Genomic Elucidation of a COVID-19 Resurgence and Local Transmission of SARS-CoV-2 in Guangzhou, China.

While China experienced a peak and decline in coronavirus disease 2019 (COVID-19) cases at the start of 2020, regional outbreaks continuously emerged in subsequent months. Resurgences of COVID-19 have also been observed in many other countries. In Guangzhou, China, a small outbreak, involving less than 100 residents, emerged in March and April 2020, and comprehensive and near-real-time genomic surveillance of SARS-CoV-2 was conducted. When the numbers of confirmed cases among overseas travelers increased, public health measures were enhanced by shifting from self-quarantine to central quarantine and SARS-CoV-2 testing for all overseas travelers. In an analysis of 109 imported cases, we found diverse viral variants distributed in the global viral phylogeny, which were frequently shared within households but not among passengers on the same flight. In contrast to the viral diversity of imported cases, local transmission was predominately attributed to two specific variants imported from Africa, including local cases that reported no direct or indirect contact with imported cases. The introduction events of the virus were identified or deduced before the enhanced measures were taken. These results show the interventions were effective in containing the spread of SARS-CoV-2, and they rule out the possibility of cryptic transmission of viral variants from the first wave in January and February 2020. Our study provides evidence and emphasizes the importance of controls for overseas travelers in the context of the pandemic and exemplifies how viral genomic data can facilitate COVID-19 surveillance and inform public health mitigation strategies.

Transmission risk of avian influenza virus along poultry supply chains in Guangdong, China.

OBJECTIVES: Avian influenza viruses (AIVs) poise significant risk to human health and the poultry industry. We evaluated the transmission risk along the poultry supply chain. METHODS: During October 2015 and July 2016, four rounds of cross-sectional surveys were performed to characterize AIV spread in farms, transport vehicles, slaughterhouses, wholesale and retail live poultry markets (LPMs). Poultry cloacal and oral swabs, environmental swabs, bioaerosol samples and human sera were collected. Poultry and environmental samples were tested for AIVs by rRT-PCR, further subtyped by next generation sequencing. Previous human H9N2 infections were identified by hemagglutination inhibition and microneutralization tests. Logistic regression was fitted to compare AIV transmission risk in different settings. RESULTS: AIVs was detected in 23.9% (424/1771) of the poultry and environmental samples. AIV detection rates in farms, transport vehicles, wholesale and retail LPMs were 4.5%, 11.1%, 30.3% and 51.2%, respectively. 5.2%, 8.3% and 12.8% of the poultry workers were seropositive in farms, wholesale and retail LPMs, respectively. The regression analysis showed that virus detection and transmission risk to human increased progressively along the poultry supply chain. CONCLUSIONS: Strengthening control measures at every level along the poultry supply chain, using a one health approach, is crucial to control AIV circulation.

[Evaluation on the risks of H5, H7 and H9 avian influenza infections in Guangzhou: using data from the 2006-2012 avian influenza surveillance program].

OBJECTIVE: To analyze the results of avian influenza surveillance program in Guangzhou from 2006 to 2012 and to evaluate the risk of infections with H5, H7 and H9 subtypes avian influenza viruses. METHODS: Avian influenza surveillance system in Guangzhou consisted five components:serum surveillance on occupational population, environmental specimen surveillance of avian influenza virus, avian flu emergency surveillance, influenza viruses surveillance on ILI patient and surveillance on pneumonia of unknown causes. Hemagglutination inhibition test was conducted to detect the antibodies against H5, H7 and H9 while RT-PCR was used to test the nucleic acid of H5, H7 and H9 viruses. RESULTS: From 2006 to 2012, 4103 serum specimens were collected from occupational populations and the overall positive rate of H5/H7/H9 antibodies was 3.82% . The antibody positive rates for H5, H7 and H9 were 0.22% ,0.00% and 3.70% respectively. 4 serum specimens for H5 and H9 simultaneously showed antibody positive. The positive rate of H9 among occupational populations(4.21%)appeared higher than that from the control population(2.16%). 2028 specimens were collected from poultry sites and 55 samples found positive for H5 nucleic acid (positive rate:2.71%), 14 samples positive for H9 nucleic acid (positive rate:0.69%), 5 specimens, simultaneously positive for H5 and H9 nucleic acids. However, none of the samples showing H7 nucleic acid positive. From 2006 to 2012, all the tested H5/H7/H9 virus were negative from the respiratory/serum specimens among those close contacts of patients or high risk groups through the avian flu emergency surveillance program,ILI patient influenza virus surveillance programs or pneumonia of unknown causes surveillance program. CONCLUSION: Contamination of H5/H9 avian influenza virus did exist in the poultry sites in Guangzhou, especially in the wet Markets. The H5/H9 avian influenza virus caused asymptomatic infection was proved to be existed within the population exposed to the poultry, suggesting that the poultry occupational population in Guangzhou was under the risk of avian influenza virus infection.

[Risk evaluation on H7N9 avian influenza in Guangzhou, China].

OBJECTIVE: We conducted both quick surveillance and evaluation programs within one week after the novel H7N9 influenza cases had been released by the Ministry of Health (MOH), to get the basic information on H7N9 virus in Guangzhou. METHODS: We sampled live birds from food markets and the natural habitat of birds to detect H7N9, H5 and H9 viruses. We interviewed workers from both markets and natural habitats. We also reviewed records on pneumonia patients with unknown causes from the surveillance system, to find clues related to the identification of severe pneumonia. RESULTS: We sampled 300 specimens from 49 stalls in 13 food markets and a natural habitat but none showed H7N9 positive result. A chopping block was detected positive of carrying H5 avian influenza virus, while another 4 specimens including a chicken cage, a duck cage, a chopping block and a pigeon cage were detected positive of carrying H9 avian influenza virus. In the past month, no sick, dead birds or ILI cases among the workers were discovered. 21.2% (7/33) of the stalls did not follow the set regulations for prevention. 10.3% (4/39) of the stalls had the cages cleaned, 4 days after the inspection. 3.7% (2/54) of the workers wore masks and 40.7% (22/54) of them wore gloves during the slaughtering process. 102 bird feces specimens were tested negative on H7N9 virus. No pneumonia cases with unknown reason were identified. From April 3(rd) to 17(th), we found 26 severe pneumonia cases but with negative results on influenza A (H7N9). CONCLUSION: According to the data and information from 1) lab tests, 2) pneumonia cases with unknown reasons under the surveillance system, 3) the identification of severe pneumonia cases, and 4) preventive measures and actions taken by the workers, we inferred that no H7N9 virus or related cases were found prior to April in Guangzhou. However, the risk of H7N9 epidemic does exist because of the following reasons:1) improper market management process, 2) negligent behavior of the workers and 3) potential trend of the national situation, suggesting strategies related to poultry markets management, health education and preventive measures against the avian influenza need to be strengthened.

[Investigation on the source of infection regarding an avian influenza (H5N1) case in Hong Kong that returning from Guangzhou].

OBJECTIVE: We conducted an epidemiologic investigation to determine the source of infection on an avian influenza (H5N1) case who returned from Guangzhou, in Hong Kong. METHODS: Data related to epidemiologic investigation, medical observation on close contacts, Syndromic Surveillance on poultry salesmen, emergency monitoring, detection of the samples, source tracing on potential Avian influenza virus (H5, H7, H9) infected people, situation on environment pollution by avian influenza virus in the markets etc. were gathered. The determination of infection source was through comparing the different genes between the case and positive environmental samples. RESULTS: The infected case witnessed the procedure of how a live duck was killed, in market A in Guangzhou during May 17(th) to 19(th). The case was diagnosed as respiratory tract infection in 2 Third-grade-Class A hospitals in Guangzhou on May 23(th) and 24(th). The diagnosis was made as Avian influenza cases on May 26(th) after going back to Hong Kong. 23 close contacts and 34 markets poultry salesmen did not show any ILI related symptoms. However, 2 poultry salesmen from the markets nearby the place where the Avian influenza case stayed, were detected having positive H9 avian influenza antibody, with the H9 positive rate as 6.06% (2/33). Among the environmental samples in the 2 markets nearby home of the patient, chopping block was found to have carried H5, with positive rate as 9.8% (5/51) while poultry cage was found to carry H9, with the positive rate as 2.0% (1/51). A H5 positive sample was found with clade 2.3.2.1, same to the case, from a chopping block at the market B where the sources of poultry was the same as market A. CONCLUSION: The source of infection seemed to come from the markets in Guangzhou, that calling for the strengthening of poultry market management, for avian influenza prevention. History related to contact of poultry should be gathered when a diagnosis of respiratory tract infection was made. Timely sampling and testing should be made to improve the sensitivity of diagnosis.

[Epidemiological characteristic of first case of locally identified A/H1N1 secondary cases caused by imported source of infection in China].

OBJECTIVE: To study the first locally identifed A/H1N1 secondary cases outbreak in China. METHODS: Interview and field investigation were integrated to describe the whole process of transmission on each case and to illustrate the relationships between the onset of the disease and the retated factors. RESULTS: Two contact persons appearanced fever and whose throat swabs were tested positive to H1N1 viral nucleic acid. The two had a history of contact in a short distance with the initial imported case without any protective measure in the poor air ventilation. The patients clinical situation was slight. The incubation was between 37 hours and 57 hours. No other new case was found after intervention as isolation and antisepsis were taken. CONCLUSION: This event was proved to be an outbreak of local A/H1N1 secondary cases caused by the imported case. The main mode of transmission was personal contact in a short distance without protection, through air and droplet. The locus with poor air ventilation was high risk place. Contact persons should be observed seven days and tested continuously. Infectivity and pathogenicity of the A/H1N1 virus were limited and appeared weakened by generations. Patient's condition was related with persistence and frequency of contact with the infection sources. Enhancing management of contact persons, health education, early diagnose, early treatment and early insulation were effective measures of controling and prenventing the spread A/H1N1.

[Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation.

[Study on the immunogenicity of adeno-vector vaccine against H5N1 influenza A virus].

OBJECTIVE: To construct adenovirus vector vaccine against H5N1 influenza virus and study on the immunogenicity. METHODS: In this study, we amplified hemagglutinin (HA) gene sequence of H5N1 influenza virus (A/Anhui/1/2005), then constructed an adenovirus vector vaccine (Adv-HA), followed by tests in BALB/c mice for the immunogenicity with the vaccine and immunization strategies. RESULTS: The recombinate Adv-HA vaccine could effectively induce both humoral and cellular immunity against human H5N1 influenza virus. CONCLUSION: The Adv-HA vaccination against H5N1 influenza is a potential strategy and worthy of further investigation.

[Study on immunogenicity of a recombinant adenovirus vaccine containing neuraminidase gene of H5N1 influenza virus (A/Anhui/1/2005) in mice].

OBJECTIVE: To investigate immunity of a recombinant adenovirus vaccine (rAdV) containing codon-modified neuraminidase (Mod. NA) gene of H5N1 influenza virus in BALB/c mice and to screen for appropriate dose. METHODS: BALB/c mice were immunized with the rAdV-Mod.NA vaccine intramuscularly twice (double injection at 0 and 4th week) in three groups, low dosage (10(5) TCID50 per dose), medium dosage (10(7) TCID50 per dose) and high dosage (10(9) TCID50 per dose). The effect of humoral and cell-mediated immunity were analysed at 5th week. RESULTS: (1) The rAdV-Mod.NA vaccine could elicit both humoral and cell-mediated robust NA specific immunity in mice by neuraminidase inhibitor assay and IFN-gamma ELISpot assay; (2) 10(7) TCID50 per dose was the appropriate dose; (3) Peptide NA(109-124): CRTFFLTQGALLNDKH and peptide NA(182-199): AVAVLKYNGIITDTIKSW were the dominant epitopes for neuraminidase-immunized BALB/c mice, which was screened out from the whole length of neuraminidase of an H5N1 virus, A/Anhui/1l/2005. CONCLUSION: The recombinant adenovirus NA could induce specific humoral and cellular immune responses in BALB/c after immunization, which suggest rAdV-Mod.NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation.

[Influence of avian influenza virus NS1 protein on the expression of IP-10 in BEAS-2B cells].

OBJECTIVE: To investigate the influence of avian influenza virus (AIV) NS1 protein on the expression of interferon-inducible protein 10 (IP-10). METHODS: NSI gene from virus A/Anhui/1/2005 (H5N1), NS1 gene inserted with 80-84 amino acids from virus A/Anhui/1/2005 (H5N1) and NS1 gene from virus A/Puerto Rico/8/1934 (H1N1) were cloned into the eukaryotic expression vector pEGFP-N1, and transfected into BEAS-2B cells, IP-10 expression level in transfected cells was detected by flow cytometry. RESULTS: Compared with the control group pEGFP-N1, expression of these three different NS1 genes can down-regulate the expression of IP-10 in BEAS-2B cells, but there is no significant difference as to the lower level among them. CONCLUSION: NS1 protein of A/Anhui/1/2005 (H5N1) can down-regulate the expression level of IP-10, but this may not clarify its relationship with the virulence of AIV.

[Codon optimization of the H5N1 influenza virus HA gene gets high expression in mammalian cells].

In order to improve the expression of human avian influenza virus hemagglutinin (HA) and meet the pandemic influenza vaccine needs, we optimized and synthesized the whole length of HA gene of H5N1 (A/Anhui/1/2005) influenza virus in accordance with the human's codon preference, and inserted it to the eukaryotic expression vector pDC315 to construct a eukaryotic expression vector pDC315-Mod. HA. This plasmid and the eukaryotic expression vector pDC315-Wt. HA containing wild HA gene were transfected into 293T cells respectively to compare the expression of HA protein. The results showed that according to the comparison and identification by indirect immunofluorescence assay and Western blot test, the expression of HA protein in 293T cells was significantly improved after codon optimization. This laid a foundation for pandemic influenza vaccine research.