊断提供科学依据.方法连续、系统地采集2003年12月至2004年1月广东省新发4例SARS怀疑病例不同发病时间的各类标本,采用酶联免疫吸附测定(ELISA)、免疫荧光测定(IFA)、中和试验等血清学检测技术和逆转录聚合酶链反应(RT-PCR)、荧光定量聚合酶链反应等分子生物学技术检测采集的标本,并对检测结果进行分析.结果血清学检测结果显示,4例病例在发病后6～8d,均可检测到SARS-CoV IgM和IgG抗体;前3例病例SARS-CoV抗体有4倍或4倍以上增长,第4例病例SARS-CoV IgG抗体虽无4倍增长,但却有明显的抗体阳转过程.采用ELISA、IFA和中和试验的检测结果基本一致.用荧光定量PCR检测咽拭等标本的SARS-CoV核酸,结果只有病例1的3份咽拭标本阳性,其他标本均阴性.对阳性标本,采用RT-PCR扩增出SARS-CoV的M、N、S基因并测序,S基因序列和种系发生分析提示,该病毒与从广东省果子狸中分离到的SARS毒株最接近.未能从这4例SARS病例的任何标本中分离到病毒.结论检测结果证明,该4例病例报告可以确认为SARS实验室确诊病例。

Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan hospital and the 309th Hospital of the Chinese People's Liberation Army

A worldwide outbreak of severe acute respiratory syndrome (SARS) had been reported. Over 8439 SARS cases and 812 SARS-related deaths were reported to the World Health Organization from 32 countries around the world up to 5 July 2003. The mechanism of transmission of SARS-CoV has been limited only to close contacts with patients. Attention was focused on possible transmission by the sewage system because laboratory studies showed that patients excreted coronavirus RNA in their stools in Amoy Gardens in Hong Kong. To explore whether the stool of SARS patients or the sewage containing the stool of patients would transmit SARS-CoV or not, we used a style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing, as well as cell culture, semi-nested RT-PCR and sequencing of genes to detect and identify the viruses from sewage. There was no live SARS-CoV detected in the sewage in these assays. The nucleic acid of SARS-CoV was found in the sewage before disinfection from both hospitals by PCR. After disinfection, SARS-CoV RNA could be detected from some samples from the 309th Hospital of the Chinese People's Liberation Army, but not from Xiao Tang Shan Hospital after disinfection. In this study, we found that the virus can survive for 14 days in sewage at 4 degrees C, 2 days at 20 degrees C, and its RNA can be detected for 8 days though the virus had been inactivated. In conclusion, this study demonstrates that the RNA of SARS-CoV could be detected from the concentrates of sewage of both hospitals receiving SARS patients before disinfection and occasionally after disinfection though there was no live SARS-CoV; thus much attention should be paid to the treatment of stools of patients and the sewage of hospitals receiving SARS patients.

Chemokine up-regulation in SARS-coronavirus–infected, monocyte-derived human dendritic cells

AbstractLymphopenia and increasing viral load in the first 10 days of severe acute respiratory syndrome (SARS) suggested immune evasion by SARS-coronavirus (CoV). In this study, we focused on dendritic cells (DCs) which play important roles in linking the innate and adaptive immunity. SARS-CoV was shown to infect both immature and mature human monocyte-derived DCs by electron microscopy and immunofluorescence. The detection of negative strands of SARS-CoV RNA in DCs suggested viral replication. However, no increase in viral RNA was observed. Using cytopathic assays, no increase in virus titer was detected in infected DCs and cell-culture supernatant, confirming that virus replication was incomplete. No induction of apoptosis or maturation was detected in SARS-CoV–infected DCs. The SARS-CoV–infected DCs showed low expression of antiviral cytokines (interferon α [IFN-α], IFN-β, IFN-γ, and interleukin 12p40 [IL-12p40]), moderate up-regulation of proinflammatory cytokines (tumor necrosis factor α [TNF-α] and IL-6) but significant up-regulation of inflammatory chemokines (macrophage inflammatory protein 1α [MIP-1α], regulated on activation normal T cell expressed and secreted [RANTES]), interferon-inducible protein of 10 kDa [IP-10], and monocyte chemoattractant protein 1 [MCP-1]). The lack of antiviral cytokine response against a background of intense chemokine up-regulation could represent a mechanism of immune evasion by SARS-CoV.

Effects of early corticosteroid treatment on plasma SARS-associated Coronavirus RNA concentrations in adult patients

Background:The effect of corticosteroid treatment on the viral load of Severe Acute Respiratory Syndrome (SARS) patients is unknown.Objective:To compare the plasma SARS-CoV RNA concentrations in ribavirin-treated patients who received early hydrocortisone therapy with those who received placebo.Study design:Serial plasma SARS-CoV RNA concentrations measured in the setting of a prospective, randomized double-blinded, placebo-controlled trial designed to assess the efficacy of “early” (<7 days of illness) hydrocortisone use in previously healthy SARS patients were analyzed. SARS-CoV RNA was quantified using a one-step real-time RT-PCR assay targeting the nucleocapsid gene.Results:Among 16 non-ICU cases, SARS-CoV RNA was detected in plasma since day 3–4 after fever onset; viral concentration peaked in the first week, which then rapidly declined in the second week of illness. On days 8, 12, 16, and 20, the cumulative proportion of patients with undetectable virus in plasma was 31%, 69%, 92%, and 100%, respectively. Plasma SARS-CoV RNA concentrations in the second and third week of illness were significantly higher in patients who received initial hydrocortisone treatment (n = 9), as compared to those who received placebo (n = 7)(AUC; Mann–Whitney, P = 0.023). The median time for SARS-CoV to become undetectable in plasma was 12 days (11–20 days) versus 8 days (8–15 days), respectively.Conclusion:Our findings suggested “early” corticosteroid treatment was associated with a higher subsequent plasma viral load.

Detection of RNA of SARS coronavirus in hospital sewage

In order to explore the existence of SARS coronavirus (Co-V) and/or its RNA in sewage of hospitals administered SARS patients. A novel electropositive filter was used to concentrate the SARS-CoV from the sewage of two hospitals administered SARS patients in Beijing, including twelve 2,500 ml sewage samples from the hospitals before disinfection, and ten 25,000 ml samples after disinfection; as well as cell culture, RT-PCR and sequencing of gene to detect and identify the viruses from sewage. There was no live SARS-CoV detected in the sewage in this study. The nucleic acid of SARS-CoV had been found in the 12 sewage samples before disinfection from both hospitals by semi-nested PCR. After disinfection, SARS-CoV RNA could only be detected from the samples from the 309th Hospital, and the others were negative. It provides evidence that there is no live SARS-Cov in the sewage from hospitals with SARS patients though SARS-CoV RNA can be detected.

Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection

Background & Aims : Severe acute respiratory syndrome (SARS) is a recently emerged infection from a novel coronavirus (CoV). Apart from fever and respiratory complications, gastrointestinal symptoms are frequently observed in patients with SARS but the significance remains undetermined. Herein, we describe the clinical, pathologic, and virologic features of the intestinal involvement of this new viral infection. Methods : A retrospective analysis of the gastrointestinal symptoms and other clinical parameters of the first 138 patients with confirmed SARS admitted for a major outbreak in Hong Kong in March 2003 was performed. Intestinal specimens were obtained by colonoscopy or postmortem examination to detect the presence of coronavirus by electron microscopy, virus culture, and reverse-transcription polymerase chain reaction. Results : Among these 138 patients with SARS, 28 (20.3%) presented with watery diarrhea and up to 38.4% of patients had symptoms of diarrhea during the course of illness. Diarrhea was more frequently observed during the first week of illness. The mean number of days with diarrhea was 3.7 ± 2.7, and most diarrhea was self-limiting. Intestinal biopsy specimens obtained by colonoscopy or autopsy showed minimal architectural disruption but the presence of active viral replication within both the small and large intestine. Coronavirus was also isolated by culture from these specimens, and SARS-CoV RNA can be detected in the stool of patients for more than 10 weeks after symptom onset. Conclusions : Diarrhea is a common presenting symptom of SARS. The intestinal tropism of the SARS-CoV has major implications on clinical presentation and viral transmission.

Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection.

Severe acute respiratory syndrome (SARS) is a recently emerged infection from a novel coronavirus (CoV). Apart from fever and respiratory complications, gastrointestinal symptoms are frequently observed in patients with SARS but the significance remains undetermined. Herein, we describe the clinical, pathologic, and virologic features of the intestinal involvement of this new viral infection. A retrospective analysis of the gastrointestinal symptoms and other clinical parameters of the first 138 patients with confirmed SARS admitted for a major outbreak in Hong Kong in March 2003 was performed. Intestinal specimens were obtained by colonoscopy or postmortem examination to detect the presence of coronavirus by electron microscopy, virus culture, and reverse-transcription polymerase chain reaction. Among these 138 patients with SARS, 28 (20.3%) presented with watery diarrhea and up to 38.4% of patients had symptoms of diarrhea during the course of illness. Diarrhea was more frequently observed during the first week of illness. The mean number of days with diarrhea was 3.7 +/- 2.7, and most diarrhea was self-limiting. Intestinal biopsy specimens obtained by colonoscopy or autopsy showed minimal architectural disruption but the presence of active viral replication within both the small and large intestine. Coronavirus was also isolated by culture from these specimens, and SARS-CoV RNA can be detected in the stool of patients for more than 10 weeks after symptom onset. Diarrhea is a common presenting symptom of SARS. The intestinal tropism of the SARS-CoV has major implications on clinical presentation and viral transmission.

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<h3>ABSTRACT</h3> The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal β-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.