摘要
马流感疫苗最早出现在19世纪60年代,并广泛应用于马流感的防控,使马流感疫情在全球得到了有效地控制。而我国至今未能有自主研发、生产的马流感疫苗上市,我国内陆广大地区的绝大多数马匹也未接种过马流感疫苗,致使我国的养马业持续地受到马流感的威胁。2007年到2008年间,我国内陆地区经历了一次马流感大流行,再一次给我国养马业带来巨大的灾难,对我国即将兴起的赛马业以沉重的打击,威胁着08年北京奥运会马术竞技项目的举行。随着与国际赛事的频繁接触及国内赛马业的兴起,我国的马流感防控工作任重道远,疫苗的研制势在必行。
本研究对本实验室分离到的2株美洲系疫苗代表株A/equine/xinjiang/3/2007(H3N8)(简称XJ株)和A/equine/Hubei/6/2008(H3N8)(称简HB株)进行了生物学特性的研究,并对HB株进行了HA基因的遗传分析,为我国的马流感防控提供参考。XJ株在SPF鸡胚中连续传至7代,血凝价能够稳定在27~28之间,而HB株能够在鸡胚中稳定的繁殖,血凝价均在28左右。两株马流感病毒均能被H3N8亚型马流感病毒特异性血清所中和;均能凝集猪、马、牛、绵羊、山羊、驴、兔、豚鼠、大鼠、小鼠、鸡的红细胞;均为热稳定型流感病毒;酸、碱、氯仿和乙醚处理后感染性依然存在;感染幼驹后,均能出现完全的马流感临床症状,感染后排毒可持续1周左右,并在感染后1周左右即可检测到HI抗体。
用RT-PCR法克隆了HB株的HA基因,并进行了HA基因核苷酸序列的测定。经NCBI上Blast同源性比较,发现与A/equine/Newmarket/5/2003(H3N8)同源性较高,为98.7%。HA蛋白遗传进化分析表明该毒株隶属于H3N8亚型马流感病毒中的美洲系佛罗里达亚系。
经终浓度为0.05%甲醛和0.025% BPL于4℃摇床灭活H3N8亚型马流感病毒尿囊液48h后,病毒灭活程度检查结果表明均能完全灭活马流感病毒;HA试验表明在灭活前后病毒的HAU不变。同时对BPL灭活病毒的时间进行优化,结果发现灭活时间与病毒的HAU相关,综合各因素判定马流感病毒灭活需48~72h。采用分子量切割技术提纯马流感病毒发现,与分子孔径大小为350kD的超滤膜相比,用1000kD提纯后的病毒悬液总蛋白含量较低,纯化效果较好。以1000kD的超滤膜包反复纯化三次即可达到纯化效果;以HAU粗略计算,病毒回收率可达100%。用经过提纯后的疫苗和未经提纯的疫苗免疫幼驹,发现经Millipore切向式过滤纯化后的疫苗免疫效果远好于不经纯化的疫苗。
OIE推荐疫苗应该含有美洲系和欧洲系的马流感病毒的疫苗株。本实验选用代表美洲系的XJ株和代表欧洲系的A/equine/Qinghai/1/94(H3N8)株(简称QH株),经过1000kD的超滤膜提纯后,将两株马流感病毒等量混合。然后与10%的铝胶佐剂制成HAU为210、29、28、27、26的H3N8亚型马流感病毒灭活疫苗,进行相关的动物实验。试验表明,制备的疫苗对豚鼠和幼驹安全、有效。豚鼠效力试验表明,各HAU的灭活疫苗能够刺激机体产生的相似的HI抗体。马的效力试验表明,各HAU的灭活疫苗在二免后第二周产生的HI抗体均在6log2以上,均能达到OIE要求,且几何平均抗体水平都比ProteqFlu疫苗产生的要高。对抗体消长规律的检测表明,各类疫苗均能在首免后第1周开始即产生抗体,并在二免后1~2周抗体水平达到高峰,随后缓慢下降,并在25周内抗体持续存在;三免后抗体产生及消长规律基本同第二次免疫。
Equine influenza vaccines were first developed in the 1960s, and are used widely for control of equine influenza. Therefore, equine influenza outbreak has been effectively controlled in the word. Until now, the commercial equine influenza vaccines developed by our country are unavailable, and majority horses in the mainland of China have never been vaccinated. Thus, Chinese horse industry continues to be threatened by equine influenza. From 2007 to 2008, Chinese mainland areas experienced a horse flu pandemic, which brought great disaster once again to the equine industry, gave a heavy blow to Chinese horse racing industry which will rise, and threatened the 2008 Beijing Olympic Equestrian Competition held at the project. With frequent contacts with the international competition and the development of the Chinese horse racing industry, the equine influenza prevention and control work in China has a long way to go, and vaccine development in China is imperative.
This dissertation studied the biological characteristics of two American lineage vaccine prototype, A/equine/xinjiang/3/2007(H3N8) (XJ isolation) and A/equine/Hubei/6/2008(H3N8) (HB isolation), which were isolated by our laboratory, and analyzed genetic characterization of HA gene of HB isolation, which provided references to the control of equine influenza in China. After 7 continuous passages in SPF chicken embryos, the HAU of XJ isolation stabilized at 27 to 28, while the HB isolation can be stabilized at 28 starting from the 1st passage. Two isolations can be neutralized by H3N8 subtype special antiserum; can agglutinate the red blood cell of pigs, horse, cattle, sheep, goat, donkey, rabbit, guinea pig, rat, mice and chicken; both were thermostabilization influenza virus; the infectivity still existed after treated by acid, alkali, chloroform and ether. After infected XJ isolation and HB isolation, foals can appear complete influenza clinical symptoms. The virus can be isolated from nasal swabs about during 1 week after infection, and specific antibody can be detected by HI assay about 1 week later after infection.
HA gene of HB isolation was cloned using RT-PCR and sequenced by Invitrogen Company. Comparing homology of H3N8 EIV HA gene By NCBI Blast, we found that the HA gene of HB isolation and A/equine/Newmarket/5/2003 (H3N8) showed a homology as high as 98.7%. HA protein amino acids sequence phylogenetic analysis showed that HB isolation belonged to Florida sub-lineage in American lineage equine H3N8.
The virus was inactivated by adding formalin to a final concentration of 0.05% and adding BPL to a final concentration of 0.025% shaking at 4°C for 48 to 72 hours. The results showed that equine H3N8 can be completely inactivated by both of two virus inactivators, and the virus HAU detected by HA assay unchanged during inactivation. The result of optimizing virus inactivation time of BPL implied that the inactivation time was relevant to HAU of EIV. Comprehensive considering all factors, we suggested that EIV inactivation time needs 48 to 72 hours. Using molecular weight cut off technology to purify equine H3N8, the results indicated that total protein content of viral suspension purified by 1000kD ultrafiltration membrane was lower than by 350kD ultrafiltration membrane. The aim of purification can be achieved by repeated purified three times using 1000kD ultrafiltration membrane, and virus recovery was up to 100% roughly estimated by HAU. When ponies were vaccinated purified vaccine and unpurified vaccine, the results suggested that the potency of vaccines purified by Millipore tangential flow filtration is much better than non-purified vaccines.
OIE suggests that both American lineage prototype and European lineage prototype should be included in the equine influenza vaccine. Equine H3N8 inactivated vaccine was prepared by combing 10% alhydrogel adjuvant with viruses mixture which was equally mixed American lineage vaccine prototype A/equine/xinjiang /3/2007(H3N8) and European lineage vaccine prototype A/equine/Qinghai/1/94(H3N8) (QH isolation) when both of virus isolations were purified by 1000kD ultrafiltration membrane. Inactivation vaccines had a graded HAU of 210, 29, 28, 27 and 26, The animal experiments showed that the inactivated vaccine was safety to guinea pigs and ponies. Serological responses of guinea pigs indicated that similar HI antibody level which was stimulated by graded level of XJ isolation and QH isolation can be produced. Serological responses in horses implied that the HI antibody titers were above in 6log2 at the second week after the second vaccination of all inactivated vaccines, which can meet the requirements of OIE that horses can be completely protected when the HI antibody titers were not less than 6log2. All inactivated vaccines can stimulate higher geometric antibody titers than Virus-based vector ProteqFlu vaccine which has been shown previously to protect horses against challenge infection. Antibody dynamics showed that antibody which was stimulated by all kinds of vaccines can be produced at the first week after the first vaccination, and reached peak at the first or second week after the second vaccination. Then, the antibody was slowly down, and persisted in 25 weeks after the second vaccination. Antibody dynamics after the third vaccination was the similar to the second vaccination.
引文
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