Epidemiologic investigations showed that 2 of 4 patients with severe acute

Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003–2004 were a waitress at a restaurant in Guangzhou China that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages. severe acute respiratory syndrome (SARS) epidemic emerged in 2003 in 6 municipalities in the Pearl River delta region in Guangdong China. Early case-patients were more likely to be persons with occupational exposure to animals such as animal sellers or restaurant cooks (1 2). Tracing the source of infection has been complicated given the sporadic nature of index cases without a clear history of contact with animals. After the World Health Organization (WHO) declared the end of the SARS epidemic 4 new cases of SARS were reported from December C 75 16 2003 to January 1 2004 in Guangzhou in Guangdong Province. These cases JTK13 were not linked to any laboratory accidents. All patients had a temperature >38°C radiographic evidence of pneumonia and serologic evidence of SARS infection. Fever lasted from 6 to 18 days (median 7) no mechanical ventilation was required and the clinical course of the disease ranged from 21 to 24 days with full recovery. All 4 patients had community-acquired infections without any apparent epidemiologic link. A total of 257 contacts including 113 close contacts of these patients were observed for 2 weeks with no secondary transmission identified. These patients had mild symptoms and no secondary transmission which was remarkably different from patients in the 2003 epidemic. Since potential reemergence of SARS leading to epidemic spread was possible identification of the infectious source was a high priority. The S gene sequence of SARS-associated coronavirus (SARS-CoV) isolated from 2 of these 4 patients was found to be closely related to the sequence of virus isolated from palm civets (3). However 1 of these patients reported no contact with palm civets or other animals in the preceding 2 months. The second patient was a 20-year-old waitress from a restaurant that served palm civets as food (4 5). Based on the virologic and epidemiologic findings provincial officials took aggressive action on January 5 2004 ordering a sweep through farms and C 75 food markets to destroy any animals that might harbor SARS-CoV. No additional SARS cases have since been reported. This information highlights the necessity for investigating restaurants as a possible source of infection understanding that the virus can be transmitted from animals or environmental sources to humans and clarifying the genetic basis of pathogenicity and infectivity of SARS-CoV from animal sources. Methods Specimen Collection Serial nasopharyngeal fecal and serum specimens of patients were collected at hospitals by Guangzhou Municipal Centers for Diseases Control and Prevention. When possible SARS was diagnosed C 75 in the waitress on January 2 2004 serum throat and rectal swabs were obtained from all 6 palm civets at the restaurant. It was reported that the animals were purchased from Xinyuan live animal wholesale market in Guangzhou. Serum samples from employees of the restaurant were C 75 obtained on January 4. Persons with positive results provided additional samples as needed. All specimens were stored at –80°C. Laboratory Diagnosis and Direct Sequencing of Primary Specimens Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) immunofluorescent antibody (IFA) test and Western blot for specific immunoglobulin G (IgG) and IgM. Nasopharyngeal throat and rectal specimens were tested by reverse transcription–polymerase chain reaction for polyprotein (P) and nucleocapsid (N) genes of SARS-CoV. Gene sequences were determined directly from original samples. RNA was transcribed into cDNA (SuperScript Invitrogen Carlsbad CA USA) and subsequently used for PCR amplification. Complete spike (S) gene and whole genome sequencing of SARS-CoV virus was conducted by using 48 primer sets based on the sequence data of a SARS-CoV SZ3 isolate from palm civet (6) and an ABI 3730 Genetic Analyzer (Applied Biosystems Foster City CA USA). Assembled genome sequences were compared with those of the first virus isolates of human (TOR2) and animal (SZ3) origin. Any nucleotide (nt) differences were double-checked and confirmed. Sequences from this study were deposited in GenBank (accession nos. {“type”:”entrez-nucleotide” attrs :{“text”:”AY572034″ term_id.