Comparison of Rapid Influenza Diagnostic Tests With Digital Readout Systems and Conventional Rapid Influenza Diagnostic Test for Influenza Virus Detection

@#Introduction: Rapid diagnosis for influenza virus infection is essential for proper patient management, delivering prompt treatment and reducing unnecessary antiviral therapy. Early diagnosis helps in disease prevention and control. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay yields high sensitivity and specificity in detecting influenza virus infection. However, it is relatively expensive and requires trained personnel and special equipment. In this study, we compared two rapid influenza diagnostic tests (RIDTs): digital readout systems (STANDARD™ F Influenza A/B FIA, fluorescence immunoassay) and conventional visual confirmation (QuickNavi™-Flu2, chromatography immunoassay) with the real-time RT-PCR assay. Methods: Two hundred ninety-eight respiratory samples were obtained from patients suspected of influenza infection at Siriraj Hospital from December 2018 to December 2019. Results: Real-time RT-PCR results showed the detection of influenza A virus in 99 samples (60%), influenza B virus in 61 samples (37%) and co-infection by both viruses in 5 samples (3%) by the real-time RT-PCR assay. The QuickNavi™-Flu2 sensitivity for detecting influenza A and B viruses were 81.73% and 84.85%, and the specificity was 100%. The STANDARD™ F Influenza A/B FIA sensitivity for detecting influenza A and B viruses were 84.62% and 83.33%, respectively. The specificity for influenza A virus detection was 99.25% and 94.74% for influenza B virus. Conclusion: The STANDARD™ F Influenza A/B FIA and the QuickNavi™-Flu2 showed acceptable and comparable sensitivity and specificity. Both RIDTs are potential alternative methods of real-time RT-PCR for rapid screening of influenza virus infection.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Antiviral Drugs For Influenza Viruses

Antiviral drugs have significant action against influenza viruses A and B. Virus spread deadly disease in which many people die, and the country economy greatly suffer. Presently, most of the people need to get vaccination, which is depending on the dose limit in humans. It reacts directly or sometimes indirectly in the form of metabolites. However, it is mandatory to know how much drug is absorbed or metabolites concentration after administered. Therefore, pharmacokinetics data is very crucial for all drugs. Our review discusses the mechanism of drugs action and their activity and also describes how antiviral drugs and their metabolites is determined using a highly sensitive instrument such as high-performance liquid chromatography (HPLC), ultra-pressure liquid chromatography (UPLC), mass spectrometry (MS). Therefore, the present review gives brief information about antiviral drugs, their activity, biotransformation and analytical methods for quantification and this information will be helpful for any future studies done by experts in this field and will be beneficial for research scientists and influenza experts of all over the globe.

High expression and immunogenicity analysis of HA globular head domain of H5 subtype avian influenza virus produced in Pichia pastoris / 生物工程学报

To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218) and the optimal dosage of the recombinant H5HA was 50 μg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.

Inhibition of the replication of H9N2 influenza virus in vivo by short-term repeated oral administration of chicken interferon α / 生物工程学报

To evaluate the optimal administration frequency for interferon-α (IFN-α) and the effect of its combined use with inactive virus on chicken flocks, the prokaryotic expression plasmid pET-22b-ChIFN-α was constructed and transferred into Escherichia coli BL21(DE3) host bacteria to induce the expression of chicken IFN-α and to harvest recombinant proteins inclusion bodies. The expression of recombinant chicken IFN-α was confirmed by SDS-PAGE, and the results demonstrated that the chicken IFN-α (20 kDa) was highly expressed using the prokaryotic expression vector with a concentration of 0.2 mg/mL in the medium. Chicken IFN-α was diluted to 2.5×10⁴ U/fowls and administered to immunized specific-pathogen-free chickens orally in combination with inactivated H9N2 subtype influenza virus. Chicken that received chicken IFN-α were safe after three repeated immunizations (96 h). In addition, chicken IFN-α could induce higher levels of antiviral-related inducible genes in peripheral blood, spleen, and thymus of chicken flocks. The results of a challenge assay revealed that the lowest detoxification rates of chicken IFN-α ranged from three to five days, suggesting a higher capacity to resist H9N2 subtype avian influenza virus. The present study obtained the optimal immune frequency and immunization period for chicken IFN-α to provide theoretical support for the optimal clinical application of IFN-α.

Genetic evolution of HA and NA genes of H9N2 influenza viruses isolated in regions of Hunan Province, China, in 2015 / 生物工程学报

The high prevalence of influenza A virus is identified in Hunan Province because of the high density of poultry farms. To survey the variations of H9N2 subtype avian influenza virus in Hunan province, we analyzed HA and NA genes of 10 virus strains isolated from different areas of Hunan Province. All these strains belong to the Eurasian lineage, Y280-like sub-lineage. The cleavage sites in their HA genes were all RSSR↓GLT, corresponding to the feature of low pathogenic AIV. All strains had an L (Leu) at the site 234 in the HA genes, indicating the ability of binding with the SAα-2,6 receptor. NA gene stalk deletions at aa 63-65 were also detected from all the isolates, indicating a possibility of increased virus replication in mammals. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

Universal Oral Vaccine for Influenza Infections / Jurnal Sains Kesihatan Malaysia

Each year, influenza A infections have caused tremendous death rate as high as 300,000-500,000 globally. Althoughthere are effective anti-influenza agents and vaccines, high mutational rate among influenza A viruses renders dramaticdecline in the effectiveness of anti-influenza agents or vaccines in certain individuals. The situation is further complicatedby limitations in influenza vaccine production, for instance, long production period, limited vaccine capacity and lackof cross-protection against various influenza A virus strains. To solve these issues, development of universal influenzavaccine based on conserved antigens such as non-stuctural protein 1 (NS1) has been endeavoured. NS1 protein is highlyconserved in all influenza A virus strains known by far, produced abundantly on infected cell surfaces and responsible formaintaining virulence. Furthermore, cytotoxic T-lymphocytes that are active against NS1 were also reported to be ableto avoid shedding of influenza in hosts. To better inhibit influenza infections, oral immunization has long been proposeddue to feasibility of this method to be implemented and safer for recipients while able to target influenza A viruses fromthe entry point. Lactobacillus has been vastly studied for its roles as bacterial carrier in oral vaccine development dueto its significant probiotic properties. For examples, stimulation of immune responses in oral and airway mucosal layers,high colonization in oral and airway mucosal layers and great natural adjuvant effects. In this light, influenza universaloral vaccine developed using NS1 dan Lactobacillus should be further studied in influenza oral vaccine design.

Reseach Progress on Molecular Detection Technology of Influenza Virus / 现代检验医学杂志

Influenza viruses are global epidemic and diversely difficult to distinguish,which threaten human’s survival and development very much.In recentyears,the frequent outbreaks of influenza prompt the rapid development of Influenza virus detection.Compare with the traditional isolated culture and immunological detection,molecular diagnostic technology is of high detection speed,high sensitivity and specificity,that gradually play an important role in the current Influenza virus de-tection.In order to provide a theoretical basis for the rapid diagnosis of Influenza virus in the clinic,the article summarize the update progress of molecular biology and diagnostic techniques of Influenza viruses.

Quantitative assessment of the virucidal activities of three commercial disinfectants against human in-fected highly pathogenic avian influenza viruses subtype H5 / 中华微生物学和免疫学杂志

Objective To quantitatively assess the virucidal activities of three commercial disin-fectants against human infected highly pathogenic avian influenza viruses subtype H5. Methods The 50%tissue culture infective dose ( TCID50 ) of avian influenza viruses was calculated. Quantitative suspension test was performed to evaluate the efficacy of three disinfectants. In that test, 105 TCID50 of avian influenza viru-ses were exposed to different disinfectants at different concentrations for different times with or without the in-terference with fetal bovine serum ( FBS) simulating the contaminated condition. The residual infectivity was determined by endpoint titration in Madin-Darby canine kidney ( MDCK) cells. The detail steps were that the mixture of viruses and disinfectants was inoculated at 37℃ with 5% CO2 for 1 hour. Then, it was re-placed by virus dilution medium and further incubated for 18 to 20 hours. ELISA was performed for the cal-culation of TCID50 . The titers of residual viruses were calculated according to Reed and Muench method. The low pathogenic avian influenza virus H9N2 was chosen as the control in this study. Results The re-mained infectivities of three viruses after 1 minute exposure to 1% Virkon solution were below the limit of de-tection (1. 0 lgTCID50/100 μl). Exposing to 0. 5% Virkon solution decreased the viral titers of H5N1 and H9N2 viruses below the detection limit and reduced the titer of H5N6 virus to 1. 75 lgTCID50/100 μl. The virucidal efficacy of 0. 25% Virkon solution against some of the detected viruses was achieved by increasing the exposure time to 5 minutes. The 84 Disinfectant solutions at concentrations of 10%, 5% and 2. 5% low-ered the viral titers of three viruses below the detection limit of 1. 0 lgTCID50/100 μl, but the 1. 25% 84 Disinfectant solution only lowered the viral titers to 1. 25-2. 5 lgTCID50/100 μl. The similar results were ob-served in groups treated with SOLARSEPT solutions. 1% 84 Disinfectant solution didn′t show any virucidal activity against the three viruses after 1 minute of exposure even when the exposure time was extended to 5 minutes. Under the contaminated condition, 1% Virkon solution, 10% and 5% 84 Disinfectant solutions as well as 100% and 50% SOLARSEPT solutions lowered the viral titers below 1. 0 lgTCID50/100μl. Conclu-sion The three commercial disinfectants (1% Virkon solution, 10% 84 Disinfectant solution and SOLAR-SEPT solution) were efficient virucides for highly pathogenic avian influenza viruses subtype H5 even under the contaminated condition. Increasing the exposure time had no significant effects on the efficacy of three disinfectants after the virucidal activities were neutralized by enough viruses. No significant differences in vi-rucidal activities of three disinfectants against HPAI H5 viruses and LPAI H9 virus were observed.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012 / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization.</p><p><b>METHODS</b>RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed.</p><p><b>RESULTS</b>Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus.</p><p><b>CONCLUSION</b>Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.</p>

The aetiological analysis of common viruses of acute lower respiratory infection among the hospitalized children 0 to 7 years old in Nantong of Jiangsu / 中国基层医药

Objective To investigate the infection status and clinical features of common viruses in acute lower respiratory infection (ALRI) among the hospitalized children 0 to 7 years old in Nantong of Jiangsu.Methods 1 376 swab samples from pharynx nasalis in the ALRI inpatients 0 to 7 years old were collected.The human respiratory syncytial virus (RSV),adenovirus (ADV),influenza virusA,B (IVA,B),parainfluenza virus Ⅰ ～ Ⅲ (PIV Ⅰ ～ Ⅲ)were detected by direct immunofluorescence assay,and the results were analyzed.Results In 1 376 respiratory tract samples,there were 577 cases(41.93％) of positive samples.In all positive samples,there were 376 cases of RSVpositive (65.16％),42 ADV-positive (7.28％),63 IVA-positive (10.92％),24 IVB-positive (4.16％),20 PIV Ⅰ-positive(3.47％),19 PIV Ⅱ-positive (3.29％),108 PIV Ⅲ-positive (18.72％),68 cases with mixed infection (11.79％) [two virus-positive ones in 59 cases (86.76％),three virus-positive ones in 9 cases (13.24％)].In different age group,the highest positive rate was in 0 ～ 6-month-old group(53.32％),with the lowest in 5-7 years old group(6.90％).Virus detection rate was higher in March 2012 (58.67％),December 2012 (53.33％),and January 2013(53.63％)than the rest months,including the lowest June 2012(33.33％).Bronchiolitis virus detection rate was the highest(69.23％)among ALRI.Conclusion The virus is major pathogen of children 0 to 7 years old with ALRI in Nantong of Jiangsu,and with difference among different ages,seasons and diseases.Infants and young children are the main affected population.

Pathogenesis of influenza A (H5N1) virus-induced viral pneumonia in the rhesus macaque / 中国人兽共患病学报

At present ,the mechanism of highly pathogenic avian influenza H5N1 virus causing human infection or death is still not fully clear .In order to better understand the pathogenesis of the disease ,the rhesus macaques were infected with H5N1 virus (AF148678/ACGoose/Guangdong/11961H5N1) .We analyzed the clinical symptoms ,characteristics of the virus invades body ,pathological changes ,and immune response to discuss the pathogenesis of viral pneumonia induced by H 5N1 virus infection from the early time to the recovery time .The rhesus macaques were infected with H5N1 virus through nasal .Clinical signs were assessed daily ,and major organs and blood were collected for detection of blood routine analysis ,viruses were isola-ted and titrated from organs ,and pathologic and immunohistochemical were also conducted .As a result ,the rhesus macaques in-fected with H5N1 virus experienced fever ,dyspnea ,and anorexia .The respiratory tract was the major target of the virus and the virus could not replicate in organs outside the respiratory tract .Positive staining cells by immunohistochemistry were bronchial epithelial cells and alveolar macrophages .Rhesus macaques experienced temporary severe pneumonia after 1-3 days ,mainly be-cause of neutrophils infiltration ;gradual recovery 6 days later ,mainly with macrophage infiltration ;lung tissue presented recov-ery state after 14 days ,mainly with T lymphocytes infiltration .Finally ,we concluded that the predilection of the H 5N1 virus to infect the lower airway suggests that it may be a limiting factor in human-to-human transmissibility of the H5N1 virus .The pathogenesis may include virus invasion ,replication and immune injury .

Host Immune Responses Against Type A Influenza Viruses

The influenza viruses are divided into 3 different types, A, B and C, all of them are known as human pathogens. However, only type A influenza viruses cause both epidemic and pandemic influenza. Typically, influenza virus infects a respiratory tract, targets a lung and causes an acute infectious disease. Influenza infection can be identified by a high fever, headache, body ache and extreme fatigue. Host immune system against Influenza infection consists of innate immune response and adaptive immune response. Innate immune responses include recognition of influenza viruses by alveolar macrophages and natural killer cells. Adaptive immune responses contain influenza virus specific antibody production by B cells and killing infected cells by cytotoxic T cells. Initially, influenza viruses are recognized by pattern recognition receptors (PRRs) on respiratory epithelial cells and alveolar macrophages, which can induce efficient anti-viral immune responses. Host immune responses play crucial roles in defense against influenza virus infection but sometimes these may contribute to immuno-pathology, which results in serious tissue damage. In this review, we went over the understanding of current literature on subtypes of influenza A viruses, important viral antigens and anti-viral immune mechanisms.

The difference in IL-1β, MIP-1α, IL-8 and IL-18 production between the infection of PMA activated U937 cells with recombinant vaccinia viruses inserted 2004 H5N1 influenza HA genes and NS genes.

Background: The severity of avian influenza H5N1 disease is correlated with the ability of the virus to induce an over production of pro-inflammatory cytokines from innate immune cells. However, the role of each virus gene is unknown. To elaborate the function of each virus gene, the recombinant vaccinia virus inserted HA and NS gene from the 2004 H5N1 virus were used in the study. Methods: U937 cells and PMA activated U937 cells were infected with recombinant vaccinia virus inserted with HA or NS gene. The expressions of HA and NS proteins in cells were detected on immunofluorescence stained slides using a confocal microscope. The cytokine productions in the cell supernatant were quantitated by ELISA. Results: The recombinant vaccinia virus inserted with HA genes induces the production of IL-1β, MIP-1α, IL-8 and IL-18 cytokines from PMA activated U937 cells significantly more than cells infected with wild type vaccinia, whereas the recombinant vaccinia virus inserted with NS genes it was similar to that with the wild type vaccinia virus. However, there was no synergistic nor antagonistic effect of HA genes and NS genes in relation to cytokines production. Conclusion: Only the HA gene from the 2004 H5N1 virus induces IL-1β, MIP-1α, IL-8 and IL-18 cytokine productions from activated U937 cells. The same HA gene effect may or may not be the same in respiratory epithelial cells and this needs to be explored.

Molecular evolution of HA1 in pandemic H1N1 influenza viruses isolated in Guangdong during 2009 to 2011 / 中华微生物学和免疫学杂志

Objective To understand the evolutionary characterization of hemagglutinin (HA)gene of pandemic H1N1 influenza virus in Guangdong during 2009-2011. MethodsWe selected 83 pandemic H1N1 strains isolated in Guangdong during 2009-2011. The HA1 genes were sequenced and analyzed comparatively by Bioedit 7.0 and MEGA 4.0. ResultsThe evolutionary rate of Hal gene of pandemic H1N1 and seasonal H1N1 viruses was 5.2×10-3 substitutions/site/year, higher than that of seasonal H1N1 viruses. Most amino acid changes in HA1 molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Two fatal infections were detected with a mutation at HA residue 222, in one virus with a change D222G, and in one virus D222N. ConclusionThe phylogenetic analysis demonstrates that the influenza epidemic in Guangdong at the beginning of 2011 are due to occurrence of genetic changes of pandemic H1N1 virus. The amino acid change at residue 222 of the HA1 are likely to be associated with severe or even fatal illness.

Clinical study on influenza viruses infection detected by gold immunchromatographic assay in hospitalized patients / 中国综合临床

Objective To study the common influenza viruses infection of hospitalized patients admitted for acute respiratory tract infections, using gold immunchromatographic assay ( GICA ) to detect influenza viruses. Methods The result of FluA/B antigen detection in 1145 patients with various types of respiratory diseases from two class-A hospitals were analyzed. Influenza virus detection rates of patients in different seasons,with different gender,age,types of respiratory diseases and whether with foundation diseases were analyzed to identify the common rules and characteristics. Results There were significant differences for Flu A/b detection rate between first quarter and the second or third quarter,P <0.05 by x2 test( FluA x2 = 17. 735, P = 0.000;X2 = 14.855,P = 0. 000;FluB x2 =5. 326,P = 0. 021;x2 = 4.349, P = 0.037 ) . The result was repeated in the comparison between Flu A/B detection rate in the fourth quarter and the second or third quarter,P <0. 05 by x2 test (FluA x2 =19. 480,P= 0.000;x2 =16.771,,P=0. 000;FluB X2 = 6. 885.P = 0. 009;x2 =5. 959,P =0.015). These results indicated the detection rates of the first and fourth quarter were higher than the second and third quarter. Elderly patients (≥65 years old) had higher Flu A/ B detection rate compared with patients below 65 years ( FluA x2 =55. 362,P = 0.000;FluB x2 = 8.984,P = 0.003). The detection rate of Flu A/B in patients without foundation diseases or with one,two or three kinds of foundation diseases had significant differences, which showed with an increase in the number of types of the foundation diseases, FluA/B-positive detection rate increased. In patients with various foundation diseases, the FluA antigen detection rate in group of AECOPD patients was 18.2% and 17.1% in pneumonia group, which were higher than in all other diseases. Conclusions Sporadic cases of influenza were found in general wards, incidence rate was higher in the first and the fourth quarter. There is a higher risk of influenza virus infection for elder patients and patients with foundation diseases.

Molecular characteristics of the hemagglutinin genes of avian influenza virus subtype H5N1 in the boundary region of Yunnan province / 中国人兽共患病学报

To elucidate the molecular characteristics of the hemagglutinin (HA) genes of H5N1 subtype of avian influenza viruses in the boundary region of Yunnan province. Of 420 samples were collected from the foreign poultry in boundary region of Yunnan province during 2003 to 2008 and these samples were subjected to screening by H5/N1 subtype-specific and multiplex RT-PCR. testing. The HA genes of H5N1 viruses from positive samples were amplified by RT-PCR and cloned into vector pMD18 T for subsequent sequencing. The alignment with sequences of the known reference strains and phylogenetic analysis were then performed. The genes from 21 representative positive samples with 4 different sequences at the cleavage site were obtained and all of them possessed the molecular characteristic of highly pathogenic avian influenza virus. The mutation of key amino acids had been found among receptor-binding sites, potential glycosylation sites and neutralizing epitopes.-Phylogenetic analysis showed those positive samples could be divided into 5 distinct clades, including clade 1, 2.4. 2.3.2, 2.3.4 and 7. It is evident that H5N1 viruses from the foreign boundary region of Yunnan province in 2003 to 2008 show genetic divergence and clade 2,3,4 is the dominant clade in this region.

Comparative advantages of different methods of collecting nasopharyngeal secretions for rapid detection of influenza virus / 中国综合临床

Objective To compare the effectiveness of different methods of collecting nasopharyngeal secre-tions by nasopharynx swab and nasopharyngeal underpressure suction catheter for rapid detection of influenza virus. Methods Nasopharyngeal secretions as the experimental samples of 1042 patients with acute respiratory tract disea-ses were collected by nasopharynx swab and nasopharyngeal suction catheter, and gold immunochromatographic assay (GICA) kit was applied for the detection of influenza viruses. Results The use of the above two methods collecting nasopharyngeal secretions as samples for rapid detection of influenza virus would get the same results. The difference between the two methods had no statistical significance( P > 0.05 ). Conclnsions Nasopharynx swab is a reliable method for rapid detection of influenza virus, which is fast and convenient, compared with nasopharyngeal suction catheter.