Source: UNIV OF ARKANSAS submitted to
MOLECULAR TECHNIQUES FOR THE RAPID DIAGNOSIS OF VIRAL DISEASES IN CYPRINIDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0183483
Grant No.
(N/A)
Project No.
ARX05014
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 1999
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Goodwin, A. E.
Recipient Organization
UNIV OF ARKANSAS
(N/A)
PINE BLUFF,AR 71601
Performing Department
AQUACULTURE & FISHERIES
Non Technical Summary
Viral diseases in cyprinids are difficult to detect PCR technology will allow us to provide fast and an accurate diagnoses
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31137151101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3715 - Baitfish;

Field Of Science
1101 - Virology;
Goals / Objectives
1) To develop rapid diagnostic methods for newly discoveres viral diseases of cyprinids. 2) To validate those new assays using tissue cultures and experimentally-infected fish. 3) To determine the significance and outcome of viral infections of cultured bait and ornamental fish. 4) To determine the prevalence of these viruses in the aquaculture industry.
Project Methods
1) Development of PCR primers for the detection of important cyprinid viruses. 2) Validation of the primers and PCR protocols using experimentally infected tissue cultureed cells. 3) Identification of appropriate sample types for PCR. 4) Validation of PCR methods through comparison with traditional tissue culture techniques. 5) Diagnostic surveys of sick baitfish to determine clinical signs and the course of viral infections. 6) Diagnostic surveys of the baitfish industry to determine prevalence of viruses.

Progress 10/01/99 to 09/30/06

Outputs
In this project developed standard PCR assays for Chinese Grass Carp Reovirus (CGRV: know as Golden Shiner virus prior to our study), Koi Herpes Virus (KHV), Goldfish Hematopoetic Necrosis Virus (GHNV), Fathead Minnow Rhabdovirus (FHMRV), and Carp Edema Virus and used these assays to determine the incidence of these viruses in the US. We then designed and validated new quantitative PCR assays for CGRV and GHNV and are using these assays to differentiate between carriers and actively infected fish. These efforts led to the discovery of the American Grass Carp Reovirus (AGCRV) and a new goldfish reovirus (GRV). All of these assays are now used in routine clinical work in our laboratory and in other laboratories world wide. From this we have learned that 1) GHNV is an important pathogen of goldfish, 2) that CGRV is an important pathogen of grass carp and fathead minnows, but probably not of golden shiners, 3) that KHV can be harbored by other fish species and that the virus is not in AR aquaculture, 4) that FHMRV is present in the northern US, but not AR, 5) that AGCRV is a relative of the Golden Ide reovirus and can cause heavy mortality in grass carp during the winter, and 6) that there is a previously undescribed goldfish reovirus.

Impacts
The development of PCR assays for these important diseases has allowed us to determine which diseases are important, their geographic distributions, and how farms should be managed to avoid viral disease problems. As a result of these studies, farmers have made significant changes in biosecurity and culture methods that will reduce the risk of fish disease. Our findings regarding the rarity of CGRV and FHMRV and our development of sensitive detection methods has allowed farmers to access markets that might otherwise be limited due to disease concerns. Coupled with our Extension efforts, our work has helped to keep Arkansas, which produces the bulk of the cyprinid (carp and minnow) fish grown in North America, free of exotic diseases like KHV, Spring Viremia of Carp, and FHMRV. The work has resulted in 11 peer reviewed journal articles.

Publications

  • Janet V.Warg, Audrey L. Dikkeboom, Andrew E. Goodwin, Kevin Snekvik, John Whitney. (2007 currently e-pub, printed version upcoming) Comparison of multiple genes of spring viremia of carp viruses isolated in the United States. Virus Genes


Progress 01/01/05 to 09/30/05

Outputs
In previous years of this project we have developed standard PCR assays for Chinese Grass Carp Reovirus (CGRV: know as Golden Shiner virus prior to our study), Koi Herpes Virus (KHV), Goldfish Hematopoetic Necrosis Virus (GHNV), Fathead Minnow Rhabdovirus (FHMRV), and a Carp Edema Virus and used these assays to determine the incidence of these viruses in the US. This year we have designed and validated new quantitative PCR assays for CGRV and GHNV and are using these assays to differentiate between carriers and actively infected fish. Completed work has shown that GHNV is vertically transmitted through eggs and that a high percentage of broodstock are carriers of the virus. Improved assays for CGRV have demonstrated that CGRV is also a pathogen in fathead minnows, and we have shown that many of the aquareoviruses isolated from cyprinid fishes are not GHNV but instead are undescribed viruses of limited pathogenicity. Work is currently underway to characterize these viruses and to develop PCR assays for edemiological studies.

Impacts
The development of PCR assays for these important diseases has allowed us to determine which diseases are important, their geographic distributions, and how farms should be managed to avoid viral disease problems. As a result of these studies, farmers have made significant changes in biosecurity and culture methods that will reduce the risk of fish disease. Our findings regarding the rarity of CGRV and FHMRV and our development of sensitive detection methods has allowed farmers to access markets that might otherwise be limited due to disease concerns. Coupled with our Extension efforts, our work has helped to keep Arkansas, which produces the bulk of the cyprinid (carp and minnow) fish grown in North America, free of exotic diseases like KHV, Spring Viremia of Carp, and FHMRV.

Publications

  • Dikkeboom, A. L., Craig, R., Kurth, K. T., Marcquenski, S., Engel, M., Goodwin, A. E., Way, K., Stone, D.M. and C. Longshaw (2006) First Report of Spring Viremia of Carp Virus in feral Common Carp (Cyprinus carpio) in North America. Journal of Aquatic Animal Health. In Press.
  • Goodwin, A. E., Khoo, L., LaPatra, S. E., Bonar, C., Key, D. W., Garner, M., Hanson, L. (2006) Goldfish Hematopoietic Necrosis Herpesvirus (Cyprinid herpesvirus 2) In the USA: Molecular Confirmation of Isolates from Diseased Fish. Journal of Aquatic Animal Health 18:11 - 18
  • Andrew E. Goodwin, D. K. Nayak, and R. S. Bakal (2006) Natural infections of wild creek chubs (Semotilus atromaculatus ) and cultured fathead minnows (Pimephales promelas) by Chinese grass carp reovirus (Golden Shiner Virus). J. of Aquat. Anim. Health, in press.
  • A.E. Goodwin, G.E. Merry, J. Sadler (2006) Detection of the herpesviral hematopoietic necrosis disease agent (Cyprinid herpesvirus 2) in moribund and healthy goldfish: Validation of a quantitative PCR diagnostic method. Diseases of Aquatic Organisms.In press


Progress 01/01/04 to 12/31/04

Outputs
In previous years of this project we have developed PCR assays for Chinese Grass Carp Reovirus (CGRV: know as Golden Shiner virus prior to our study) and koi herpes virus (KHV). This year we have added (with collaborators) assays for Goldfish Hematopoetic Necrosis Virus (GHNV), Fathead Minnow Rhabdovirus (FHMRV), and a virus known in Japan as Fushu-Sho. Using these assays we have found that fathead minnows are impacted by CGRV and that infections by CGRV are quite rare in the baitfish industry. Using a published KHV assay we have shown that survivors of KHV carry the viral genome for several months. Using the assay for GHNV we have shown that this disease is present in the US and that it has been responsible for a number of major fish kills in private goldfish collections in several states of the northern US. Our work with FHMRV has shown that this virus is not present in the farmed baitfish industry. Our more recent work with Fushu-sho has demonstrated that the virus is present in the US, but its importance is not yet known.

Impacts
The development of PCR assays for these important diseases has allowed us to determine which diseases are important, their geographic distributions, and how farms should be managed to avoid viral disease problems. As a result of these studies, farmers have made significant changes in biosecurity and culture methods that will reduce the risk of fish disease. Our findings regarding the rarity of CGRV and FHMRV and our development of sensitive detection methods has allowed farmers to access markets that might otherwise be limited due to disease concerns. Coupled with our Extension efforts, our work has helped to keep Arkansas, which produces the bulk of the cyprinid (carp and minnow) fish grown in North America, free of exotic diseases like KHV, Spring Viremia of Carp, FHMRV, and GHNV.

Publications

  • Mathew E. McEntire, Luke R. Iwanowicz, A. E. Goodwin (2003) Molecular and clinical evidence that Golden Shiner Virus and Chinese Grass Carp Reovirus are a single promiscuous, cosmopolitan pathogen with multiple identities. Journal of Aquatic Animal Health. 15:175-181
  • Goodwin, A. E. (2002) First report of spring viremia of carp virus (SVCV) in North America. Journal of Aquatic Animal Health 14:161-164.
  • Andrew E. Goodwin, James E. Peterson, Theodore R. Meyers, David J. Money (2004) Transmission of exotic fish viruses: The relative risks of wild and cultured bait. Fisheries 29:19-23.


Progress 01/01/03 to 12/31/03

Outputs
Development of molecular methods (PCR) for the detection of Golden Shiner Virus has allowed us to investigate a group of Aquareoviruses isolated from golden shiners, grass carp, and fathead minnows. By DNA sequencing of the PCR products, we have shown that all of these are variants of GSV and that GSV is actually the Chinese Grass Carp Reovirus (CGRV). The best evidence is that GSV/CGRV is an exotic virus that was introduced into the US with grass carp imported by a Federal Agency in 1968. Results from this assay have also demonstrated that several other viruses that we have recently isolated are not GSV and may be novel. Work is currently underway to identify these viruses. We are also assisting USDA APHIS by culturing thousands of ornamental fish for viruses as part of a nationwide survey for SVCV. During that survey, we have identified several novel viruses and are now working toward developing diagnostic techniques for these potential pathogens. In the summer of 2004, we will use our GSV/CGRV assay to help farmers find management methods to prevent GSV-related losses.

Impacts
Our discovery that GSV is CGRV and that the virus produces significant mortality in several cultured fish species has led to changes in farm biosecurity that should reduce fish losses. Application of our diagnostic methods for GSV/CGRV, Koi Herpes Virus, and Spring Viremia of Carp, and our associated biosecurity education programs, have protected the Arkansas bait and ornamental fish industries from all incidence of these important pathogens and our test results are instrumental in facilitation interstate and international fish shipments.

Publications

  • Audrey L. Dikkeboom, Craig Radi, Kathy Toohey Kurth, Susan Marcquenski, Marty Engel, Andrew E. Goodwin, Keith Way, David M. Stone And Clare Longshaw ( 2004, in press) First Report of Spring Viremia of Carp Virus in Feral Common Carp (Cyprinus carpio) in North America. Journal of Aquatic Animal Health
  • Mathew E. McEntire, Luke R. Iwanowicz, and Andrew E. Goodwin. (2004, in press) Molecular, physical and clinical evidence that Golden Shiner Virus (GSV) and Grass Carp Reovirus (GCRV) are variants of the same virus. Journal of Aquatic Animal Health.
  • W. L. Gray, L. Mullis, S. Lapatra, and A.E. Goodwin (2002) Detection of koi herpesvirus DNA in tissues of infected fish. Journal of Fish Diseases 25:171-178.
  • Goodwin, A.E., and J. R. Winton (2003) Virology Chapter 10: Spring Viremia of Carp in American Fisheries Society, Fish Health Section. 2003. Suggested Procedures For The Detection And Identification Of Certain Finfish And Shellfish Pathogens, 5th edition. American Fisheries Society, Bethesda, Maryland.


Progress 01/01/02 to 12/31/02

Outputs
The molecular methods that were developed in 2001 are now being applied to the epidemiology of viral fish diseases. The Golden Shiner Virus (GSV) PCR has allowed us to investigate a group of reoviruses isolated from cyprinid fish. These viruses appeared to be GSV-like by electron microscopy, but do not react with our GSV primers. We now recognize that these isolated are novel. As part of our research project, we accept fish from all over the US for viral screening. In July we discovered Spring Viremia of Carp Virus in samples from North Carolina. This is the first isolation of this OIE reportable virus in North America. We established the identity of this isolate, characterized its pathogenicity, and collaborated with others to look for SVCV in wild fish.

Impacts
The KHV PCR developed last year is now widely used in KHV diagnosis. This is an extremely important disease of ornamental koi. Our discovery of SVCV in the US led to an immediate quarantine of the infected farm and efforts to prevent the spread of this important pathogen. Farmers in Arkansas have initiated a biosecurity and screening program to prevent the introduction of SVCV into Arkansas.

Publications

  • A.E. Goodwin (2002) First Report of Spring Viremia of Carp Virus (SVCV) in North America. Journal of Aquatic Animal Health. 14:161-164
  • L. R. Iwanowicz and A. E. Goodwin (2002) A New Bacilliform Fathead Minnow Rhabdovirus That Produces Syncytia In Tissue Culture. Archives of Virology 147 (5):899-915


Progress 01/01/01 to 12/31/01

Outputs
We isolated a group of viruses from baitfish and grass carp and identified them according to type. We found one rhabdovirus (FHMRV), several reoviruses, and a virus of type unknown (as of December of 2001). Using a published sequence for Chinese Grass Carp Reovirus, we developed PCR primers that are useful in the detection of GSV and all of our other reoviruses. We then sequenced the cDNA of the reoviruses to determine their relatedness and to verify that the PCR primers were amplifying the correct target sequences. We have also collaborated with another laboratory (Dr. Wayne Gray of UAMS) to develop a PCR method for Koi Herpes Virus (KHV). Summary of key findings and results by study and overall. A. Our isolates of shiner viruses collected from small fish and during the winter are actually golden shiner virus (GSV), a virus that has been described as a pathogen of large shiners during the summer. B. The golden shiner virus and carp reovirus isolates are almost identical to each other and to the Chinese Grass Carp Reovirus (an important virus in China). C. That GSV and Koi Herpes virus can be reliable detected in tissue culture using PCR. D. That there are at least two additional viruses (the rhabdovirus and recent unknown) that may be important in Arkansas aquaculture.

Impacts
Using traditional methods, viral diseases are so difficult and time consuming to diagnose that we have been unsure as to how important viral diseases may be in our cultured baitfish and Asian carps. Our new methods reduce the time required to diagnose these viral diseases from 6 weeks to 1 week and allow us to identify the particular virus involved. In the case of very infectious viruses like KHV and FHMRV, this new capability has proven important in preventing the introduction and spread of these diseases. With GSV, we know the virus to be widespread, but we are unsure as to its impact on fish health. These new PCR tools will allow us to determine if farmers need to manage for this disease.

Publications

  • Wayne L. Gray, Lisa Mullis, Scott Lapatra, and Andy Goodwin (2002:in press) Analysis of the Koi Herpesvirus (KHV) Genome and Detection of Viral DNA in Tissues of Infected Fish. Journal of Fish Diseases.
  • Iwanowicz, L, and Goodwin, A.E. (2002: in press) A New Bacilliform Fathead Minnow Rhabdovirus That Produces Syncytia In Tissue Culture. Archives of virology.


Progress 01/01/00 to 12/31/00

Outputs
We have completed the first year of a 5-year study. Thus far we have completed the characterization of our cyprinid viruses and have produced and purified quantities of each virus that should be sufficient to do the rest of our work. This step was very laborious and involved several hundred tissue culture flasks and purification of the virus using sucrose gradient centrifugation. Because the viral material is so expensive to produce, we have been thoroughly testing the reagents and protocols that will be involved in the rest of our study. Development of PCR assays for these RNA viruses involves RNA purification, reverse transcription, cloning, and sequencing. We have been working to verify that all of the protocols and reagents work on purified catfish mRNA, then we will move onto the viral material. This work is almost complete and we anticipate that cloning of the viral RNAs will be accomplished in the early months of 2001.

Impacts
Current tissue culture-based methods for diagnosing baitfish viruses require 2-4 weeks and only indicate that a virus is present. New PCR techniques will allow us to diagnose a viral infection in one day and will also identify the virus involved. This will allow us to study the incidence of viral diseases in the industry and help farmers to prevent outbreaks.

Publications

  • No publications reported this period


Progress 11/01/99 to 12/31/99

Outputs
This project has been underway for two months. During that time we have purified large quantities of several novel aquareoviruses and a rhabdovirus. These will provide the raw material for sequencing.

Impacts
PCR techniques, based on the results of this study, will allow us to diagnose and manage viral diseases in bait and ornamental fish

Publications

  • No publications reported this period