Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

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.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.

Effect of Different Culture Media on the Proliferation of Avian Influenza Virus H9 Subtypes in MDCK Cells

[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

H9亚型禽流感病毒的分离与鉴定

从临床表现呼吸困难并伴有啰音、肺部和支气管黄色干酪样栓塞为特征的病鸡肝脾肺混合匀浆中分离到1株病毒(YD株),该病毒能凝集鸡红细胞并能被AIV H9标准阳性血清抑制,对热不稳定且对热敏感;对鸡胚和番鸭胚的最小致死量分别为10-7和10-6,其MDT分别为78h和70h;鸡胚尿囊液毒的HA效价和EID50明显高于番鸭胚尿囊液毒;能扩增出大小约为1 027bp的特异性目的片段。结果表明,该分离毒为H9亚型禽流感病毒。

Detection of avian influenza virus H9N_(2) based on self-driving and self-sensing microcantilever piezoelectric sensor

In this article, we used the self-excitation and self-inductance characteristics of polyvinylidene fluoride(PVDF) piezoelectric materials, combined with the powerful signal processing and calculation analysis capabilities of integrated circuits, for the first time to explore a set of microcantilever sensor "readout system" without additional driver(self-driving) and can realize self-sensing external signal(self-sensing).It was successfully applied to the unlabeled detection of avian influenza virus(AIV) H9N_(2). The specific force of the antigen-antibody complexes on the surface of the microcantilever leads to the change of the stress of the cantilever, which drives the constructed detection device, and does not require an additional excitation source to drive it, that is, the self-driving part. At the same time, due to the movement of piezoelectric charges in the film caused by the positive piezoelectric effect of the PVDF film, self-inductive charges are generated on the surface of the sensor dielectric. The charge signal is converted into a voltage signal, and the sensing part is completed, that is, self-sensing. The immunosensor has a linear range of100-1000 ng/m L with a detection limit of 2.9 ng/m L. The method will also open up a new avenue for the detection of other analytes based on antigen-antibody responses.

Maternal-derived antibodies hinder the antibody response to H9N2 AIV inactivated vaccine in the field

The H9N2 subtype avian influenza virus(AIV)inactivated vaccine has been used extensively in poultry farms,but it often fails to stimulate a sufficiently high immune response in poultry in the field,although it works well in laboratory experiments;hence,the virus still causes economic damage every year and poses a potential threat to public health.Based on surveillance data collected in the field,we found that broilers with high levels of maternal-derived antibodies(MDAs)against H9N2 virus did not produce high levels of antibodies after vaccination with a commercial H9N2 inactivated vaccine.In contrast,specific pathogen-free(SPF)chickens without MDAs responded efficiently to that vaccination.When MDAs were mimicked by administering passively transferred antibodies(PTAs)into SPF chickens in the laboratory,similar results were observed:H9N2-specific PTAs inhibited humoral immunity against the H9N2 inactivated vaccine,suggesting that H9N2-specific MDAs might hinder the generation of antibodies when H9N2 inactivated vaccine was used.After challenge with homologous H9N2 virus,the virus was detected in oropharyngeal swabs of the vaccinated and unvaccinated chickens with PTAs but not in the vaccinated chickens without PTAs,indicating that H9N2-specific MDAs were indeed one of the reasons for H9N2 inactivated vaccine failure in the field.When different titers of PTAs were used to mimic MDAs in SPF chickens,high(HI=12 log2)and medium(HI=log 9 log2)titers of PTAs reduced the generation of H9N2-specific antibodies after the first vaccination,but a booster dose would induce a high and faster humoral immune response even of PTA interference.This study strongly suggested that high or medium titers of MDAs might explain H9N2 inactivated vaccine failure in the field.

Human infection with avian influenza A（H7N9） virus in Shanghai： current status and future trends

Avian influenza A（H7N9） virus is one subgroup among the larger group of H7 viruses,which normally circulate among birds.The H7N9 subtype of avian influenza viruses has not been known to infect humans until only recently.On March 31,2013,China confirmed the first three human cases of novel avian influenza A（H7N9）infection in Shanghai and Anhui,two of these patients died.1 As of February 27,2014,367 laboratory-confirmed human cases have been reported from 15 provinces/municipalities in China's Mainland,

2014 update of the Chinese guidelines for diagnosis and treatment of avian influenza A（H7N9） virus infection

The National Health and Family Planning Commission of China published an updated guidance （2014 version） on clinical management in Chinese on January 26, 2014.The guidelines come as human cases of avian influenza A（H7N9） virus infection undergo a seasonal spike.

禽类养殖场人员禽流感病毒抗体水平调查

目的 调查珠海地区养殖场人员中禽流感H5和H9二种亚型血清中抗体水平 ,以了解上述 2种禽类甲型流感病毒亚型以往的接触感染情况 ,为防治人间禽流感提供科学依据。方法 珠海地区养殖场人员血清 2 77份 ,正常出入境人员血清 2 70份 ,通过血凝抑制试验HI实验进行抗体检测。结果 养殖场人群中H9亚型抗体阳性率为2 17% ,同时发现有 1份血清H5抗体阳性 ,H5抗体阳性率为 0 36 %。而正常出入境人群H9亚型抗体阳性率为2 96 % ;未见H5亚型抗体的存在。结论 H9亚型在养殖场人群和出入境人群中的存在隐性感染 ,但感染率不高 ;养殖场人群H5亚型抗体的存在也应值得注意。

Acute lung injury induced by H9N2 virus in mice

Background H9N2 avian influenza viruses （AIVs） have repeatedly caused infections in mammals even humans in many countries. The purpose of our study was to evaluate the acute lung injury （ALl） caused by H9N2 viral infection in mice. Methods Six- to eight- week-old female SPF C57BL/6 mice were infected intranasally with lx104 MIDso of A/HONG KONG/2108/2003 [H9N2 （HK）] virus. Clinical signs, pathological changes, virus titration in tissues of mice, arterial blood gas, and cytokines in bronchoalveolar lavage fluid （BALF） and serum were observed at different time points after AIV infection. Results H9N2-AIV-infected mice exhibited severe respiratory syndrome, with a mortality rate of 50%. Lung histopathological changes in infected mice included diffuse pneumonia, alveolar damage, inflammatory cellular infiltration, interstitial and alveolar edema, and hemorrhage. In addition, HgN2 viral infection resulted in severe progressive hypoxemia, lymphopenia, and a significant increase in interleukin 1, interleukin 6, tumor necrosis factor, and interferon in BALF and serum. Conclusions The results suggest that H9N2 viral infection induces a typical ALl in mice that resembles the common features of ALl. Our data may facilitate the future studies of potential avian H9N2 disease in humans.