Source: UNIVERSITY OF MAINE submitted to NRP
MOLECULAR DIAGNOSTICS AND GENETIC CHARACTERIZATION OF FISH VIRUSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0192656
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Oct 1, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
BIOCHEMISTRY MICROBIOLOGY AND MOLECULAR BIOLOGY
Non Technical Summary
Control of infectious diseases is one of the most important factors in successful aquaculture. Diseases caused by viruses are particularly difficult to control. This project applies recent developments in biotechnology to the development of improved diagnostic assays for several fish viruses which cause serious diseases in aquaculture. Also the phylogenetic relationships of aquatic birnaviruses will be investigated based on genome sequence comparisons.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113711104050%
3113712110150%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3711 - Trout; 3712 - Salmon;

Field Of Science
1040 - Molecular biology; 1101 - Virology;
Goals / Objectives
Objective 1: Determine nucleotide and deduced amino acid sequences of genome segment B of a variety of isolates worldwide representing each serotype of aquatic birnavirus serogroup A. Investigate the genomic relationships among these viruses based on variations in genome segment B sequences. Objective 2: Conduct a collaborative investigation of the prevalence, geographical distribution, and indigenous strains of aquatic birnaviruses in Mexico with Dr. Cesar Ortega Santana, Universidad Autonoma Del Estado De Mexico. Objective 3: Continue to receive new aquatic birnavirus isolates from different hosts and geographical areas worldwide for genome sequence analysis and, with viruses obtained under Objective 2, expand the genomic sequence database and phylogenetic tree of this group of viruses. Objective 4: Continue to provide our genomic sequence database, RT-PCR and sequencing capabilities, and genomic typing expertise for aquatic birnaviruses to diagnostic laboratories and researchers who routinely require these services and/or research collaborations. Objective 5: Develop a RT-PCR assay for simultaneous general identification of ISAV and discrimination between the European and North American strains. Objective 6: Investigate genomic sequence variations among existing and new isolates of ISAV in Maine and Canada to provide information and specific molecular tools for use in molecular epidemiology to track the spread of specific strains of ISAV. Objective 7: Continue to provide RT-PCR, genomic sequencing and genomic typing assistance to federal and state governments as well as commercial fish disease diagnostic laboratories in detecting and identifying ISAV. Objective 8: Maintain the only laboratory in New England specializing in the identification and characterization of new fish viruses and new strains of existing viruses to assist government fish health management programs, commercial diagnostic laboratories, and the aquaculture industry.
Project Methods
Objective 1. Primers for RT-PCR amplification of the 2784 bp genome segment B of aquatic birnaviruses and the RT-PCR protocol conditions will be designed based on the published cDNA sequences and several partial sequences already obtained in our laboratory. PCR products from all viruses will be sequenced and nucleotide and amino acid sequence similarities will be compared using appropriate software programs. Objectives 2, 3, 4. New virus isolates will be sent to the University of Maine for genomic sequence analysis. Nucleotide and deduced amino acid sequences will be determined for the VP2 coding region (1,611 bp) of these viruses and their phylogenetic relationships to known virus strains determined using the procedures previously described. Objective 5. Two ISAV-specific oligonucleotide primer sets will be designed using the Primer Select Program of the Lasergene DNASTAR software package for ISAV based on the published genomic sequences of isolates from Norway, Scotland, and North America. One primer set will be designed to identify a genomic sequence unique to the European strains of ISAV and the other primer wet will be designed to recognize a sequence unique to the North American strains of ISAV. An initial protocol for a RT-PCR assay using these two primer sets will be designed using the DNASTAR program which will result in the amplification of separate, distinct PCR products for European and North American isolates. Objective 6. RT-PCR will be used to amplify cDNA from the HA gene of a variety of ISAV isolates from different geographic areas in New Brunswick, Canada, and Maine, United States. These PCR products will be sequenced and the sequences analyzed for small unique sequence differences comparable to those reported for Norwegian isolates. This information will be used to determine the likelihood of the occurrence of "substrains" or "genotypes" among ISAV isolates in North America. Objectives 7,8. Continue to provide RT-PCR, genomic sequencing and genomic typing assistance to federal and state governments as well as commercial fish disease diagnostic laboratories in detecting and identifying ISAV.

Progress 10/01/02 to 10/01/05

Outputs
The investigator has left the University and is unavailable to submit termination report.

Impacts
The investigator has left the University and is unavailable to submit termination report.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
A Reverse-transcriptase Polymerase Chain Reaction (RT-PCR) assay was developed and optimized for detecting and distinguishing both the North American and European strains of infectious salmon anemia virus (ISAV). ISAV is a serious pathogen of Atlantic salmon aquaculture in North America and Europe.

Impacts
The ability to rapidly distinguish between the European and North American strains of ISAV will enhance the diagnostic and monitoring programs established for the control of infectious salmon anemia(ISA) in Atlantic salmon aquaculture.

Publications

  • Ahne W, S. Blake, S. Essbauer, and B. L. Nicholson. 2003. Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Diseases of Aquatic Organisms 56:201-206.


Progress 10/01/02 to 09/30/03

Outputs
An RT-PCR assay was developed for the simultaneous general identification of infectious salmon anemia virus (ISAV) as well as the discrimination between the European and North American strains of ISAV.

Impacts
The ability to rapidly distinguish between the European and North American strains of ISAV will enhance the diagnostic and monitoring programs established for the control of infectious salmon anemia(ISA) in Atlantic salmon aquaculture.

Publications

  • Ahne, W., S. Blake, S. Essbauer, and B. L. Nicholson. 2003. Isolation and identification of aquatic birnaviruses from flounder (Pseudopleuronectes americanus) and mummichog (Fundulus heteroclitus) in the Chesapeake Bay, Virginia, USA. Diseases of Aquatic Organisms. Diseases of Aquatic Orgainisms 56:201-206
  • Ortega, Cesar S., R. Montes de Oca, David Groman, Carmencita Yason, Bruce Nicholson and Sharon Blake. 2002. Viral infectious pancreatic necrosis virus in farmed rainbow trout from Mexico. Journal of Aquatic Animal Health 14: 305-310.