The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from−45 to 1469, designated Ad-SN), which encoded a truncated Sprotein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-SN once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV Spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

We have previously reported that the subgroup C adenovirus E2 early (E2E) RNA polymerase 11 promoter can specify efficient in vitro transcription by RNA polymerase m. We now show that promoter proximal sequences of the E2E transcription unit are also transcribed by RNA polymerase Ill in nuclei isolated from adenovirusinfected cells. Small E2E RNA species that possessed the same properties as in vitro synthesized RNA polymerase m E2E transcripts were detected in cytoplasmic RNA populations from infected cells by using blotting, primer extension, and RNase protection assays. The 3' termini of these RNAs were mapped to thymidine-rich sequences typical of RNA polymerasemtermination sites. These results demonstrate that a single gene can be transcribed by both RNA polymerase II and RNA polymerase m in vivo.

To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402 xenografted tumors.

The immune spectrum of severe acute respiratory syndrome (SARS) is poorly understood. To define the dynamics of the immune spectrum in SARS, serum levels of cytokines, chemokines, immunoglobulins, complement and specific antibodies against SARS-associated coronavirus (SARS-CoV) were assayed by enzyme-linked immunosorbent assay (ELISA), and phenotypes of peripheral lymphocytes were analyzed by flow cytometry in 95 SARS-infected patients. Results showed that interleukin (IL)-10 and transforming growth factor β (TGF-β) were continuously up-regulated during the entirety of SARS. Regulated on activation normally Tcell-expressed and secreted (RANTES) levels were decreased, while monocyte chemoattractant protein-1 (MCP-1) was elevated in acute patients. Immunoglobulins and complement were elevated during the first month of SARS. Both serum-positive rates and titers of specific IgM and IgG antibodies responding to SARS-CoV peaked at days 41-60 from the onset of SARS. CD4+ and CD8+Tlymphocytes decreased significantly in acute-phase. CD3+CD8+CD45RO+Tlymphocytes were decreased by 36.78% in the convalescent patients. Conclusion: SARS-CoV seemed to elicit effective humoral immunity but inhibited cellular immunity, especially CD8+memory Tlymphocytes over time. Prolonged overproduction of IL-10 and TGF-β may play an important role in the disease.

The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase 11 in high salt nuclear extracts, but by RNA polymerase Ⅲ in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of a-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase Ill transcripts, unlike those synthesized by RNA polymerase 11, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase Ⅲ. Such RNA polymerase Ⅲ dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIIDdepleted extract to restore RNAp Ⅲ transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.

In adenovirus type 5-infected cells, RNA polymerase Ⅲ transcription of a gene superimposed on the 5 end of the E2E RNA polymerase Ⅱ transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91: 1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T1 protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase Ⅲ was found to be at least as efficient as that by RNA polymerase Ⅱ. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase Ⅱ and Ⅲ in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase Ⅱ transcriptional machinery in infected cells limits transcription by RNA polymerase Ⅲ, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase Ⅲ suggest that the function of this viral RNA polymerase Ⅲ transcription unit is unusual.