DEVELOPMENT, STANDARDIZATION AND USE OF INFLUENZA VIRUS VACCINES

A/Shangdong/9/93（S93 [H3 N2]）和A/Beijing/32/92的血凝素 (B32[H3 N2]）通过动物和人的血清学应答鉴定 血清与以前的H3 N2毒株明显不同，因此 新疫苗的候选人。 流感病毒与 使用A/Puerto产生增加的在卵中复制的能力 Rico/8/34（PR 8 [H1N1]）与S93或B32结合以提供表面 来源于血凝素（HA）和神经氨酸酶（NA）的糖蛋白 野生型菌株的基因。 为了提高确定性， 鉴定回复突变体中单个基因的起源， 改良的聚合酶链反应方法，使所有八个 病毒基因片段可以用一组DNA同时扩增， 用于逆转录病毒RNA的寡核苷酸引物 和随后的互补DNA的扩增。 的相对 大基因片段的扩增效率显著提高 通过调节加入到反应混合物中的二甲基亚砜， 几乎等摩尔浓度的每一个基因片段， 恢复 尽管HA和NA基因对于 通过琼脂糖迁移率对来源菌株进行阳性鉴定， 其他六个基因在大多数甲型流感病毒株中变异较小。 因此，开发了单个基因片段的限制性作图 关键基因 例如，具有针对一种或多种蛋白质的特异性的限制性内切酶， 基因片段7（[GS 7]）中的一个位点， 在鸡蛋中的复制）被鉴定为PR 8，现代H1N1毒株， 目前的H3 N2病毒株，以便能够对病毒株进行阳性鉴定 混元 作为限制性位点分析的补充， GS 7的5-端可变区序列为 测定 相比 与其他两个实验室报告的PR 8菌株GS 7的序列相比， 这里用于引用的PR 8是介于公布的 序列的 A/Beijing/353/89和B 92的相似序列分析（其中 代表H3 N2 HA的最新变化）与已公布的 20世纪70年代初和70年代末H_3N_2病毒株分离情况 表明H3 N2毒株中的GS 7在25年内保持了很好的保守性。 该方法的快速、灵敏和相对简单， 它们用于制备用于疫苗的流感抑制剂， 潜在的混合人群或流感病毒株的污染。 限制性酶切作图及可变基因组测序策略 这些基因正在扩展到其他基因。

The hemagglutinins of A Shangdong/9/93 (S93 [H3N2]) and A/Beijing/32/92 (B32 [H3N2]) were identified by serologic responses with animal and human sera to be markedly different from previous H3N2 strains, and therefore candidates for new vaccines. Reassortant influenza viruses with increased ability to replicate in eggs were produces using A/Puerto Rico/8/34 (PR8 [H1N1]) combined with S93 or B32 to provide surface glycoproteins derived from the hemagglutinin (HA) and neuraminidase (NA) genes of the wild type strains. In order to improve certainty of identification of the origin of individual genes in the reassortants, a modified polymerase chain reaction method was developed so that all eight viral gene segments could be amplified simultaneously with one set of oligonucleotide primers for both reverse transcription of the viral RNA and subsequent amplification of the complementary DNA. The relative efficiency of amplification of larger gene segments was markedly improved by adjustment of dimenthylsulfoxide added to the reaction mixture, so that nearly equimolar concentrations of each gene segment could be recovered. Although HA and NA genes are sufficiently different for positive identification of strain of origin by mobility in agarose, the other six genes are less variable among most influenza A strains. Therefore, restriction mapping of individual gene segments was developed for key genes. For example, restriction enzymes with specificity for a single site in gene segment 7 ([GS7]) one of the genes critical to replication in eggs) were identified for PR8, for modern H1N1 strains and for current H3N2 strains to permit positive identification of the strain of origin. As a supplement to the restriction site analyses, the sequence of the variable 5~ end of GS7 was determined. In comparison to the sequences reported by two other labs for GS7 from PR8, the strain of PR8 used here for reassorting is intermediate between the published sequences. Similar sequence analysis for A/Beijing/353/89 and B92 (which represent recent change in the H3N2 HA) in comparison to published information for H3N2 strains isolated during early and late 1970~s indicated that GS7 in H3N2 strains has well been conserved over 25 years. The rapidity, sensitivity, and relative simplicity of the methods suits them to preparing influenza reassortants for vaccines and to identifying potential mixed populations or contamination of influenza virus strains. The strategy of restriction mapping and sequencing variable genomic regions is being extended to other genes.