In this study ,we established Cross Priming Amplification (CPA ) technology for detection of influenza A virus (H1N1) approach ,and evaluated the method through clinical specimens .A set of specific primers were designed for CPA ac‐cording to the conservative gene sequences ,designed and realized in the same temperature reverse transcription of RNA and DNA amplification . The amplification products can be totally enclosed nucleic acid detection device for testing . Fourteen healthy pharyngeal swab specimens ,seven other respiratory viruses ,and six arboviruses strains were used as the controls .We used a method that application of gradient dilution to the H 1N1 virus strain as the control to test the sensitivity of the CPA .We also used 102 clinical pharyngeal swab specimens of H1N1 patients for detection object to evaluate the feasibility of CPA clinical detection .Results showed that the CPA reaction did not appear cross reaction on health cases samples and other viruses .The sensitivity of the CPA was approximately 10 copies/uL in the established method that exactly titer H1N1 virus strain gradient dilution test .As to the positive results among the clinical pharyngeal swab samples collected from patients at different stages after onset ,the CPA had the highest positive detection rate during the first three days after onset (100% ) .While the detection rate from day 4 to day 6 after onset was 79 .31% .After 7 days ,the detection rate was 9 .09% .The established CPA assay was a highly sensitive ,specific and reproducible approach for rapid detection of H1N1 virus ,which is conducive to the early diagno‐sis of influenza A virus (H1N1) for basic medical units .

Objective:To analyze the epidemiological characteristics of novel coronavirus positive cases including confirmed cases with clinical symptoms and asymptomatic infected cases in Guangzhou.Methods:Epidemiological data were collected on the nucleic acid positive cases of COVID-19 in Guangzhou from January to September 2020. The epidemiological characteristics, the distribution of time intervals between the confirmed/isolation date and the date of the first positive detection were analyzed, at last the influencing factors for the confirmed cases and asymptomatic infected persons were discussed.Results:From January 7 to September 4 in 2020, a total of 1 097 nucleic acid positive cases were identified, including 658 confirmed cases (59.98%) and 439 asymptomatic infected cases (40.02%). Among the 658 confirmed cases, the median age was 42 years old, the cases indicated two significant peaks. one of the peaks was related to the imported and associated cases from Hubei province, and the other peak was connected with individuals from overseas. In terms of 439 asymptomatic infected cases, the median age was 32 years old. There were two stages in these cases. The first stage followed the second peak of confirmed cases, and the second stage overlapped with the confirmed cases in Guangzhou when the epidemic was in a period of normal prevention and control, mainly related to imported cases from abroad. The asymptomatic infected persons accounted for 57.32% in all the imported infected cases. In both of asymptomatic and symptomatic cases, the positive rate of pharyngeal swabs was higher than that of nasopharyngeal swabs and anal swabs. There were statistically significant differences in age, source of infection and gender composition between confirmed cases and asymptomatic infected persons ( P<0.05). Older age groups were more likely to have clinical symptoms, with ≥40 years being the risk factor for confirmed cases (OR=2.334, P=0.001), and 20-39 years less likely to have clinical symptoms (OR=0.620, P=0.047), compared with the 0-19 years old group. Compared with those infected in China, those infected abroad were less likely to develop clinical symptoms and became confirmed cases (OR=0.723, P=0.013). Women were more likely to have clinical symptoms than men (OR=1.574, P=0.001). Conclusions:At present, asymptomatic infected persons and confirmed patients with clinical symptoms co-existed, and the number of asymptomatic infected patients was higher than that of confirmed cases in Guangzhou. High age, domestic infection and female may be risk factors for confirmed cases. It was of great value to further explore these underlying mechanisms for the prevention and treatment of the COVID-19.

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.
Blotting, Western ; Cell Line ; Codon ; Fluorescent Antibody Technique, Indirect ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; Plasmids

OBJECTIVETo analyze the results of nine-round environmental specimen surveillance programs in five live poultry markets pre-, during and post the 'closing days' and to evaluate the effects of 'closing days' on live poultry markets regarding the control against avian influenza pollution.

METHODSIn January 2014, control measures including culling poultry, completely cleaning and disinfecting and a 'three-day-closing' measure were conducted in five live poultry markets which were found positive for H7N9 nucleic acid in the 1(st) round environmental specimen surveillance program. Second surveillance program was conducted after a thorough disinfection campaign was launched. Several times surveillance were conducted in one week, after the markets were reopened. RT-PCR was used to test the nucleic acid of HA, H5, H7 and H9 viruses.

RESULTS654 specimens from the environment were collected and tested. During the first round surveillance program, positive rates for influenza A and H5/H7/H9 nucleic acid of poultry stalls appeared to be 94.44% and 61.11% respectively. The positive rates of poultry stalls reduced to 0 after the disinfection campaign but increased again after the markets reopened. The positive rate for influenza A of poultry stalls slightly increased from 50.00% in the third surveillance to 72.22% in the ninth surveillance (P > 0.05). The positive rate for H5/H7/H9 of poultry stalls showed a significantly increasing trend, from 0 in the third surveillance to 44.44% in the ninth surveillance (P < 0.01). The positive rates for influenza A and H5/H7/H9 nucleic acid of specimens were 28.89% and 17.78% respectively. The positive rate of specimens reduced to 0 after disinfection while increased again after reopening of the markets. The positive rate for influenza A of specimens slightly increased from 19.67% in the third surveillance to 27.54% in the ninth surveillance programs (P > 0.05). The positive rate for H5/H7/H9 of specimen showed a significant increasing trend, from 0 in the third surveillance to 8.70% in the ninth-round surveillance programs (P < 0.01). The positive rate for influenza A was the highest for slaughter- related specimens of 22.4% (35/156). The positive rates for influenza A from sewage and drinking water being collected on the later stage after the markets reopened (25.9%, 12.4%)were higher than those on the early stage (8.3%, 8.6%) (P > 0.05). The positive rate for influenza A of poultry stalls with overnight poultry storage (91.7%) was significant higher than that of poultry stalls without the overnight storage (33.3%). The positive rate for influenza A of poultry stalls in which simultaneously selling different kinds of poultry (85.7%) was significant higher than that of poultry stalls in which selling only one kind of poultry at one time (25.0%) (P < 0.05).

CONCLUSIONSlaughter in live poultry markets posed a large risk of pollution diffusion. Sewage and drinking water showed an accumulation effect for avian influenza virus. Overnight poultry storage and selling different kinds of poultry at one time at the poultry stalls seemed the risk factors for avian influenza virus transmission. Complete cleaning, disinfecting and several 'closing days' for live poultry markets seemed effective in eliminating avian influenza virus. Once the markets were reopened, they seemed to be soon polluted again.

Animals ; China ; Commerce ; Disinfection ; Environmental Microbiology ; Environmental Monitoring ; Influenza A virus ; isolation & purification ; Influenza in Birds ; prevention & control ; Poultry ; virology

OBJECTIVETo evaluate the effect of supply of fresh poultry products on reducing environment contamination of avian influenza virus (AIV) in markets in Guangzhou.

METHODSA total of 40 markets, including 20 selling alive poultry and 20 selling fresh poultry products, were selected randomly in Guangzhou to conduct environment surveillance in 80 poultry stalls every 4 months from July 2014 to April 2015. Four smear samples were collected from different sites of each poultry stall to detect nucleic acid of AIV. The positive samples were further detected for AIV subtype H5, H7 and H9 nucleic acids.

RESULTSAmong 40 alive poultry stalls, 95.0% (38/40) kept alive poultry overnight, 25.0% (10/40) were disinfected daily, 95.0% (38/40) were cleaned up weekly, 95.0% (38/40) were closed for one day every month. Among 40 fresh poultry product stalls, 20.0% (8/40) were disinfected daily, 90.0% (36/40) were cleaned up weekly, and 96.0% (38/40) ever sold dressed poultry from alive poultry markets. The positive rate of AIV in alive poultry markets was 40.4% (252/623), higher than that in fresh poultry product markets (32.3%, 197/610), the difference was significant (χ(2)=8.85, P=0.003), and the positive rate of subtype H9 virus in alive poultry markets was 28.6% (178/623), higher than that in fresh poultry product markets (16.2%, 99/610), the difference was significant (χ(2)=26.95, P<0.001). In fresh poultry product markets, the positive rate of AIV in stalls selling dressed poultry was 37.3% (180/482), higher than that in stalls selling no dressed poultry (13.3%, 17/128), the difference was significant (χ(2)=26.78, P<0.001), and the positive rate of subtype H9 virus in stalls selling dressed poultry was 19.1% (92/482), higher than that in stalls selling no dressed poultry (5.5%, 7/128), the difference was significant (χ(2)=13.80, P<0.001). Both the positive rate of AIV and the positive rate of subtype H9 virus were highest in the second round surveillance (October 2014). The differences in AIV and its subtype H5, H7 and H9 virus positive rates of environmental samples from four different sites were not significant, respectively. In the same sample site, the positive rate of subtype H9 virus in alive poultry markets was higher than that in fresh poultry product markets the difference was significant (P<0.05).

CONCLUSIONSThe supply of fresh poultry products could effectively reduce the level of environment contamination of AIV in markets. Dressed poultry supplement caused the risk of AIV spread in fresh poultry product markets.

Animals ; China ; Commerce ; statistics & numerical data ; Disinfection ; statistics & numerical data ; Environmental Microbiology ; Influenza A virus ; isolation & purification ; Poultry ; Poultry Products ; supply & distribution