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BackgroundCOVID-19 pandemic is underway. Some COVID-19 cases re-tested positive for SARS-CoV-2 RNA after discharge raising the public concern on their infectivity. Characterization of re-positive cases are urgently needed for designing intervention strategies. MethodsClinical data were obtained through Guangdong COVID-19 surveillance network. Neutralization antibody titre was determined using a microneutralization assay. Potential infectivity of clinical samples was evaluated after the cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. ResultsAmong 619 discharged COVID-19 cases, 87 were re-tested as SARS-CoV-2 positive in circumstance of social isolation. All re-positive cases had mild or moderate symptoms in initial diagnosis and a younger age distribution (mean, 30.4). Re-positive cases (n=59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n=150) parallel-tested in this study. No infective viral strain could be obtained by culture and none full-length viral genomes could be sequenced for all re-positive cases. ConclusionsRe-positive SARS-CoV-2 was not caused by the secondary infection and was identified in around 14% of discharged cases. A robust Nabs response and a potential virus genome degradation were detected from nearly all re-positive cases suggesting a lower transmission risk, especially through a respiratory route.
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COVID-19 is caused by the SARS-CoV-2 coronavirus and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, Chinas most populous province, during early 2020 resulted in 1,388 reported RNA positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain due to low virus genetic variation early in the pandemic. Our results illustrate how the timing, size and duration of putative local transmission chains were constrained by national travel restrictions and by the provinces large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required as the number of cases imported from other countries is increasing. HighlightsO_LI1.6 million molecular diagnostic tests identified 1,388 SARS-CoV-2 infections in Guangdong Province, China, by 19th March 2020 C_LIO_LIVirus genomes can be recovered using a variety of sequencing approaches from a range of patient samples. C_LIO_LIGenomic analyses reveal multiple virus importations into Guangdong Province, resulting in genetically distinct clusters that require careful interpretation. C_LIO_LILarge-scale epidemiological surveillance and intervention measures were effective in interrupting community transmission in Guangdong C_LI
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Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.
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Objective@#To develop a micro-neutralization test for determination of neutralizing antibody against ZIKA virus (ZIKV) in human sera and to verify the acute and convalescent serum samples of 10 ZIKA virus-infected cases diagnosed by nucleic acid detection and/or virus isolation.@*Methods@#ZIKV isolated from ZIKA cases was used to determine micro-neutralization antibody. The virus solution was prepared by infecting BHK21, VERO and VERO-E6 cell lines and viral titer was tested; 100 TCID50 viral solution and 4 times diluted sera which were inactivated at 56 ℃ for 30 min were neutralized, then added the cell suspension and incubated in 5% CO2 incubator at 37 ℃ for 7 d. The CPE was observed every day.@*Results@#The sensitivity of BHK21, VERO and VERO-E6 was different after infection with ZIKA virus. VERO cell line was the most sensitive and showed typical CPE. VERO cell line was used to establish a micro-neutralization test for determination of neutralizing antibody against ZIKA virus in sera.@*Conclusions@#The neutralizing antibody test for zika virus in sera is a special and usefulmethod to diagnose human infection of ZIKV and to conduct population based epidemiological investigation.
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Objective To establish a method for the isolation of Zika virus and to gather experi-ences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples posi-tive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nu-cleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The se-quences of NS5 gene were analyzed by using MEGA6. 0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle thresh-old (Ct) values of 24. 4 and 25. 3, respectively. The second generation of Zika virus was identified in suck-ling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus ( GDZ16002 strain) be-longed to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage.
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Objective To provide scientific evidences for Zika virus detection by clarifying the means by which Zika virus was discharged and the duration of corresponding processes. Methods Various samples of Zika cases were collected at different times and detected by using real-time RT-PCR. The positive samples were inoculated into cells and suckling mice through intracranial injection. The whole genome se-quences of those isolated Zika virus strain were sequenced and the results were further analyzed by comparing with the sequences of Zika virus from GenBank. Results The positive rates of Zika virus in urine, saliva and serum samples were 82. 4% (14/17), 82. 4% (14/17) and 52. 9% (9/17) respectively. The longest period of detected presence of Zika virus was found in urine samples amongst the three types of samples, fol-lowed by saliva and serum samples. Six Zika virus strains were isolated from 9 positive serum samples. Phy-logenetic analysis showed that the six genomes of Zika virus all belonged to Asia lineage, but located in two branches by Samoa and Venezuela strains. Conclusion This study indicated that urine, saliva and serum all could be used as the samples for routine detection of Zika virus. Urine and saliva samples showed higher detection rates of Zika virus RNA in comparison to serum samples, while Zika virus could be easily isolated from positive serum samples. Suckling mice were better for Zika virus isolation than cell lines.
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Objective To understand the distribution and the characteristics on molecular typing of Salmonella (S.) typhimurium isolates gathered from the surveillance program and to construct the standard S.typhimurium databank in the laboratory through surveillance network PulseNet,in Guangdong province to improve the capability of detection on laboratory-based foodbome outbreaks.Methods With the application of standard pulse-field gel electrophoresis (PFGE) and multiple loci VNTR analysis (MLVA) including seven VNTRs loci protocols on PulseNet International Network,275 isolates of S.typhimurium from ten cities in Guangdong province were typed and their patterns analyzed.The molecular typing databank was constructed by BioNumerics.Results With S.typhimurium the most common serotypes,the average annual positive rate of Salmonella strains and S.typhimurium were 4.03％ and 1.38％ respectively.The positive rate and proportion presented a double-peak trend,appearing in May and September.The chromosomal DNA of S.typhimurium was digested with Xba Ⅰ restricted endonucleotidase and 124 PFGE patterns were observed after pulse-field gel electrophoresis,with the discrimination index (D) as 0.928 6.The patterns including more than three S.typhimurium isolates and were further digested with the second enzyme Bln Ⅰ to achieve 174 patterns,with the D value as 0.989 1.Under MLVA method,143 variant patterns were obtained,with the D value reaching 0.966 5.Data showed that the discriminatory ability of the MLVA typing method in S.typhimurium was superior to PFGE-Xba Ⅰ but equal to PFGE-Xba Ⅰ-Bln Ⅰ.In addition,when S.typhimurium strains were respectively analyzed by PFGE under double enzymes digestion and MLVA,the results appeared coincident and relative.Conclusion The variant patterns showed by the two molecular typing methods indicating a genetic diversity existed among the clinical S.typhimurium isolates in Guangdong province.Databank of S.typhimurium was constructed and could be used in laboratory surveillance programs.Under the characterization of analyzing similarity and evolution among S.typhimurium isolates,MLVA was suitable for cluster analysis on early detection of outbreaks caused by S.typhimurium.
