Sequence analysis for genes encoding nucleoprotein and envelope protein of a new human coronavirus NL63 identified from a pediatric patient in Beijing by bioinformatics / 病毒学报

The aim of this study was to characterize the N and E protein encoding genes of a new human coronavirus (HCoV-NL63) which was identified from one of the clinical specimens (BJ8081) collected from a 12 years-old patient with acute respiratory infection in Beijing. The complete N and E gene sequences of HCoV-NL63 were amplified from clinical sample by RT-PCR, then were cloned into the pCF-T and pUCm-T vectors respectively and sequenced. The complete sequences of N and E genes were submitted to GenBank by Sequin and compared with N and E genes of prototype HCoV-NL63 and the other coronaviruses published in GenBank. The secondary structure and the characteristics of sample BJ8081 N and E proteins were predicted by bioinformatics. It was indicated that the N and E genes amplified from sample BJ8081 were 1134 bp and 234 bp in length and the predicted proteins including 377 amino acids and 77 amino acids, respectively. The data suggested that the region of amino acids 78-85 within N protein probably was the conserved region for all coronaviruses identified so far including HCoV-NL63. The region of amino acids 15-37 for E protein was probably the transmembrane domain. In conclusion, the recombinant plasmids pCF-T-8081 N and pUCm-T-8081 E were successfully constructed and sequenced, and the data predicted by bioinformatics are helpful for the further analysis of HCoV-NL63.

Sequence analysis and prokaryotic expression of nucleocapsid protein genes of human respiratory syncytial viruses isolated from children in Beijing / 病毒学报

:To characterize nucleocapsid (N) protein genes of human respiratory syncytial viruses isolated from children in Beijing and express the N genes in E. coli,seven HRSV strains (three subtype A and four subtype B) were isolated from clinical samples of infants and children with acute respiratory infections and visited the Children's Hospital affiliated to Capital Institute of Pediatrics in Beijing during the period of Jan. 2006 to Mar. Full length of N genes from seven HRSV strains were amplified by reverse-transcription PCR (RT-PCR). The seven PCR amplicons were sequenced after cloning into pUCm-T and the sequences were compared with the N genes from HRSVs in GenBank. N gene was amplified from recombinant plasmid pUCm-N9968 by PCR and then sub-cloned into the prokaryotic expression vector pET30a(+) after digestion with EcoR I and Xho I . The pET30a-N9968 was transformed into E. coli BL21 (DE3) and expressed by inducing with IPTG. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. The amplified N genes were 1 176 bp in length and the deduced N proteins were 391 amino acids in length. The nucleotide identities of N genes among these seven strains were 85.4%-99.7% and the de-duced amino acid similarities were 95.4%-100%. The recombinant plasmid pET30a-N9968 had correct open reading frame confirmed by dual-enzyme digestion and sequence analysis. The fusion protein 6 x HisN was produced after inducing by 1 mmol/L IPTG at 37 degrees C. A unique protein band with molecular weight 49 kD was characterized by SDS-PAGE and purified by Ni2+ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein had specific binding reaction to specific monoclonal antibody and human sera, indicating that the expressed protein is of specific antigenicity.

Study on the association of hand, foot and mouth disease and enterovirus 71/CA16 among children in Beijing, 2007 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To reveal the etiological agent of hand, foot and mouth disease in children in Beijing.</p><p><b>METHODS</b>Throat swabs were collected from 6 infants and young children with hand, foot and mouth disease who visited the affiliated Children's Hospital from May to June 2007. Aspirated fluid from tracheal intubatton, serum and cerebral spinal fluid (CSF) were collected from a 9 years old girl (No.4243) having central neural system complication of severe hand, foot and mouth disease and admitted to the hospital from the Emergency Department. Throat swab and aspirated fluid were inoculated into the cell lines Hep-2, MDCK and Vero for virus isolation. RNAs were extracted by Trizol from 6 throat swab specimens and aspirated fluid, serum while CSF was from that severe case (No.4243). The gene fragment from 5' UTR of enterovirus was amplified from throat swabs and aspirated fluid by reverse transcription-polymerase chain reaction (RT-PCR) with the primer pairs located at the untranslated region of all enterovirus. EV71 was identified by RT-PCR with the 2 and half primer pairs located at different parts of VP1 gene of EV71. The PCR products for VP1 encoding gene of EV71 from the specimens were sequenced and sequence analysis was performed by comparing those published VP1 genes of EV71. EV71 and CA16 specific primers were used to identify the isolates by RT-PCR and the sequences were directly determined from PCR products.</p><p><b>RESULTS</b>Gene fragments with expected molecular weight were amplified from all 6 throat swabs and the aspirate by the primer pairs universal for the 5' UTR of enterovirus, suggesting that these patients with hand, foot and mouth disease were infected by entorovirus. Out of these 7 specimens, 2 throat swabs and the aspirate were also showing positive for the VP1 of the EV71 by different primer sets. Sequence analysis revealed that the sequences for the amplicons from 1 throat swab (No. F4211) and the aspirate shared highest homology with those published EV71, indicating that these specimens were truly positive for EV71. The sequences amplified from these specimens shared 100% and 98.9% homology to each other and were closer to the sequences of EV71 identified from Zhejiang province than those from Taiwan and strain BrCr. Gene fragments for 5' UTR of enterovirus were obtained by RT-PCR after CPE appeared in 6 out of 7 inoculations including that aspirate fluid in Vero cell, indicating that enteroviruses were isolated from these specimens. Virus isolates from one throat swab (No. F4211) and the aspirate (No. 4243) were positive by RT-PCR with the primer pairs for EV71, which was consistent with RT-PCR amplification directly from specimens. Virus isolates from other 4 specimens were CA16 by RT-PCR and sequence analysis.</p><p><b>CONCLUSION</b>These data suggested that hand, foot and mouth disease recently appeared in children in Beijing was related with EV71 and CA16. EV71 could cause severe clinical manifestations with central nerve system complications even in the child older than 5 years.</p>