Study on the COVID-19 infection status, prevention and control strategies among people entering Shenzhen.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) confirmed cases overseas have continued to rise in the last months, and many people overseas have chosen to return to China. This increases the risk of a large number of imported cases which may cause a relapse of the COVID-19 outbreak. In order to prevent imported infection, the Shenzhen government has implemented a closed-loop management strategy using nucleic acid testing (NAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and requiring 14 days of medical observation for individuals with an overseas tour history (Hong Kong, Macao, Taiwan province and other countries). Our study aims to describe the status of COVID-19 infection among people entering Shenzhen, and to evaluate the effect of the closed-loop management strategy. METHODS: We undertook a descriptive study and risk analysis by the entry time, time of reporting, and local confirmed cases in countries of origin. The NAT were completed in Shenzhen Center for Disease Control and Prevention (CDC), ten district-level CDCs, and fever clinics. RESULTS: A total of 86,844 people from overseas entered Shenzhen from January 1 to April 18, 2020; there were 39 imported COVID cases and 293 close contacts. The infection rate of people entering was 4.49‰ [95% Confidence interval (CI): 3.26‰-6.05‰]. Fourteen imported cases (35.9%) came from the UK, and nine (23.08%) came from the USA. People entering from the USA since March 9 or from the UK since March 13 are the high-risk population. As of July 17, there have been no new confirmed cases in Shenzhen for 153 days, and the numbers of confirmed case, close contacts, and asymptomatic cases are 0. CONCLUSIONS: The closed-loop management has been effective in preventing imported infection and controlling domestic relapse. The distribution of entry time and report time for imported cases overseas was similar. This shows that it is important to implement closed-loop management at the port of entry.

[Identification and sequence analysis of E gene of Dengue virus type 2 strain isolated from patient serum in Shenzhen].

OBJECTIVE: To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin. METHODS: IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed. RESULTS: Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90. CONCLUSION: Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.

[Epidemiological and molecular characterization of seasonal influenza A/H3N2 viruses circulating in Shenzhen, 2005 - 2007].

OBJECTIVE: To determine the epidemiological characteristics of seasonal influenza in Shenzhen from 2005 to 2007 and the molecular variation of HA1 domain of influenza H3N2 viruses. METHODS: The consultation rate for influenza-like illness (ILI) were calculated weekly for indicating the influenza activities (the Shenzhen Influenza Surveillance System mainly consisted of 16 institutions with 9 hospitals, 6 districts and one municipal centers of disease control and prevention). Pharyngeal swabs from the cases of ILI, which were collected during 2005 to 2007 from the city-wide and quality-controlled surveillance network, were used to propagate the viruses. The HA1 region of the influenza A/H3N2 viruses were detected by RT-PCR and sequenced subsequently. The analyses of pairwise amino acid variations, genetic clustering and phylogenetics was performed. RESULTS: The activity levels of influenza showed certain changes during each year from 2005 to 2007, and there were summer peaks from May to July in 2006 and 2007. The positive rates of influenza virus were 4.78% (114/2385), 5.77% (212/3674) and 12.12% (343/2831) from 2005 to 2007 respectively. The weekly isolating rates changed accordingly with the trend of the percentages of ILI. The proportions of influenza H3N2 virus were 25.46% (28/114) and 2.83% (6/212) in 2005 and in 2006 respectively, but the proportion increased to 62.68% (215/343), which indicated that H3N2 virus became the predominant strain in 2007. Phylogenetic clustering analysis of influenza H3N2 virus revealed that there were 5 clades. The viruses which were isolated in 2005 contained in the clade I and II, the viruses in 2006 were comprised in clade III, and clade IV and V included the viruses isolated in 2007. Although the stem of cladogram developed with one accord of the time isolated viruses, the viruses which were similar to vaccine strains had circulated in Shenzhen before a given strain was determined as vaccine strain by WHO. It was also noticed that more amino acid changes at antigenic sites, especially at sites A and B in the H3N2 viruses, were found in 2007 than that in 2005 and in 2006. But the sequences at the receptor-binding sites and disulphide bond sites were conserved and no new circulating strain for genetic reassortment had been found in the period. CONCLUSION: Shenzhen might be one of areas where the ongoing genetic drift of influenza H3N2 viruses appeared earlier in China. The changes of influenza H3N2 virus showed the active status in the population. The results suggested that monitoring seasonal influenza viruses by sequence analysis could provide important and timely information on the appearance of strains with epidemiologic significance.

[Study on the molecular characteristic of natural infection of rodents with Hantaviruses in Shenzhen city].

OBJECTIVE: In order to investigate Hantavirus (HV) infection of captured rodents and to understand the genotypes and the molecular characteristic of Hantaviruses in Shenzhen. METHODS: The captured rodents were classified and the density of distribution was calculated. A total of 472 animals were captured, among which Rattus norvegicus was the dominant group. The total viral RNA was extracted from the lung tissues positive with HV antigens by immunofluorescent assay and gene sequence of M fragment was amplified with RT-nested-PCR by using the Hantavirus genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis. RESULTS: The results of genotype analysis showed that the Hantaviruses taken from twenty-one lung specimens in Rattus norvegicus in Shenzhen city belonged to the Hantavirus type II (SEOV). Results in homology analysis suggested that the homology among twenty-one samples should be rather high with 95.4% of nucleotide sequence identity and they belonged to the same subtype. Phylogenetic tree analysis showed that they were branched into at least six different lineages, and were highly homologized with SZ2083. We also found that these virus strains had not shown more highly homology of nucleotide sequence in nearest district, whereas revealed consistency in farther district. CONCLUSION: The major hosts of Hantaviruses in Shenzhen city were Rattus norvegicus and the epidemic strains were genotyped as SEO-type. Nucleotide sequence and deduced amino acid sequence from different rodents were highly homologous, while nucleotide mutation had also been observed. Further studies are required to explore the possible viruses' sequence mutation.

[Rapid detection of influenza virus A by fluorescence quantitative RT-PCR].

OBJECTIVE: To develop a method to rapidly detect influenza virus type A by fluorescence real-time quantitative RT-PCR. METHODS: According to conservative sequence of influenza virus type A M2 gene, a pair primers and Taqman probe were designed, respectively. After the quantitative curve of the assay were established using tenfold serial dilution of TCID50, the sensitivity and the specificity of clinic samples were determined. RESULTS: The detection sensitivity of the assay was 2.56 x 10(-5) TCID50 and the regression coefficient of the quantitative curve was 0.999. The specificity was determined by testing five other specimens, all of which yielded negative results. CONCLUSION: The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.

[Study on the rapid detection of Dengue viruses by Taqman MGB real-time fluorescent PCR].

OBJECTIVE: To develop the Taqman MGB real-time fluorescent PCR assay for rapid detection of Dengue viruses. METHODS: Using Taqman MGB technique, a pair of universal primers and Taqman MGB probe were designed according to a highly reserved sequence of the 3'-noncoding region of dengue viruses type 1-4. Dengue virus strains were used as standard and Japanese encephalitis virus strains were used as control, the real-time PCR assay for specific and sensitive detection of the dengue viruses was established. While 8 serum specimens of ELISA positive were detected by the RT-PCR and fluorescent PCR. RESULTS: The sensitivity of real-time PCR was 0.17pg/microl (cDNA)or 10(-5)TCID50. There was no cross-reaction with Japanese encephalitis virus. Of 8 specimens, 2 were positive by RT-PCR and 5 were positive by real-time PCR. The test could be completed in 4 hours. CONCLUSION: The Taqman MGB real-time PCR assay was fast, sensitive and specific. It could be applied to the quick early diagnosis of dengue viruses.

[Expression and assay in vitro of phage single chain antibody against nucleoprotein of influenza virus A type].

OBJECTIVE: To expression the phage single chain antibody against nucleoprotein of influenza virus A type in E. coli strain HB2151 by screening the positive clone from the phage antibody library against nucleoprotein, which will prepare for the construction of fast leak kit of Influenza virus A type. METHODS: The positive clone ratio in the phage antibody library was enriched by three-round continual solid panning, ELISA, SDS-PAGE and Western-blot methods were used to analyze the protein secreted into the supemants and periplasmic. Affinity chromatography was used to purified the protein expressed in periplasmic and the titer was also analyzed using ELISA method. RESULTS: 10 clones were screened from96 clones and among the 10, there were 3 stronger positive with OD450nm value of 0.469, 0.582 and 0.507, respectively. The phage single chain antibody against Influenza virus A type was primary expressed in periplasmic. The ELISA results were positive even if the single chain antibody purified by affinity chromatography was diluted 4096 folds. CONCLUSION: Using phage antibody library technique, phage single antibody against nucleoprotein of influenza virus A type was preliminary expressed in E. coli.

[Evaluation on construction and expression in vitro of nucleic acid vaccine of nucleoprotein of influenza virus A].

OBJECTIVE: Constructing nucleic acid vaccine of influenza virus A to study the protective effects. METHODS: NP gene was reverse transcripted from influenza virus A and linked with pSECTAG 2/Hygro A to constructed nucleic acid vaccine of influenza virus A. The constructed nucleic acid vaccine was transinfected into VERO cell resorting to lipofectamineTM2000. NP gene of influenza virus A was expressed in VERO cell by the method of ELISA. RESULTS: The results showed that the NP gene was expressed to the highest level at 36h after transinfection with the A40 value of 0.382. From 36 h to 60 h after transinfection, the expression of NP gene was stable (the A45, value at 48h and 60h was 0.385 and 0.387, respectively) . CONCLUSION: The nucleic acid vaccine of influenza virus A was successfully constructed, which sets up groudworks for the research of effection of nucleic acid vaccine in vivo.

[Analyses of serological and genetic characteristics on novel H1N1 influenza A virus from the infected patient in Shenzhen].

Analysis of serological and genetic characteristics on 2009 swine-origin influenza A (H1N1) virus (S-OIV) isolated from four patients with severe disease in Shenzhen were performed. Microneutralization assay showed that the neutralizing antibody titers of the infected patients did not exceed 1 : 20 in a short term post infection, which could not neutralize the viruses efficiently. Hemagglutination inhibition (HI) tests confirmed that the antigenicity of S-OIV from the patients was distinct from the seasonal influenza A virus, but similar to the reference strains of S-OIV. Phylogenetic and molecular analysis showed that S-OIV from the patients still belonged to the classical swine lineages and did not have the genetic characteristics of highly pathogenic influenza virus. Several amino acid residue mutations on HA protein were detected, which seemed not to affect the virulence and pathogenicity of the viruses. Further, A His 275 Tyr mutation on NA protein of a virus strain was detected, which induced the oseltamivir resistance of the virus.

[Laboratory diagnosis and molecular characterization analysis of the H5N1 influenza virus isolated from the first human case in Shenzhen, China].

The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza (firstly found in Shenzhen, China) were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient's tracheal aspirates on MDCK cell and was named A/Guangdong/2/06(H5N1). The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1 : 160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.

[Study on molecular epidemiological characteristics of influenza H1N1 viruses circulating in Shenzhen, China from 2005 to 2007].

OBJECTIVE: To study the genetic and epidemiological characteristics of HA1 of influenza H1N1 viruses circulating in Shenzhen from 2005 to 2007. METHODS: The HA1 region was analyzed by RT-PCR and subsequently sequenced to analyze the HA1 genetic evolution. Phylogenetic analysis was confirmed on the homology of nucleotide comparing with the reference viruses of vaccines recommended by WHO and representative virus confirmed by China CDC. Relationship between isolation rates and genetic evolutions was explored. RESULTS: The average isolation rate from 2005 to 2007 was 7.16%. Of the isolates, the proportions of influenza H1N1 viruses in 2005, 2006 and 2007 were 56.14%, 66.03%, 3.61%, respectively. Data from HA1 phylogenetic analysis showed that there were at least three clades circulated in Shenzhen. Different viruses isolated during January to April were clustered with A/New Caledonia/20/1999 viruses isolated in the latter months of 2005 clustered with A/Solomon Island/3/2006 and viruses from 2006 to 2007 were in the same clade with A/GDLH/219/2006. Results showed that most viruses had a deletion of lysine at position 130. Compared with A/New Caledonia/20/1999, the virus isolated after May of 2005 occurred T82K, Y94H, R146K, R209K, T267N amino acid substitution, while some virus isolated after May 2006 took place the amino acid substitutions of A190T, H193Y, E195D (located at antigenic site B) and R146K (antigenic site A). The sequences at the receptor-binding sites and glycosylation sites were conserved. Compared with referring viruses, A/SZ/68/2007 had 50 amino acid substitutions in the HA1 region. Of these, eleven and six were located at antigenic sites and receptor-binding sites, respectively. Four amino acid substitution resulted in the deletion of glycosylation site. CONCLUSION: Three different genetic lineages of influenza H1N1 virus were circulated in the population in Shenzhen during 2005 - 2007. The special virus named A/SZ/68/2007 should be paid further attention on its antigenic and epidemiological characteristics.

[A case of human highly pathogenic avian influenza in Shenzhen, China: application of field epidemiological study].

OBJECTIVE: Based on analyzing the characteristics of a case with human avian influenza and the effects of field epidemiological study. METHODS: An emergency-response-system was started up to follow the probable human Highly Pathogenic Avian Influenza case initially detected by the "Undefined Pneumonia Surveillance System of Shenzhen". Public health professionals administered several epidemiologic investigations and giving all the contacts of the patient with a 7-day-long medical observation for temporally related influenza-like illness. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers for H5 and N1 was applied to test respiratory tract samples and/or throat swabs of the patient and all his contacts specific for the hemagglutinin gene of influenza A H5N1. Activities and strategies such as media response,notification in the public, communications with multiple related sectors, social participation and information exchange with Hong Kong were involved in field control and management. RESULTS: The patient was a male, 31 years old,with an occupation as a truck driver in a factory,and had been residing in Shenzhen for 7 years. Started with an influenza-like syndrome, the patient received treatment on the 4th day of the onset, from a clinic and on the 6th day from a regular hospital. On the 8th day of the disease course, he was confirmed by Shenzhen Center for Disease Control and Prevention as human avian flu case and was then transferred to Intensive Care Unit (ICU). On the 83rd day of commence, the patients was healed and released from the hospital. The patient had no significant exposure to sick poultry or poultry that died from the illness before the onset of the disease. The patient and five family members lived together, but no family member was affected and no contact showed positive results for H5N1. A small food market with live poultry, which was under formal supervision and before illness the patient once visited, located near his apartment. Totally, 35 swabs from live birds and bird's coops in the market for H5 nucleic acid were tested and all were negative. The influenza H5N1 virus isolated for the case was named as A/Guangdong/02/2006 (H5N1) or GD/2/06. Phylogenetic relationships and molecular characterization analysis revealed that all the segments of the H5N1 virus named GD/2/06 still belonged to avian segments. Investigation process and control measures were released to the general public through the media. Soon after the laboratory confirmation, information was released to the society, as well as Hong Kong Center for Health Protection. Local Departments of Agriculture, Industries & Business, and Entry-Exit Inspection & Quarantine Bureau together with the Public Health Department put up combined actions. A computer-based telephone survey was initiated to investigate attitudes and knowledge of residents in town, revealing that positive atmosphere dominated and no panic existed. CONCLUSION: Rapid laboratory diagnosis of the virus was the key for successful treatment and survival result of the case. Still, the pathogen was from birds resources. No human-to-human transmission was observed, however, source of infection was unclear. Field epidemiological study could offer special methods for the responses of emergency public health problems.

[Surveillance on natural infection of rodents with hantavirus in Shenzhen city and identification of a hantavirus strain SZ2083].

OBJECTIVE: For clarifying the situation of the natural infection of rodents having hemorrhagic fever with renal syndrome (HFRS) virus and to type Hantavirus (HV) using molecular technique in Shenzhen city in 2005, and offering guidance for prevention and control of HFRS. METHODS: Data on the host animals was collected from the city of Shenzhen. ELISA and indirect immunofluorscent antibody(IFA) test were applied to the specific antibodies against HV in the sera of captured rats. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues of the HV infected rats were inoculated into Meriones unguiculata to isolate HV. The whole viral RNA was extracted from the lung tissues of the HV infected rats and amplified the partial M fragments with RT-nested-PCR, using the HV genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis. RESULTS: 472 rodents were captured from Shenzhen in 2005. Surveillance on rats demonstrated 9.96% rats carrying HV (with a density of 8.25%) and the main host was Rattus norvegicus. In the blood samples of rats, anti-HV IgG antibodies were detectable in 56 cases by IFA, and proved to be positive in 76 cases by ELISA. We successfully isolated a HV strain designated as SZ2083 from Rattus norvegicus for the first time in Shenzhen and was identified to SEO type by RT-nested-PCR. Compared with the coding region of the M gene of HV L99 virus strain, the homologies of nucleotide among them were 97%, but the homology was 76% of the SZ2083 with HTN 76-118 virus strain. CONCLUSION: Results showed the existence of natural epidemic areas of HFRS in Shenzhen city. Based on the results of sequencing, it is possible that the Seoul strain of HV might be the predominant serotype of virus harbored.

[Application of fluorescent real-time reverse transcriptase-polymerase chain reaction in detecting influenza viruses].

OBJECTIVE: To apply fluorescent real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting influenza viruses. METHODS: A total of 207 oral swab samples were obtained in 16 collections from SARS patients and suspected influenza outbreak cases. They were subjected to influenza virus detection by fluorescent real-time RT-PCR, MDCK cell culture, and hemagglutinin inhibition assay. RESULTS: Out of 207 samples, 79 (38.16%) were positive for influenza viruses when tested by fluorescent real-time PCR, and 62 (29.95%) positive when tested by MDCK cell culture. There was a statistically significant difference between them (chi square=8.64, P less than 0.005). From 104 cases in 9 collections dual serum samples were obtainable. When tested with hemagglutinin inhibition assay, 64 cases (61.54%) showed a 4-fold increase against H3N2 antigen. CONCLUSION: This study showed that fluorescent real-time PCR is a reliable, sensitive, and fast method for detecting influenza viruses.