INFLUENZA REASSORTANT VIRUS--BIOLOGY AND GENETICS

具有更大产量潜力的流感病毒疫苗候选株 比自然发生的野生型甲型流感病毒株 通过将野生型菌株用一株 流感A/波多黎各/8/34（PR 8），能够赋予“高” 产量”表型。本实验测定了一种具有神经氨酸酶（NA）和血凝素（HA）活性的 野生型菌株是通过抑制携带 通过在抗血清存在下孵育PR 8的HA和NA 生产PR 8。有了这种策略， 所得病毒体的次要组分可能仍然包括基因型 或PR 8 HA或NA的表型。为了开发一种灵敏的方法， 鉴定PR 8基因组是否可以是持久的，聚合酶 PCR技术已被用于HA的鉴定 以及来自H1N1和H3 N2病毒的NA基因。引物的特异性 首先确认了HA和NA的DNA聚合选择 使用一系列H1N1和H3 N2病毒。随后，H1N1和 H3 N2病毒以不同比例混合，以确定限度 检测方法的NA。一次30个周期的 聚合，可以在1000个N1中检测到1个N2病毒粒子 病毒粒子使用相同的方法进行额外的30个周期 一组引物或一组用于包含在所述引物中的序列的引物。 从第一组引物的产物中，可以增加 灵敏度提高了10倍，但并不一致。通过PCR， 能够以1：1000的比例具体识别次要组分- 1：10000。与此形成鲜明对比的是， 神经氨酸酶通过用“特异性”抑制酶活性 抗体，并不总是能够识别N1和 N2病毒体，即使在1/10的比例。结果表明，PCR 是一种更灵敏的检测基因混合流感的方法 病毒种群，PCR技术可以是非常有用的， 程序来生产revertant病毒。

Influenza virus vaccine candidate strains with greater yield potential than the naturally occurring wild type strains of influenza A virus have been produced by reassorting the wild type strain with a strain of influenza A/Puerto Rico/8/34 (PR8) with the ability to confer the "high yield" phenotype. The hemagglutinin (HA) and neuraminidase (NA) of a wild type strain are selected by suppressing the progeny virions bearing the HA and NA of PR8 by incubation in the presence of an antiserum produced against PR8. With this strategy, the possibility exists that a minor component of the resulting virions may still include the genotype or phenotype of PR8 HA or NA. In order to develop a sensitive method for identifying whether the PR8 genome may be persistent, the polymerase chain reaction (PCR) technique has been adapted for identification of HA and NA genes from H1N1 and H3N2 viruses. The specificity of the primers chosen for DNA polymerization was first confirmed for both HA's and NA's using a battery of H1N1 viruses and H3N2 viruses. Subsequently, H1N1 and H3N2 viruses were mixed in varying ratio in order to determine the limit of detection of the method for NA. With a single session of 30 cycles of polymerization, it was possible to detect 1 N2 virion in 1000 N1 virions. With an additional session of 30 cycles using either the same set of primers or a set of primers for a sequence contained within the product from the first set of primers, it was possible to increase the sensitivity by 10 fold, although not consistently. Thus, by PCR it was possible to specifically identify a minor component at ratios of 1:1000- 1:10000. In marked contrast, the standard method of identification of neuraminidase by inhibition of enzymatic activity with a "specific" antibody, was not consistently capable of identifying mixtures of N1 and N2 virions even at ratios of 1 part in 10. The results indicate that PCR is a more sensitive method of detection of genetically mixed influenza virus populations, and that PCR techniques can be very useful in a program to produce reassortant viruses.