Application evaluation of a rapid fluorescence quantitative PCR method for the detection of SARS-CoV-2 / 中华微生物学和免疫学杂志

Objective:To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE ? Flash20 real-time fluorescent quantitative PCR instrument. Methods:A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2, and the sensitivity and specificity of the system were verified. At the same time, 108 clinical samples of COVID-19 were used to evaluate the application of this method.Results:The detection method did not require nucleic acid extraction, and the manual operation time was only one minute. After the sample was sent to the system, the test could be completed in 30 minutes. The detection limit of this method was 4×10 2 copies/ml. It had no cross-reactivity with other human coronaviruses (including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) and other respiratory viruses. The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%. Conclusions:Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2, it was found that the method was simple, fast, specific and sensitive, and it was suitable for real-time and rapid detection needs in varieties of situations.

Serotypes of human rhinoviruses isolated from hospitalized children with severe acute respiratory in-fection in Shanghai during the autumn and winter of 2016-2017 / 中华微生物学和免疫学杂志

Objective To investigate the etiological characteristics of common viral respiratory tract infections and to analyze the distribution of human rhinovirus(HRV) serotypes in children with severe acute respiratory tract infection (SARI) in Shanghai. Methods Totally 199 nasopharyngeal aspirate speci-mens were collected from children with SARI in Shanghai from October 2016 to March 2017. A nuclear acid test was performed to detect 15 common respiratory viruses in these specimens. HRV strains were screened out using the primer pairs derived from the 5′UTR of HRV and the serotypes of them were identified based on the VP4-VP2 gene sequencing. Results Among the 199 specimens,HRV-positive specimens accounted for 26.1%,followed by those positive for influenza A(6.5%),adenovirus(6.5%),respiratory syncytial vi-rus(6.5%) and Boca virus(5%). Fifty-two HRV-positive specimens were typed by the VP4-VP2 gene se-quencing with 30 belonging to species A(18 serotypes,predominant serotypes:A21,A12,A38,A78,A88 and A96),seven belonging to species B (five serotypes, predominant serotype: B72) and 15 belonging to species C (nine serotypes,predominant serotypes:C27 and C40). There were two cases of HRV co-infec-tion. Two HRV-positive specimens could not be typed. HRV mixed serotype infections and co-infections of HRV with other viruses were existed. No significant difference in infection rates of different age groups and clinical characteristics was found between HRV-A and HRV-C infection groups. Conclusion HRV-A and HRV-C were the predominant pathogens causing SARI in children in Shanghai. Thirty-two HRV serotypes were detected and the predominant types were A21,A12,A38,A78,A88,A96,B72,C27 and C40.

Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue / 中华微生物学和免疫学杂志

Objective To investigate the feasibility of using recombinant infectious clones of hu-man coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids ( pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12. 9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12. 9 genes with the reporter gene en-coding the green fluorescent protein ( GFP ) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12. 9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12. 9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12. 9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43 FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors.

Sequencing and Phylogenetic Analyses of Structural and Accessory Proteins of Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China, 2015 / 病毒学报

To investigate the genetic character and origin of the first imported infection case of middle East respiratory syndrome coronavirus (named as MERS-CoV_China GD01), RNA was extracted from swabs of this patient followed by RT-PCR amplification. All coding gene of structural (S, E, M, E) and accessory (ORF3, ORF4a, ORF4b, ORF5, ORF8b) proteins were sequenced and analyzed. Phylogenetic analyses of structural protein coding genes of MERS-CoV_ China GD01 indicates that several substitutes exists in S coding gene and its origin belong group 5 of MERS-CoV, which were recent circulated in Saudi Arabia area, while other three structural genes (N, E, M) were very conserved. Phylogenetic analyses of accessory protein coding genes of MERS-CoV China GD01 indicates that several substitutes exists among ORF3, ORF4a, ORF4b and ORF5, while ORF8b was conserved. In conclusion, genome of MERS-CoV_ China GD01 was general conserved although several genetic variations were found among structural and accessory protein coding genes. This is the first report on sequencing and phylogenetic analyses of the first imported MERS case in China, which may pay the way for prevention and control of imported MERS-CoV infection.

A novel one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop an one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses (HCoVs).</p><p><b>METHODS</b>Genbank sequences of six human coronaviruses, including HCoV-NL63, HCoV-229E, SARS-CoV, HCoV-OC43, MERS-CoV, and HCoV-HKU1, were included as reference sequences. Primers were designed based on multiple alignment of reference sequences, targeting the conserved regions of each species of HcoV. Virus strains and nucleic acid preserved in our lab were used as template in developing this automatic-electrophoresis-based one-tube multiplex RT-PCR assay. Detection limits and reproducibility were also evaluated with these templates. Samples with infection of other respiratory viruses preserved in our lab were used to evaluate specificity of this assay. Finally, we tested this assay with 140 clinical samples that were validated by real-time PCR in parallel.</p><p><b>RESULTS</b>This automatic-electrophoresis-based multiplex RT-PCR assay was able to detect six human coronaviruses simultaneously. All positive samples in this study were detected with at least one specific fragment of anticipated length (195, 304, 332, 378, 415, 442 bp) . No fragment was detected in negative controls. Detection limits of 1.0×10(1-1.0)×10(2) copies/µl were achieved in tests of single virus. No cross reaction was observed with other respiratory viruses. This multiplex RT-PCR assay showed the same sensitivity and specificity to that of individual real-time RT-PCR validated with clinical samples. Both methods detected 28 positive samples (20%) .</p><p><b>CONCLUSIONS</b>Six HCoVs can be detected in one tube by this novel multiplex RT-PCR assay with high sensitivity and specificity.</p>

Characterization of complete genome sequences of human coronavirus NL63 strains derived from Chinese patients / 中华微生物学和免疫学杂志

Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .

Expression of IL-6 in recombinant non-replicating vaccinia virus and studying its effects on immunogenicity / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.</p><p><b>METHODS</b>The recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.</p><p><b>RESULTS</b>The recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.</p><p><b>CONCLUSIONS</b>IL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.</p>

CpG-ODN Enhances Anti-Tumor Effects of Non-Replicating Vaccinia Virus-Modeified Oncolysates / 中国肿瘤生物治疗杂志

Objective:To investigate the antitumor effect of CpG-ODN combining non-replicating recombinant vaccinia virus in tumor immunotherapy.Methods: CpG-ODN and constructed vaccinia virus v△11?75 were combined to study the antitumor effects in the walker′s rat tumor model.Results: Recombinant vaccinia virus v△11?75 lost the replicating capacity on human cell line,143TK - cell.The genes between HindⅢ C & K fragment of vaccinia virus TianTan strain were deleted,verified with Southern-blot. In the Walker′s tumor model of Wistar rats,Combinational immunotherapy with CpG-ODN and non-replicating vaccinia virus-modified WRC256 oncolysates resulted in prolonged life span and reduced tumor hyperplasia. Conclusion: CpG-ODN can enhance the antitumor effects of non-replicating vaccinia virus-modified oncolysates,providing a new route of tumor therapy.