In addition, 64 of the 108 positive samples were amplified and sequenced for the S gene of SARS-CoV, and we confirmed that all of these samples contained S gene fragments of SARS-CoV [22]

In addition, 64 of the 108 positive samples were amplified and sequenced for the S gene of SARS-CoV, and we confirmed that all of these samples contained S gene fragments of SARS-CoV [22]. Syndrome Patient == Introduction == Severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease that has resulted globally in 774 deaths from 8,096 probable cases to date [1,2]. A novel coronavirus has been identified as the etiological agent of SARS and been designated as the SARS-associated coronavirus (SARS-CoV) [37]. SARS-CoV contains a single-stranded plus-sense RNA genome approximately 30 kb in length, which typically has five major open reading frames (ORFs) that encode the replicase (R) polyproteins, and the spike (S), the envelope (E), the membrane (M), and the nucleocapsid (N) proteins. SARS-CoV can infect African green monkey kidney (Vero E6) cells in Rabbit Polyclonal to RPL3 vitro and experimentally cause a similar disease in cynomolgus macaques (macaca fascicularis) [8]. It has been reported that around 90% (83.3100%) of patients with probable SARS generated antibodies against SARS-CoV [911], while the figure was negligible in a healthy population [12]. For early laboratory diagnostics, assays based on conventional nested and/or real-time reverse transcription-polymerase chain reactions (RT-PCR) have been developed to detect viral RNA in the clinical specimens of SARS patients [3,13,1416]. Even though the viral RNA and the antibody profiles in SARS patients have been well studied, the simultaneous detection of both profiles in sera or plasma has rarely been documented. In particular, there is a paucity of data concerning viral vitality in the excretions of SARS patients in the convalescent phase. In the 6-(γ,γ-Dimethylallylamino)purine study presented here viral RNA was detected in different types of clinical specimens. Dynamic profiles of SARS-CoV RNA in plasma and antibodies against SARS-CoV were simultaneously investigated in patients with probable SARS. The viral RNA-positive stool and urine samples from some patients were studied further for viral vitality. The results obtained further the available knowledge regarding the laboratory diagnostics and prophylactic control of this emerging infection. == Materials and methods == == Participants and specimens == A total of 486 clinical specimens from 54 patients with a clinical diagnosis of SARS were tested for the presence of both viral RNA and specific antibodies against SARS-CoV. The specimens included 213 plasma samples (isolated from citrate sodium-anticoagulated peripheral blood), 115 blood cell samples (remaining part of the blood from the plasma), 81 sputum/throat swab samples, 47 stool specimens, and 30 urine specimens. When possible, specimens were collected weekly. Moreover, 101 plasma specimens from an additional 19 patients were tested solely for the presence of SARS-CoV antibody. The patients were all hospitalized in our unit from March to June 2003, they all had a clinical diagnosis of SARS, and all of them received ribavirin/steroid combination therapy. In addition, plasma samples from 278 healthy subjects were analyzed for the presence of antibodies specific against SARS-CoV. All 6-(γ,γ-Dimethylallylamino)purine specimens were stored at 70C until tested. == RNA extraction == RNA extraction was performed in the biosafety level 3 (P3) laboratory at the Beijing 302 Hospital. RNA was extracted directly from plasma and urine samples. Sputum samples were shaken for 30 min with an equal volume of 1.0% acetylcysteine and 0.9% sodium chloride, and the supernatant was isolated by centrifugation (10,000g 3 min). Throat swabs and stool samples were suspended with phosphate-buffered saline (PBS) containing 10 U/ml RNasin (Promega, Madison, WI, USA), shaken for 10 min, and the supernatant was isolated by centrifugation as mentioned above. Blood cells (from 1.8 ml of blood) were treated with an equal volume of Trizol (Invitrogen, Carlsbad, CA, USA), followed by isolating the aqueous phase by centrifugation (12,000g 15 min). RNA was extracted using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. == Nested RT-PCR == Two groups of nested primers were used for amplifying the viral RNA. One set of BNI primers recommended by the Word Health Organization was used to target the R polyprotein encoding 6-(γ,γ-Dimethylallylamino)purine region (ORF1b) of SARS-CoV [17]. For this, the outer sense/antisense primers were 5-ATGAATTACCAAGTCAATGGTTAC-3/5-CATAACCAGTCGGTACAGCTA-3, and the inner sense/antisense primers were 5-GAAGCTATTCGTCACGTTCG-3/CTGTAGAAAATCCTAGCTGGAG-3, resulting in a 109-bp-long fragment complementary to nucleotide 18182-18290 of the BJ01 strain viral genome (GenBank accession no.AY278488). Another set of primers was used to target the N protein-encoding region of SARS-CoV designed using sequence analysis software, MacVector (Accelrys, San Diego, CA, USA). For this, the outer.