西安市人民政府关于加强高致病性禽流感防控工作的通知

近期,我国周边多个国家和地区出现较大规模的高致病性禽流感疫情。国内部分省区高致病性禽流感疫情也呈流行趋势,发生的地区增加,疫点增多。高致病性禽流感疫情的发生不仅严重影响畜牧业发展,而且对人民生命安全构成了威胁。目前,又值高致病性禽流感高发季节和候鸟大规模迁徙季节。

从人类养禽史审视“禽流感”

猴年伊始．禽流感在亚洲肆行．短短几周中，已有数人因染“鸡病”而亡，面对严峻事态，人们不得不“杀鸡儆猴”．以控制疫情。对此，很多学者已从流行病角度做了分析，那么．让我们再从人类驯养动物，特别是养禽历史，来做一次回顾吧。

对美国物流及其猪禽冷链物流的考察和启示

美国物流运输网络覆盖范围广,成本低,信息化程度高。猪禽的冷链物流整体化程度高;设备设施先进,能够控制货物温度和环境;监控体制完善,物流运输企业采用信息技术对冷链运输进行全面动态监控,并且有政府主管部门进行检验检疫;物流基础服务完善,并提供第四方物流服务;物流标准体系全面、规范。我国冷链物流起步晚,通过考察,感到我国物流虽然发展很快,但冷链物流与美国相比还存在差距,其中冷链运输设备设施技术条件差,甚至没有,监管体系和标准体系存在缺陷,国际化较欠缺,尚待完善。

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Sequence Comparison and Analysis of HA Gene of Four H9N2 Avian Influenza Virus Isolates

[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

饲料中添加α-硫辛酸和α-生育酚乙酸酯可增强鸡肉的氧化稳定性

肉品质下降是肉发生脂质过氧化反应引起的。饲料中添加抗氧化剂可减少脂质过氧反应化和改善屠宰后肉的氧化稳定性。研究结果表明,给肉鸡饲喂添加α-生育酚醋酸酯(200mg·kg-1)与α-硫辛酸(25、75、150mg·kg-1)可增加肉的氧化稳定性及降低脂肪沉积。随着饲料中添加α-硫辛酸浓度的增加,肉中总酚含量和α-硫辛酸的含量增加。添加α-硫辛酸和α-生育酚乙酸酯,肉中的蛋白质含量没有改变。DPPH和TBA检测的结果表明,饲料中添加α-硫辛酸和α-生育酚乙酸酯,增强了鸡肉的抗氧化活性。给肉鸡饲喂食添加氧化油的饲料(4%),可导致其氧化稳定性降低。

前沿

关注国际爱牙日:健康口腔,幸福家庭;关爱老人,修复失牙【简介和来历】国家爱牙日为每年的9 月20 日。为了在公众中进行牙病防治知识的普及教育,增强口腔健康观念和自我口腔保健意识,帮助公众建立口腔保健的习惯,从而提高全民族的口腔健康水平,1989年,由卫生部、教委等部委联合签署,确定每年的9月20日为"全国爱牙日"。

The role of wild goose(Anser) populations of Russia and theTibet Plateau in the spread of the avian influenza virus

Wild birds of the orders Anseriformes and Charadriiformes represent a natural reservoir of low pathogenic avian influenza(LPAI) viruses(family Orthomyxoviridae).Wild geese(order Anseriformes)relating to waterfowls undertake extensive migration flights reaching thousands of kilometers.Isolation of the avian influenza virus(AIV) from wild geese is quite low or absent.The aims of this study are to monitor the AIV in different wild goose species,nesting on Russian territory and the Tibet Plateau and to analyze the derived data for the purpose of determining the role of these wild bird species in spreading pathogens.In our study 3245 samples from nine wild goose species in nine regions of Russia and on the territory of the Tibet Plateau(the Xizang Autonomous Region) were tested and no AIV were detected.Our study shows the non-essential role of wild geese in the spread of the AIV over long distances and reaches theconclusion that geese are probably not natural reservoirs for the primary viruses.However,further inquiry of AIV in wild goose populations is required.Studies of wild geese and AIV ecology will allow us to obtainmore information about pathogen-host relationships and to make arrangements for the maintenance ofwild goose populations.

Study on Piezoelectric Immunosensor for the Detection of H9-subtype Avian Influenza Virus

[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.

H5N1 influenza viruses： outbreaks and biological properties

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.

An Overview of the Highly Pathogenic H5N1 Influenza Virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.

Improvement of the efficacy of influenza vaccination (H5N1) in chicken by using extract of Cochinchina momordica seed (ECMS)

Seeds of a Chinese traditional medicine plant, Cochinchina momordica were used in the present study for the improvement of influenza vaccine （HSN 1） in chicken. Crude extraction from Cochinchina momordica seed （ECMS） was obtained by ethanol extraction method. In experiment No. 1, two weeks old chickens were immunized with influenza vaccine （HSN1） alone or combined with ECMS （5, 10, 20, 40 and 80 μg/dose）. Serum IgG antibody levels （by ELISA） as well as effects on dally weight gain were measured on 0, 7, 14 and 28th day after immunization. Results revealed that all ECMS groups numerically increased the antibody levels while 10 and 20 μg/dose groups significantly （P〈0.05） enhanced total IgG antibody on day 28, when compared with control. Average daily weight gain was also significantly higher in 20 μg/dose ECMS group. Adjuvant effect was also confirmed in experiment No. 2 when chickens were immunized with 20 μg/dose ECMS and antibody titer was measured through hemagglutination inhibition （HI）. It is concluded that ECMS has potential to improve the immune responses and deserve further study as an adjuvant.

Subtyping Animal Influenza Virus with General Multiplex RT-PCR and Liquichip High Throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.