Biorisk Management for SARS-CoV-2 Research in a Biosafety Level-3 Core Facility.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents hazards to researchers and other laboratory personnel in research settings where the live virus is stored and handled. The Biosafety Level-3 (BSL-3) Core Facility (CF) at Yong Loo Lin School of Medicine in National University of Singapore (NUS Medicine) has implemented a biorisk management (BRM) system to ensure that biorisk to employees, the public, or the environment are consistently minimized to an acceptable level while working with SARS-CoV-2. This chapter summarizes how a BRM system can be implemented in academic institutions based on international standards in the context of existing local legislations/regulations and institutional policies/guidelines to minimize the risk of laboratory-acquired infections and deliberate misuse of the newly emerged virus, SARS-CoV-2 in BSL-3 laboratories. The BRM programs prioritize performing risk assessments prior to implementation of work processes and reassessing the risk portfolio of the facilities from time to time, determining root causes and prevention of recurrences. Focusing on awareness-raising and educating the laboratory users in biosafety and biosecurity, and identifying opportunities for improvement are the other key factors for a sustainable and successful BRM system in the NUS Medicine BSL-3 CF.

Clinical features of cytomegalovirus anterior uveitis in immunocompetent patients.

PURPOSE: To describe the clinical presentation of cytomegalovirus (CMV) anterior uveitis in human immunodeficiency virus (HIV)-negative patients. DESIGN: Retrospective, interventional case series. METHODS: HIV-negative patients with anterior uveitis associated with elevated intraocular pressure (hypertensive anterior uveitis) seen at the Singapore National Eye Centre had their aqueous analyzed for viral deoxyribonucleic acid by polymerase chain reaction, and their records were reviewed for demographic data, ocular findings, laboratory results, and treatment. RESULTS: Aqueous was obtained from 105 of 106 eligible eyes. Twenty-four eyes demonstrated positive results for CMV (22.8%). Eighteen eyes had Posner-Schlossman syndrome (PSS; 75%) at presentation, five eyesba had Fuchs heterochromic iridocyclitis (FHI; 20.8%), and one eye had a presumed herpetic anterior uveitis. Twelve of the 24 eyes were treated with ganciclovir. Of the 12 who completed treatment, all responded clinically, and their aqueous demonstrated negative results for CMV on repeat testing. However, nine had recurrences within eight months of stopping treatment and required further courses of ganciclovir. The 81 CMV-negative eyes included 30 with PSS, 11 with FHI, 27 with uveitic glaucomas of unknown cause, and 13 with presumed herpetic anterior uveitis. CONCLUSIONS: CMV anterior uveitis is not uncommon in our immunocompetent patients and it may present as a recurrent acute or chronic inflammation, resembling PSS, herpetic anterior uveitis, or FHI.

Corneal endotheliitis associated with evidence of cytomegalovirus infection.

OBJECTIVE: To describe the clinical presentation of cytomegalovirus (CMV) corneal endotheliitis in human immunodeficiency virus (HIV)-negative patients. DESIGN: Retrospective interventional case series. PARTICIPANTS: Twelve consecutive patients with corneal endotheliitis diagnosed between 2002 and 2005. METHODS: Aqueous of eyes with corneal endotheliitis was analyzed for viral DNA by polymerase chain reaction (PCR), and patient records were reviewed for demographic data, medical and ocular history, best-corrected Snellen visual acuity, intraocular pressure (IOP), anterior and posterior segment findings, laboratory workup, diagnosis, and treatment. MAIN OUTCOME MEASURE: Presence of CMV DNA. RESULTS: Corneal endotheliitis was seen in 12 eyes of 10 patients during the study period. There were 8 men and 2 women, and all were Chinese. Their mean age was 49 years (range, 25-61 years). The corneal involvement ranged from small areas of focal endotheliitis to diffuse bullous keratopathy. The keratic precipitates had a variable appearance. There was only mild anterior chamber inflammation with no posterior synechiae. Two thirds of eyes had diffuse iris atrophy. All the eyes had elevated IOP. Eleven of the 12 eyes were positive for CMV DNA. None of the patients were positive for HIV. All patients had received local or systemic immunosuppression, or both, before corneal endotheliitis developed. Ten eyes of 8 patients were treated with systemic antiviral therapy. After treatment, the endotheliitis resolved completely in 7 eyes, and 3 eyes had significant improvement in corneal translucency. The IOP was normal, with no medications in all but 1 eye. Repeat PCR analysis in all the treated eyes was negative for CMV DNA. CONCLUSIONS: Cytomegalovirus infection is an important cause of corneal endotheliitis in our patients, and appropriate antiviral therapy may prevent more ocular damage.

Virus-specific RNA and antibody from convalescent-phase SARS patients discharged from hospital.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). In a longitudinal cross-sectional study, we determined the prevalence of virus in bodily excretions and time of seroconversion in discharged patients with SARS. Conjunctival, throat, stool, and urine specimens were collected weekly from 64 patients and tested for SARS-CoV RNA by real-time polymerase chain reaction; serum samples were collected weekly and tested for SARS-CoV antibody with indirect enzyme immunoassay and immunofluorescence assay. In total, 126 conjunctival, 124 throat swab, 116 stool, and 124 urine specimens were analyzed. Five patients had positive stool samples, collected in weeks 5-9. Two patients seroconverted in weeks 7 and 8; the others were seropositive at the first serum sample collection. In this study, 5 (7.8%) of 64 patients continued to shed viral RNA in stool samples only, for up to week 8 of illness. Most seroconversions occurred by week 6 of illness.

Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003.

BACKGROUND: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. METHODS: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. RESULTS: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 x 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. CONCLUSIONS: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3'--most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

Multicenter evaluation of the VERSANT hepatitis B virus DNA 3.0 assay.

The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 x 10(3) to 1.0 x 10(8) HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R(2) = 0.900; Digene, R(2) = 0.985; COBAS AMPLICOR, R(2) = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment.