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1、 0012-4966/04/0102- 2004 MAIK “Nauka/Interperiodica”0058 Doklady Biological Sciences, Vol. 394, 2004, pp. 5860. Translated from Doklady Akademii Nauk, Vol. 394, No. 4, 2004, pp. 566568. Original Russian Text Copyright 2004 by Agafonov, Guskov, Ternovoi, Ryabchikova, Durymanov, Vinogradov, Maksimov,
2、Ignatev, Nechaeva, Netesov. Severe acute respiratory syndrome (SARS) or atyp- ical pneumonia was fi rst diagnosed in Guangdong Province (China) in November 2002. According to WHO data, patients with atypical pneumonia appeared in 29 countries throughout the world by mid-2003. By June 2003, the total
3、 number of affected persons reached 8435, including 789 lethal cases 1. The SARS caus- ative agent was found to be a virus from genus Coro- navirus of the Coronaviridae family 2. Members of genus Coronavirus have been known for a long time. These are enveloped, positive-stranded RNA viruses that cau
4、se human and animal diseases 3, 4. However, such a severe coronavirus infection have been previ- ously extremely rare in humans. Because of the high risk of atypical pneumonia epidemic, the properties of SARS virus, a new member of this genus, are being studied extensively 58. Our study was the fi r
5、st to analyze the basic proper- ties of SARS virus, which permitted the creation of an original diagnostic test system based on RTPCR and development of the classical RTHA diagnosticum, a “gold standard” for many infections. The new data on the virus culture properties make it possible to develop at
6、tenuated and inactivated vaccines, as well as to screen medicinal preparations. The strain Frankfurt I of SARS virus was kindly provided by Dr. H. W. Doerr (Institute of Medical Virology, Frankfurt am Main, Germany). To study spe- cifi c reproduction of SARS virus, it was cultivated in the transpant
7、able cell lines Vero, 293, and 4647, as well as in the diploid cell line L-68 at a multiplicity of inoc- ulation of 1 : 10. Changes in the monolayer depended on the cell line. In the cultures of Vero and 4647, the cells became round-shaped, and the monolayer slipped down on the second and third days
8、 after the inoculation, respectively. Similar cell damage has was described previously, when coronavirus was isolated from a colt 9. The virus cultivation in cells 293 and L-68 led to changes in the monolayer on the third day, specifi cally, the disruption of the monolayer because of local cell lysi
9、s. Virus reproduction in cells was monitored by the RTPCR method. Amplifi cation was conducted as described previously 7 using the BNIinS and BNIi- nAs primers developed in the Institute of Tropical Med- icine (Hamburg, Germany) 7. The samples taken from all passages of the cells studied were found
10、to contain the SARS-virus RNA. Electron-microscopic examination revealed coro- navirus reproduction in cultures of the Vero and 293 lines on the second and third days after inoculation, respectively. The endoplasmic reticulum of the infected cells contained particles with a morphology typical of cor
11、onavirus: long surface projections, a coiled nucle- oid, and an electron-transparent core (Fig. 1a). Virion accumulation was also observed in the perinuclear space and smooth-membrane vacuoles. In cells of the Vero culture, the content of viral particles was signifi - cantly higher than in cells of
12、line 293. In the culture medium, spherical viral particles 8090 nm in size with specifi c surface projections forming a “crown” were revealed by negative staining (Figs. 1b, 1c). Thus, SARS virus morphology was typical of coronavirus in both ultrathin sections and suspension 10. After cultivation of
13、 SARS virus, virus-containing culture medium (VCCM) was collected, and virus titer was determined on the basis of the cytopathological effect on the Vero cell monolayer. The results shown in the table suggest that the SARS virus reproduction in Vero cells was more effi cient than in cells 293, L-68,
14、 and 4647. The rate of virus reproduction in Vero cells was also higher. The cell cultures L-68 and 4647 are approved for the use as substrate cultures for manufac- turing vaccine preparations; line 293 is currently under- going evaluation at the Tarasevich Institute of Certifi ca- tion and Control.
15、 None of the cultures used in this study contained contaminating viruses, mycoplasmas, and bacteria, and were not tumorigenic. Species affi liation of these cultures was confi rmed by karyological analy- sis. Nevertheless, virus adaptation to these cell cultures, selection for nutrient media and cul
16、tivation conditions are required to develop an anti-SARS vaccine. Coronaviruses are known to contain the protein esterase-hemagglutinin, which gives the virus the abil- GENERAL BIOLOGY Primary Characterization of SARS Coronavirus Strain Frankfurt 1 A. P. Agafonov, A. A. Guskov, V. A. Ternovoi, E. I. Ryabchikova, A. G. Durymanov, I. V. Vinogradov, N. L. Maksimov, G. M. Ignatev, E. A. Nechaeva, and Corresponding Member of RAS S. V. Netesov Received August 7, 2003 Vecto
