Development Of A Fluorogenic Quantitative PCR Technique For Quantification Of HCLV And Genomic Sequence Determination And Analysis Of NDV HB92 Strain

Classical swine fever (CSF), the causative agent of which is classical swine fever virus (CSFV), is a highly contagious disease of domestic pigs that has been responsible for large economic losses in different parts of the world. Vaccination against CSF is the central means to the disease control. Hog Cholera Lapinized Virus (HCLV) is a safe and efficacious vaccine and has played an important role in disease control all over the world.The high or low of the titers of the vaccine is the key index of whether if it can be used to pigs. Because of its noncytopathogenicity and its low proliferation in cells,the rabbit fever reaction has been used up to now to estimate virus titers. The disadvantage of the method is the result of the titers can be affected by the weather and the different status of the rabbits, and it is costly, time-consuming, and requires handling of laboratory animals, especially, it can not perform the acute quantification.A rapid and reproducible method was first established for assessment of HCLV loads in CSF vaccine using Flurogenic Quantitative PCR (FQ-PCR) combining LightCycler sequence detection system. The method contains a pair of primers and an internal daul-labled fluorogenic probe spanning the part of 5' noncoding region (5'NCR) of HCLV, the use of such a probe combined with the 5'-3' nuclease activity of Tag polymerase allows direct quantification of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase- of the PCR. The sensitivity of the assay was 102 copies per reaction. The assay is linear within 6-log dynamic rang. The coefficient of variation (CV) of the standard of Ct value is 2.3%-5.1% (n =10); The CV ofIVvaccine sample is 0.85%-2.8% in intra-assay and 2.5%-7.3% in inter-assay (n=5), respectively; The CV of the same sample in different RNA isolation and reverse transcription is 5.0 %( n=5). Nine vaccines were quantified by this method and give similar but more accurate results compared to the conventional rabbit fever reaction. The entire assay, including RNA isolation, reverse transcription, and quantification, could be completed within 4 hours. In conclusion, the high sensitivity, simplicity, and reproducibility of the HCLV RNA quantification which allows the screening of large numbers of samples, combined with its wide dynamic rang, makes this method especially suitable for evaluating the viral loads and guiding how to confect the vaccine. It also provides a novel and simple research tool for CSFV research.Abstract 2: Newcastle disease (ND) is a serious illness that primarily affects birds, particularly chickens. The causative agent of the disease is Newcastle disease virus (NDV).The disease is distributed worldwide and can cause severe economic losses in the poultry.The application of the avirulent and heat-resistance vaccine strain has played a leading role in the disease control, but the biologic background of the vaccine is still not clearly known.The sequence of the newly avirulent, heat-resistance HB92 strain which was acquired from V4 strain by heat cultivation and cytophagic purification was determined in this article. Eight pairs of primers covering the entire genome was designed on the bases of Bl strain isolated Takaaki. Eight overlapping genomic cDNA fragments were amplified with RT-PCR method and cloned into pGEM-T vectors. Each clone was sequenced and eventually spliced into a full-length genomic cDNA. The complete genomic sequence of NDV HB92 strains comprises 15186 nucleotides (nts) and includes six major open reading frames (ORF) encode the viral NP, P, M, F, HN and L proteins. The leader region, situated at the 3' end of the genomic RNA, comprises 55 nucleotides, and the trailer region, located at the 5' end, is 114 nucleotides in length. The nucleotide homology of the genomic sequence of strain HB92 to that of strain ZJ1 was found to be 83.9%, and to avirulent vaccine strain La Sota and Bl, both homology to HB92 were 94.0%. We also compared the homology of the nucleotides and amino acids sequence o...