Distribution of the COVID-19 epidemic and correlation with population emigration from Wuhan, China / 中华医学杂志(英文版)

BACKGROUND@#The ongoing new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) outbreak is spreading in China, but it has not yet reached its peak. Five million people emigrated from Wuhan before lockdown, potentially representing a source of virus infection. Determining case distribution and its correlation with population emigration from Wuhan in the early stage of the epidemic is of great importance for early warning and for the prevention of future outbreaks.@*METHODS@#The official case report on the COVID-19 epidemic was collected as of January 30, 2020. Time and location information on COVID-19 cases was extracted and analyzed using ArcGIS and WinBUGS software. Data on population migration from Wuhan city and Hubei province were extracted from Baidu Qianxi, and their correlation with the number of cases was analyzed.@*RESULTS@#The COVID-19 confirmed and death cases in Hubei province accounted for 59.91% (5806/9692) and 95.77% (204/213) of the total cases in China, respectively. Hot spot provinces included Sichuan and Yunnan, which are adjacent to Hubei. The time risk of Hubei province on the following day was 1.960 times that on the previous day. The number of cases in some cities was relatively low, but the time risk appeared to be continuously rising. The correlation coefficient between the provincial number of cases and emigration from Wuhan was up to 0.943. The lockdown of 17 cities in Hubei province and the implementation of nationwide control measures efficiently prevented an exponential growth in the number of cases.@*CONCLUSIONS@#The population that emigrated from Wuhan was the main infection source in other cities and provinces. Some cities with a low number of cases showed a rapid increase in case load. Owing to the upcoming Spring Festival return wave, understanding the risk trends in different regions is crucial to ensure preparedness at both the individual and organization levels and to prevent new outbreaks.

Risk assessment and genomic characterization of Zika virus in China and its surrounding areas / 中华医学杂志(英文版)

BACKGROUND@#Zika virus (ZIKV) has emerged as a global pathogen causing significant public health concerns. China has reported several imported cases where ZIKV were carried by travelers who frequently travel between China and ZIKV-endemic regions. To fully characterize the ZIKV strains isolated from the cases reported in China and assess the risk of ZIKV transmission in China, comprehensive phylogenetic and genetic analyses were performed both on all ZIKV sequences of China and on a group of scientifically selected ZIKV sequences reported in some of the top interested destinations for Chinese travelers.@*METHODS@#ZIKV genomic sequences were retrieved from the National Center for Biotechnology Information database through stratified sampling. Recombination event detection, maximum likelihood (ML) phylogenetic analysis, molecular clock analysis, selection pressure analysis, and amino acid substitution analysis were used to reconstruct the epidemiology and molecular transmission of ZIKV.@*RESULTS@#The present study investigated 18 ZIKV sequences from China and 70 sequences from 16 selected countries. Recombination events rarely happens in all ZIKV Asian lineage. ZIKV genomes were generally undergone episodic positive selection (17 sites), and only one site was under pervasive positive selection. All ZIKV imported into China were Asian lineage and were assigned into two clusters: Venezuela-origin (cluster A) and Samoa-origin cluster (cluster B) with common ancestor from French Polynesia. The time of most recent common ancestors of Cluster A dated to approximately 2013/11 (95% highest posterior density [HPD] 2013/06, 2014/03) and cluster B dated to 2014/08 (95% HPD 2014/02, 2015/01). Cluster B is more variable than Cluster A in comparison with other clusters, but no varied site of biological significance was revealed. ZIKV strains in Southeast Asia countries are independent from strains in America epidemics.@*CONCLUSIONS@#The genetic evolution of ZIKV is conservative. There are two independent introductions of ZIKV into China and China is in danger of autochthonous transmission of ZIKV because of high-risk surrounding areas. Southeast Asia areas have high risk of originating the next large-scale epidemic ZIKV strains.

Risk assessment and genomic characterization of Zika virus in China and its surrounding areas / 中华医学杂志(英文版)

Background@#Zika virus (ZIKV) has emerged as a global pathogen causing significant public health concerns. China has reported several imported cases where ZIKV were carried by travelers who frequently travel between China and ZIKV-endemic regions. To fully characterize the ZIKV strains isolated from the cases reported in China and assess the risk of ZIKV transmission in China, comprehensive phylogenetic and genetic analyses were performed both on all ZIKV sequences of China and on a group of scientifically selected ZIKV sequences reported in some of the top interested destinations for Chinese travelers.@*Methods@#ZIKV genomic sequences were retrieved from the National Center for Biotechnology Information database through stratified sampling. Recombination event detection, maximum likelihood (ML) phylogenetic analysis, molecular clock analysis, selection pressure analysis, and amino acid substitution analysis were used to reconstruct the epidemiology and molecular transmission of ZIKV.@*Results@#The present study investigated 18 ZIKV sequences from China and 70 sequences from 16 selected countries. Recombination events rarely happens in all ZIKV Asian lineage. ZIKV genomes were generally undergone episodic positive selection (17 sites), and only one site was under pervasive positive selection. All ZIKV imported into China were Asian lineage and were assigned into two clusters: Venezuela-origin (cluster A) and Samoa-origin cluster (cluster B) with common ancestor from French Polynesia. The time of most recent common ancestors of Cluster A dated to approximately 2013/11 (95% highest posterior density [HPD] 2013/06, 2014/03) and cluster B dated to 2014/08 (95% HPD 2014/02, 2015/01). Cluster B is more variable than Cluster A in comparison with other clusters, but no varied site of biological significance was revealed. ZIKV strains in Southeast Asia countries are independent from strains in America epidemics.@*Conclusions@#The genetic evolution of ZIKV is conservative. There are two independent introductions of ZIKV into China and China is in danger of autochthonous transmission of ZIKV because of high-risk surrounding areas. Southeast Asia areas have high risk of originating the next large-scale epidemic ZIKV strains.

Clinical indices and mortality of hospitalized avian influenza A (H7N9) patients in Guangdong, China / 中华医学杂志(英文版)

BACKGROUND@#Six epidemic waves of human infection with avian influenza A (H7N9) virus have emerged in China with high mortality. However, study on quantitative relationship between clinical indices in ill persons and H7N9 outcome (fatal and non-fatal) is still unclear. A retrospective cohort study was conducted to collect laboratory-confirmed cases with H7N9 viral infection from 2013 to 2015 in 23 hospitals across 13 cities in Guangdong Province, China.@*METHODS@#Multivariable logistic regression model and classification tree model analyses were used to detect the threshold of selected clinical indices and risk factors for H7N9 death. The receiver operating characteristic curve (ROC) and analyses were used to compare survival and death distributions and differences between indices. A total of 143 cases with 90 survivors and 53 deaths were investigated.@*RESULTS@#Average age (Odds Ratio (OR) = 1.036, 95% Confidence Interval (CI) = 1.016-1.057), interval days between dates of onset and confirmation (OR = 1.078, 95% CI = 1.004-1.157), interval days between onset and oseltamivir treatment (OR = 5.923, 95% CI = 1.877-18.687), body temperature (BT) (OR = 3.612, 95% CI = 1.914-6.815), white blood cell count (WBC) (OR = 1.212, 95% CI = 1.092-1.346) were significantly associated with H7N9 death after adjusting for confounders. The chance of death from H7N9 infection was 80.0% if BT was over 38.1 °C, and chance of death is 67.4% if WBC count was higher than 9.5 (10/L). Only 27.1% of patients who began oseltamivir treatment less than 9.5 days after disease onset died, compared to 68.8% of those who started treatment more than 15.5 days after onset.@*CONCLUSIONS@#The intervals between date of onset and confirmation of diagnosis, between date of onset to oseltamivir treatment, age, BT and WBC are found to be the best predictors of H7N9 mortality.

Potential infections of H5N1 and H9N2 avian influenza do exist in Guangdong populations of China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Southeast China is one of the sites of influenza origin. During 2003--2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.</p><p><b>METHODS</b>Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT).</p><p><b>RESULTS</b>The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case.</p><p><b>CONCLUSION</b>The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.</p>

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.</p><p><b>METHODS</b>SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.</p><p><b>RESULTS</b>The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).</p><p><b>CONCLUSION</b>N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.</p>

Immune Responses and Histopathological Changes in Rabbits Immunized with Inactivated SARS Coronavirus / 中国病毒学

To evaluate the immunogenicity of inactivated SARS coronavirus (SARS-CoV), three groups of rabbits were immunized three times at 2-week intervals with inactivated vaccine + adjuvant, adjuvant,and normal saline respectively. Eight batchs of serum were sampled from the auricular vein at day 7 to day 51, and specific IgG antibody titers and neutralizing antibody titers were detected by indirect ELISA and micro-cytopathic effect neutralizing test. Antibody specificity was identified by proteinchip assay.Histopathological changes were detected by H&E staining. The results showed that, rabbits in the experimental group immunized with inactivated SARS-CoV all generated specific IgG antibodies with neutralizing activity, which suggested the inactivated SARS-CoV could preserve its antigenicity well and elicit an effective humoral immune responses. The peak titer value of specific IgG antibody and neutralizing antibody reached 1:40960 and 1:2560 respectively. In the experimental group, no obvious histopathological changes was detected in the H&E stained slides of heart, spleen, kidney and testis samples, but the livers had slight histopathological changes, and the lungs presented remarkable histopathological changes. These findings are of importance for SARS-CoV inactivated vaccine development.

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain.</p><p><b>METHODS</b>The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.</p><p><b>RESULTS</b>The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed.</p><p><b>CONCLUSIONS</b>The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).</p>

Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed. Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper.</p><p><b>METHODS</b>The large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund's adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test.</p><p><b>RESULTS</b>Rapid and potent humoral immune responses were induced by the inactivated SARS-CoV vaccine in all the eight test rabbits. Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1:81 920 and 1:20 480, respectively. After that, serum antibody levels remained at a plateau or had a slight decrease, though two boosters were given in the succedent 4 to 5 weeks. Cross neutralization response existed between SARS-CoV F69 strain and Z2-Y3 strain.</p><p><b>CONCLUSIONS</b>The inactivated SARS-CoV vaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.</p>

Isolation and identification of SARS virus in Guangdong province / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To isolate and identify pathogen of atypical pneumonia in Guangdong.</p><p><b>METHODS</b>Pathogens were isolated from variety of samples collected from atypical pneumonia patient by using MDCK cells, and identified with serological and molecular methods.</p><p><b>RESULTS</b>A novel coronavirus was isolated from patients with atypical pneumonia, from which an RNA fragment of 279 nt was amplified by nested RT-PCR. And sequence assay showed that only 39-65 percent of sequence of the virus was homogenous to known coronavirus, but almost 100% homogenous (with one base exception, 12a to t) to SARS-associated coronavirus isolated from patients outside Guangdong, such as in Beijing, Hong Kong, Taiwan, Germany, Italy and so on. Indirect immunofluorescence test showed a specific antigen-antibody reactivity between the coronavirus and convalescent-phase sera of SARS patients.</p><p><b>CONCLUSION</b>The pathogen of the atypical pneumonia in Guangdong province was a novel type of coronavirus, which could be isolated by using MDCK cells.</p>