UNITED STATES DEPARTMENT OF AGRICULTURE

ANIMAL AND PLANT HEALTH INSPECTION SERVICE

VETERINARY SERVICES

THE CENTER FOR VETERINARY BIOLOGICS

Environmental Assessment

For Issuance of a Permit for Distribution and Sale 

of an Imported Infectious Hematopoietic Necrosis 

Virus Vaccine, DNA

August 2013

Prepared by:

United States Department of Agriculture

Animal and Plant Health Inspection Service

Veterinary Services

The Center for Veterinary Biologics

_________________________________________________________________________________________________

__________________

Environmental Assessment

Permit for Distribution and Sale of an Imported Infectious Hematopoietic Necrosis Virus

Vaccine, DNA
Environmental Assessment

For Issuance of a Permit for Distribution and Sale of an Imported 

Infectious Hematopoietic Necrosis Virus Vaccine, DNA 

I. Proposed Action

APHIS is considering granting a permit for distribution and sale in the United States of

an Infectious Hematopoietic Necrosis Virus Vaccine, DNA, for use in healthy salmonids

weighing at least 30 grams (1.058 ounce). Novartis Animal Health US, Inc., Larchwood, 

Iowa, has requested authorization to provide the vaccine as an aid in the prevention of 

disease caused by infectious hematopoietic necrosis virus (IHNV) in salmon and trout. 

Under the provisions of the Virus-Serum-Toxin Act of 1913, as amended in 1985, the 

USDA must ensure that veterinary biologics are pure, safe, potent, and efficacious and 

not worthless, contaminated, dangerous, or harmful. Biological products produced in 

other countries may be imported into the United States for research and evaluation, transit 

shipment, or general sale and distribution, in accordance with Title 9, Code of Federal 

Regulations, Part 104. Products imported under a Permit for Distribution and Sale must 

meet the same requirements as those required for licensed (domestically produced) 

products. Accordingly, APHIS has conducted a risk analysis of this product and has 

concluded that the safety risks to animals, public health, and the environment are low. A 

copy of the firm’s risk analysis with confidential business information redacted is 

available online and by request, as indicated in the Federal Register Notice announcing 

the proposed issuance of a permit. The APHIS risk analysis covers the firm’s document, 

but also includes review of other studies and tests conducted by the firm, the 

confirmatory testing completed by APHIS personnel on the Master Seed to confirm the 

vaccine’s purity, identity, and expression of immunogenic protein, and results of literary

and regulatory review regarding this and similar previously tested or licensed products.

II. Background

IHNV causes an acute rhabdovirus infection of salmonids resulting in high mortality

among fry and fingerlings (young or small fish) at water temperatures less than 12oC 

(53.6oF). IHNV is not considered to be pathogenic for people. In this vaccine, only the 

glycoprotein (G) gene of IHNV is present, expressing the surface protein of the virus 

envelope. It is not associated with virulence and does not encode a toxin, enzyme, or 

allergen.

The confirmatory testing at the Center for Veterinary Biologics (CVB) laboratory for the 

Master Seed (MS) bacterial host containing the recombinant plasmid was satisfactory. 

CVB laboratory characterization of the MS included identity testing, sequence analysis, 

gene expression, and purity testing. 

The vaccine made from the plasmid DNA is licensed in Canada. Aqua Health Ltd., a 

division of Novartis Animal Health Canada, Inc., developed the vaccine (trade name 
APEX-IHNTM). It has been commercially available in Canada since 2005 and has been 

used in field trials there since 2003. 

The imported IHNV vaccine to be used under permit for distribution and sale is a 

replication-incompetent plasmid which has been genetically modified to contain an

immunogenic gene. The route of administration for this vaccine is intramuscular, given

by trained personnel in the area immediately anterior and lateral to the dorsal fin.

Plasmid DNA has been used previously to express foreign genes for vaccine 

development. Examples of USDA-licensed plasmid DNA vaccines include West Nile 

Virus Vaccine licensed in 2005, and Canine Melanoma Vaccine licensed in 2006. No 

safety issues have arisen from the use of these plasmid DNA vaccines. 

III. Need for the Proposed Action

There are no infectious hematopoietic necrosis virus vaccines approved for use in fish. A 

replication-incompetent DNA vaccine can potentially be both safe and efficacious, 

inducing a protective immune response (Kurath, et al. 2006; Garver, et al. 2005). 

IV. Areas of Concerns

The three areas of concern to APHIS are: 1) animal safety, 2) public health, and 3) 

environmental safety. APHIS has conducted its own risk analysis to assess whether risks 

are associated with the proposal to issue a permit for this imported vaccine. The safety 

characteristics of the vaccine have been thoroughly evaluated. The conclusions derived 

from the risk analysis for each of the areas of concern are summarized below.

A. Animal Safety

The risk to animals is low. 

 In a contained safety study conducted in Maine, nine hundred salmon weighing 

between 10 and 20 grams were vaccinated. Six buckets of 25 fish each were 

netted and placed into each of 6 holding tanks. After a 31-day acclimation period, 

the 150 fish from each tank were anaesthetized and randomly assigned to be 

injected intramuscularly with either saline control or one of two serials. The 

salmon were observed for 21 days after vaccination. There were no adverse 

events attributable to vaccination.

 There is a long history of safe use in salmonids in Canada. The firm reports that 

from 2003 to 2010, nearly 53 million fish in Canada were vaccinated without 

significant adverse effects.

 The vaccine is not a risk for target or non-target species because it cannot 

replicate in eukaryotic cells, is not infectious, and is highly purified. Reversion to 

virulence and shedding are not safety concerns applicable to this DNA vaccine. 

Should inadvertent ingestion occur, the DNA is readily degraded without negative 

effect to the host or non-target species.
 Regarding any potential for integration, it has been estimated that the probability

of a DNA molecule to integrate into the chromosome of muscle cells after 

intramuscular injection is at least 3000x less than the natural mutation rate 

(Martin, et al.1999; Ledwith, et al. 2000). 

 In persistence studies conducted by the firm, within fourteen days following a 2x 

dose vaccination, no plasmid DNA was detected in major tissues. The tissues 

tested included the liver, spleen, head, kidney, gonad, and gut lumen. In muscle 

tissue at the site of injection, the copy number of plasmid (1.8 x 1012) decreased 

greatly so that by day 251, only 0.01% of the plasmid remained. A 1x dose 

resulted in a 2.5-fold less copy number at day 252, or a 2.0x10-9 % reduction in 

copy number, a negligible amount.

B. Public Health

The risk to public health is likewise low. 

 The plasmid has no virulence-associated genes, and no known mobility or 

integration motifs which would allow integration into the human genome.

 Only qualified personnel, knowledgeable in the art of inoculation and prevention 

of accidental exposure, will be administering the DNA vaccine. Training, 

experience, and the use of finger guards attached to the repeating syringe greatly 

decrease the probability of human exposure.

 Although the safety of this specific vaccine has not been evaluated in humans and 

is therefore unknown, accidental human injection is not expected to cause adverse 

effects. Many doses of various DNA vaccines using a plasmid vector have been 

administered to animals, including dogs, mice, guinea pigs, and horses 

(Grosenbaugh, et al. 2011; Martin, et al.1999; Ledwith, et al. 2000; Manam, et al. 

2000; Davis, et al. 2001). No significant adverse events have been reported for

animals receiving those vaccines or the humans administering them. Regarding 

this IHNV vaccine, there was one case of human self-injection in 2009 which 

resulted in no negative impact to the patient.

 Numerous DNA vaccines have entered human clinical trials (Liu & Ulmer, 2005). 

There has been no evidence of autoimmunity, immunological tolerance, or 

integration in these various trials. The dose for fish (10 μg) of this vaccine is 

much lower than the dose of similar plasmid DNA vaccines used in human 

clinical trials (10x-30x dose) with no adverse events.

 Minute quantities of plasmid DNA in muscle tissue at the injection site were 

detected in the firm’s persistence studies, out to day 728 post-vaccination, past the 

time of marketing. However, it is known that plasmid DNA is rapidly degraded 

in the stomach by gastric acid, and in the duodenum by pancreatic and bile 

secretions (Maturin & Curtiss, 1977) and represents an infinitesimal amount 

relative to salmon genomic DNA that is routinely consumed. 

C. Environmental Safety

The risks to the environment are low. 
 The risks to environmental safety are low, given that the vaccine is based on 

highly purified and well-characterized plasmid DNA that does not replicate in 

vaccinated animals.

 The vaccine, which has no adjuvant, is administered to salmonids at the hatchery

in artificial tank-based rearing systems.

 In the unlikely event the vaccine is released in the environment, the plasmid will 

not be maintained without kanamycin selection pressure, an antibiotic not used in 

agricultural or human health applications. The plasmid construct cannot readily 

infect bacteria without prior lab-based chemical or physical treatment of the 

bacteria to generate cells competent to take up plasmids. 

 The plasmid is unstable in the environment and exposure to nucleases, pH 

variations, oxidation, and/or other denaturing conditions will result in degradation 

of the DNA.

The information above provides support to the claims made regarding the safety of the 

IHNV vaccine to animals, public health, and the environment. There are no apparent 

substantial issues with adverse environmental impacts concerning use of this vaccine.

V. Alternatives

Two alternatives were considered. The only alternative considered other than the 

preferred action alternative is not to approve the proposed permit for distribution and 

sale, the “no action” alternative. We have considered the applicants’ goals in light of the 

agency’s public interest and responsibilities and any potential environmental impact. 

Based upon the results of our risk analysis and the potential applications for this vaccine 

in disease control, APHIS adopts the alternative that the proposed permit be approved.

VI. Conclusion

Based upon the risk analysis documented in this EA, APHIS has determined that 

implementation of the proposal would not significantly affect the quality of the human 

environment and that the preparation of an Environmental Impact Statement is not 

required (Finding of No Significant Impact).

References

1. Kurath, G., Garver, K.A., Corbeil, S., et al. 2006. Protective immunity and lack of 

histopathological damage two years after DNA vaccination against infectious 

hematopoietic necrosis virus in trout. Vaccine 24:345-354.

2. Garver, K.A., LaPatra, S.E., Kurath, G. 2005. Efficacy of an infectious hematopoietic 

necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and 

sockeye O. nerka salmon. Dis Aquat Org 64:13-22.
3. Martin, T., Parker, S.E., Hedstrom, R., et al. 1999. Plasmid DNA malaria vaccine: the 

potential for genomic integration after intramuscular injection. Hum Gene Therapy 

10:759-768.

4. Ledwith, B.J., Manam, S., Troilo, P.J., et al. 2000. Plasmid DNA vaccines: 

investigation of integration into host cellular DNA following intramuscular injection 

in mice. Intervirol 43:258-272.

5. Grosenbaugh, D.A., Leard, A.T., Bergman, P.J., et al. 2011. Safety and efficacy of a 

xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for 

oral malignant melanoma in dogs following surgical excision of the primary tumor. 

Am J Vet Res 72:1631-8.

6. Manam, S., Ledwith, B.J., Barnum, A.B., et al. 2000. Plasmid DNA vaccines: tissue 

distribution and effects of DNA sequence, adjuvants and delivery method on 

integration into host DNA. Intervirol 43:273-81.

7. Davis, B.S., Chang, G.J., Cropp, B., et al. 2001. West Nile virus recombinant DNA 

vaccine protects mouse and horse from virus challenge and expresses in vitro a 

noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent 

assays. J Vir 75:4040-4047.

8. Liu, M.A. & Ulmer, J.B. 2005. Human clinical trials of plasmid DNA vaccines. Adv 

Genetics 55:25-40.

9. Maturin, L. & Curtiss, R. 1977. Degradation of DNA by nucleases in intestinal tract 

of rats. Science 196(4286):216-8.