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

Vega, Vinsensius B; Ruan, Yijun; Liu, Jianjun; Lee, Wah Heng; Wei, Chia Lin; Se-Thoe, Su Yun; Tang, Kin Fai; Zhang, Tao; Kolatkar, Prasanna R; Ooi, Eng Eong; Ling, Ai Ee; Stanton, Lawrence W; Long, Philip M; Liu, Edison T

SARS-CoV from the primary patient tissues were isolated by homogenizing the tissues in PBS buffer followed by a low speed centrifugation to obtain the viral particle containing supernatant. The virus-containing samples were also inoculated into Vero cell E6. The cells were maintained at 37°C using the usual viral cell culture media, and repassaged after 7 days of incubation. The virus-con-taining supernatants of homogenize or different passages of Vero cell E6 showing CPE were centrifuged at 23,000 RCF for 2.5 hours to pellet the viral particles and followed by RNA extraction using the QiAmp viral RNA mini kit (Qiagen, http://www.qiagen.com). The RNA genome templates were converted into double strand cDNA and sequenced as previously described [5] . The processing of raw sequence reads (base calling, assembly, and editing) was done using PHRED/PHRAP/CONSED (University of Washington, Seattle, WA, USA, http://www.phrap.org).