Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/19/2007
Publication Date: 4/3/2007
Citation: Kim, L.M., King, D.J., Suarez, D.L., Wong, C.W., Afonso, C.L. 2007. Characterization of Class I Newcastle disease virus isolates from Hong Kong bird markets and detection using real-time reverse transcription PCR. Journal of Clinical Microbiology. 45:1310-1314. Interpretive Summary: Newcastle disease is an important disease of poultry worldwide and is caused by a virus, avian paramyxovirus, serotype 1. In its most severe form, the virus causes a high rate of serious disease and death in infected chickens. Although this disease is considered to be a foreign animal disease in the United States, outbreaks have occurred, the most recent in California during 2002. Rapid diagnosis and monitoring are important to the control of this disease. A rapid and sensitive diagnostic test for Newcastle disease, known as a real-time RT-PCR test, has recently become available and has aided efforts to control this disease. However, some strains of the Newcastle disease virus identified in Hong Kong and the US do not appear to be consistently detected with this test. This study characterized these viruses to determine why they were not detected by the current test. Also, in order to improve methods of monitoring for the occurrence of this disease, a complementary real-time RT-PCR test was developed to detect these viruses from Hong Kong. Further application to other viruses similar to the Hong Kong viruses is discussed.
Technical Abstract: Of twenty-three viral isolates from Hong Kong bird market poultry samples (2003-05) that hemmagglutinated chicken red blood cells and demonstrated Newcastle disease virus (NDV) specific hemagglutination-inhibition activity with polyclonal antiserum, 21 tested negative for avian influenza virus and NDV by real-time RT-PCR (RRT-PCR). Primers to sequence the genomic region encoding the fusion protein cleavage site were designed and phylogenetic analysis of that region showed that the RRT-PCR negative viruses were NDV strains related to the recently described Class I group of NDV. The Class I viruses include primarily lentogenic wild bird origin viruses that are phylogenetically divergent from most domestic poultry isolates. The Hong Kong viruses were lentogenic as predicted by the fusion protein cleavage site and phylogenetically clustered separately from other previously characterized Class I viruses. Investigation of the RRT-PCR assay discrepancy was confirmed by examining the nucleotide sequence of Class I and Class II viruses, which demonstrated the presence of multiple mismatches at the assay probe site which targets the matrix (M) gene. A RRT-PCR assay targeted at the polymerase (L) gene was developed to efficiently detect these Class I viruses and to complement the existing validated United States Department of Agriculture’s (USDA) RRT-PCR NDV M gene assay. Current evaluation indicates that Class I isolates from Hong Kong are readily identified with the new L gene assay, while many are not detected by the USDA-validated M gene RRT-PCR assay.