Farsang A, Ros C, Renström Lena H M, Baule Claudia, Soós T, Belák S
Institute for Veterinary Medicinal Products, H-1107, Budapest, Szállás utca 8, Hungary.
Avian Pathol. 2002 Jun;31(3):229-36. doi: 10.1080/03079450220136530.
To improve the detection and molecular identification of infectious bronchitis virus (avian coronavirus), two reverse transcriptase-polymerase chain reaction (PCR) assays were developed. As 'diagnostic#10; PCR', a set of consensus nested primers was selected from highly conserved stretches of the nucleocapsid (N) gene. As 'phylogeny' PCR, a fragment of the spike protein gene (S1) was amplified and the PCR products were directly sequenced. To study the phylogenetic relationships of the viruses from various outbreaks, studies of molecular epizootiology were performed in Sweden, a Nordic region, where the occurrence of natural cases of the disease is relatively low and the occasional use of live vaccine(s) is well recorded and monitored. The disease appeared in the region in 1994, associated with production problems among layers of various ages. During outbreaks in 1995 and 1997, both layers and broilers were affected. To reduce losses, a live attenuated vaccine has been applied since 1997. By examining 12 cases between 1994 and 1998, molecular epizootiology revealed that, before 1997, the viruses had gene sequences very similar to strains of the Massachusetts serotype. However, comparative sequence analysis of the S1 gene revealed that the identity was not 100% to any of the strains of this serotype that we analysed. A virus related to the Dutch-type strain, D274, was also identified on one farm. Surprisingly, from 1997, the year that vaccination commenced with a live Massachusetts serotype vaccine, the majority of viruses detected had S1 sequences identical to the live Massachusetts vaccine strain. This genetic relation to the vaccine virus was also confirmed by N gene sequence analysis. The studies of molecular epizootiology reveal a strong probability that the vaccination had lead to the spread of the vaccine virus, causing various disease manifestations and a confusing epizootiological situation in the poultry population.
为提高传染性支气管炎病毒（禽冠状病毒）的检测及分子鉴定水平，开发了两种逆转录-聚合酶链反应（PCR）检测方法。作为“诊断#10;PCR”，从核衣壳（N）基因高度保守区域选取了一组共有巢式引物。作为“系统发育”PCR，扩增了刺突蛋白基因（S1）的一个片段，并对PCR产物进行直接测序。为研究不同疫情中病毒的系统发育关系，在瑞典这个北欧地区开展了分子流行病学研究，该地区自然发病病例相对较少，且对活疫苗的偶尔使用有详细记录和监测。该病于1994年在该地区出现，与各年龄段蛋鸡的生产问题有关。在1995年和1997年的疫情中，蛋鸡和肉鸡均受到影响。为减少损失，自1997年起使用了活的减毒疫苗。通过检查1994年至1998年期间的12个病例，分子流行病学研究表明，1997年之前，病毒的基因序列与马萨诸塞血清型毒株非常相似。然而，对S1基因的比较序列分析显示，与我们分析的该血清型任何毒株的同一性并非100%。在一个农场还鉴定出一种与荷兰型毒株D274相关的病毒。令人惊讶的是，从1997年开始使用马萨诸塞血清型活疫苗进行免疫接种后，检测到的大多数病毒的S1序列与马萨诸塞活疫苗株相同。N基因序列分析也证实了与疫苗病毒的这种遗传关系。分子流行病学研究表明，极有可能是疫苗接种导致了疫苗病毒的传播，在家禽群体中引发了各种疾病表现，并造成了令人困惑的流行病学情况。
