Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) To evaluate the performance of current US NDV real time diagnostics assays against new isolates of NDV. Approach (from AD-416) 1)The sensitivity and efficacy of the Matrix real time PCR test on 50 NDV isolates from diverse genotypes isolated worldwide will be tested using RT-PCR assays performed on RNA isolated from allantoic fluids under standard approved conditions. 2) The sensitivity and efficacy of the new SEPRL real time multiplex test on 50 NDV isolates from diverse genotypes isolated worldwide will be tested on RNA isolated from allantoic fluids using standard real time PCR approved conditions. 3) The sensitivity and efficacy of the fusion protein test will be tested on 50 NDV isolates using RNA isolated from allantoic fluids and standard approved real time conditions. 4) Sequencing of new isolates that are not detected with current methods will be done on PCR products obtained with NDV specific primers. This research relates to inhouse objective 2: Development of improved Newcastle disease control strategies addressing issues important to virus transmission, vaccines and vaccination, diagnostics, or international trade. Develop models to show vaccination is a viable method of controlling avian paramyxovirus outbreaks. The agreement has expired early in 2010 however as a result of the one year agreement significant progress has been made in characterizing Newcastle disease viruses. A total of 159 Newcastle disease viruses from 17 Countries (U.S., Canada, Vietnam, Russia, Pakistan, Indonesia, Korea, Belize, Mexico, Venezuela, South Africa, Peru, Honduras, Australia, Japan, Nigeria, Kenya) have been tested using three different U.S. rapid detection assays including two validated assays and one experimental test. Over 577 real time polymerase chain reaction (PCR) tests have been done. The performance of these tests for each one of the 159 isolates has been tabulated and analyzed. The fusion protein cleavage site of 132 new viruses have been evaluated and some of these sequenced. Virus that either failed the test or were likely to have significant differences in sensitivity were identified. The matrix protein gene of 4 new viruses was sequenced. All the sequences have been submitted to Genbank or are in the process of being made available. Three new real time PCR assays (primers and probes) were created and tested for identification of US pigeon, US cormorant, and Pakistani viruses that failed detection. Three new publications have been produced since the grant was approved. No further work is expected since the agreement has finished. Progress monitored via email, telephone conversations, and meetings. A final report was submitted and approved.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) To evaluate the performance of current US NDV real time diagnostics assays against new isolates of NDV. Approach (from AD-416) 1)The sensitivity and efficacy of the Matrix real time PCR test on 50 NDV isolates from diverse genotypes isolated worldwide will be tested using RT-PCR assays performed on RNA isolated from allantoic fluids under standard approved conditions. 2) The sensitivity and efficacy of the new SEPRL real time multiplex test on 50 NDV isolates from diverse genotypes isolated worldwide will be tested on RNA isolated from allantoic fluids using standard real time PCR approved conditions. 3) The sensitivity and efficacy of the fusion protein test will be tested on 50 NDV isolates using RNA isolated from allantoic fluids and standard approved real time conditions. 4) Sequencing of new isolates that are not detected with current methods will be done on PCR products obtained with NDV specific primers. Significant Activities that Support Special Target Populations The project is related to objective 2 of the in-house project: Development of improved Newcastle disease control strategies addressing issues important to virus transmission, vaccines and vaccination, diagnostics, or international trade. Develop models to show vaccine is a viable method of controlling avian paramyxovirus outbreaks. To evaluate the performance of current real time reverse transcriptase polymerase chain reaction (RT-PCR) test against novel viruses that are currently circulating in the U.S., Asia and Africa we evaluated the U.S. validated matrix gene and fusion gene real time polymerase chain reaction (PCR) assays against 50 different virulent Newcastle disease virus strains, called exotic Newcastle disease virus (ENDV), from birds in the U.S., Asia and Africa. We isolated ribonucleic acid (RNA) from all viruses above and tested them using the two current US rapid diagnostic tests: the matrix and the fusion gene real time PCR assays. Our current tests performed well by detecting virulent viruses from Asian and African origin, such as Vietnam, Korea, Indonesia, Kenya and Nigeria. The most significant discoveries from our work are the following: 1) The US cormorant samples obtained after 2005 (most significantly all the 2008 samples) failed the real time PCR fusion detection test as a result of genomic changes. Since the risk represented by those viruses is relatively high, effort are underway to develop new detection reagents for Cormorant viruses. We have communicated those findings to Animal and Plant Health Inspection Service (APHIS) National Veterinary Services Laboratories (NVSL).; 2) A virus responsible for a recent outbreak in the Dominican Republic (2008) also failed the test.; and 3) Other notable failure of the test is the inability to identify the virulent viruses that caused the outbreak in Australia. In summary, we have determined that the Newcastle disease multiplex test works very well to identify Newcastle disease virus (NDV) from waterfowl and poultry samples from Asia, Africa, Central and North America however the fusion test still needs to be improved to adapt to identify all virulent viruses. MONITORING: Continued communication between Southeast Poultry Research Laboratory and the cooperator has been accomplished through e-mail, phone calls and through a written report (six months after initiation of the agreement).
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