Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection

Hepatitis E virus RNA detection in serum and feces specimens with the use of microspin columns

This report describes the use of microspin columns for extraction of hepatitis E virus (HEV) RNA from stool and serum specimens for reverse transcription-polymerase chain reaction (RT-PCR) and compares this method with the glass powder method. The microspin column method was found to be 1- to 2-log more sensitive in detecting HEV RNA than the glass powder method and had better reproducibility. The microspin column method also detected HEV RNA in a larger number of specimens than the glass powder method from among a panel of serum and stool specimens. Use of this method may allow better assessment of viremia and fecal excretion in patients and experimental animals infected with HEV

Genetic variability of hepatitis E virus within and between three epidemics in India

Hepatitis E virus (HEV) is an important cause of epidemic and sporadic acute viral hepatitis in many developing countries, including India. We evaluated the genetic variability within two regions (a 476-nt long ORF1 segment and a 304-nt long ORF2 segment) from specimens collected during three outbreaks in the cities of Karnal (1987), Yamunanagar (1989), and Meerut (1996), India, and from one patient, residing in Lucknow, India, who had a case of sporadic hepatitis (1996). Within an outbreak, sequences in the ORF1 and ORF2 regions were 99.3–100.0% identical. However, when strains were compared between outbreaks, identity in the ORF1 and ORF2 region was 97.1–99.2 and 96.4–100.0%, respectively. A comparison of these sequences to previously published Indian ORF1 and ORF2 sequences revealed even lower similarities, 95.2–98.5 and 95.1–98.7%, respectively. One patient in the Meerut outbreak had genomic sequences that differed substantially from the other patients affected during this outbreak and probably reflected a sporadic infection. The sporadic hepatitis E strain from Lucknow clustered with a previously described HEV strain from a patient with fulminant hepatic failure (FHF). Our data suggest that the ORF1 and ORF2 segments can be used to study the molecular epidemiology of HEV infection and indicate that much remains to be determined about the genetic variability of Indian HEV strains

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses