Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing
Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing
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DOI：
10.1128/jcm.01142-12
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发表时间：
2012-09-01
影响因子：
9.4
通讯作者：
Guerin, Jean-Luc
中科院分区：
文献类型：
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作者：
Croville, Guillaume;Soubies, Sebastien Mathieu;Guerin, Jean-Luc
禽流感病毒(AIV)通过神经氨酸酶(NA)茎缺失从水禽适应家禽已有报道。然而，到目前为止，病毒是如何经历这种进化的还不清楚。我们通过对鸭和火鸡的低致病力禽流感病毒进行焦磷酸测序来解决这个问题。在H6N1疫情爆发之初，鸭子和火鸡就被采集了样本，8天后，火鸡再次被拭子擦拭。通过Sanger测序证实火鸡的NA茎缺失。为了进一步研究病毒的进化，进行了454次焦磷酸测序：对于每组样本，产生并比对了多达41,500个约400bp的读数。对鸭病毒和火鸡病毒之间的遗传多态进行了全基因组追踪。在鸭子粪便中检测到NA缺失的不到2%，但在同时收集的火鸡气管样本中检测到100%的NA缺失。8天后，在火鸡的NA中观察到了进一步的长度变化。同样，血凝素(HA)基因上出现了少数突变，替换主要发生在球形头部的受体结合部位。这些关键的变化表明火鸡的进化压力很大。下一代测序技术性能的提高应该使我们能够监测禽流感病毒的基因组多样性和禽类中潜在致病变异的早期出现。这项基于454焦磷酸测序的研究表明，NA缺失是AIV从水禽到家禽适应的一个例子，发生在病毒突变体的选择上，而不是从头出现。
Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.