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Environmental Assessment for Canadian Licensing of West Nile Virus Vaccine, Live Flavivirus Chimera

For Public Release

January 24, 2008

The information in this environmental assessment was current at the time of its preparation. It is possible that the situation may have changed since that time. Please consult the Veterinary Biologics Section if you have any questions.


Table of Contents

  • Summary
  • 1. Introduction
    • 1.1 Proposed Action
    • 1.2 Background
  • 2. Purpose and Need for Proposed Action
    • 2.1 Significance
    • 2.2 Rationale
  • 3. Alternatives
  • 4. Molecular and Biological Characteristics of Backbone and Recombinant Vaccine Organisms
    • 4.1 Identification, Sources, and Strains of Backbone Organism
    • 4.2 Source, Description and Function of Foreign Genetic Material
    • 4.3 Method of Accomplishing Genetic Modification
    • 4.4 Genetic and Phenotypic Stability of the Vaccine Organism
    • 4.5 Horizontal Gene Transfer and Potential for Recombination
    • 4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities
    • 4.7 Comparison of the Recombinant Vaccine Organisms to Backbone Organism Properties
    • 4.8 Route of Administration/Transmission
  • 5. Human Safety
    • 5.1 Previous Safe Use
    • 5.2 Probability of Human Exposure
    • 5.3 Possible Outcomes of Human Exposure
    • 5.4 Pathogenicity of Parent Microorganisms in Humans
    • 5.5 Effect of Gene Manipulation on Pathogenicity in Humans
    • 5.6 Risk Associated with Widespread Use of the Vaccine
  • 6. Animal Safety
    • 6.1 Previous Safe Use
    • 6.2 Fate of the Vaccine in Target and Non-Target Species
    • 6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals
    • 6.4 Reversion to Virulence Resulting from Back Passage in Animals
    • 6.5 Effect of Overdose in Target and Potential Non-Target Species
    • 6.6 The Extent of the Host Range and the Degree of Mobility of the Vector
    • 6.7 Safety in Pregnant Animals and to Offspring Nursing Vaccinated Animals
  • 7. Affected Environment
    • 7.1 Extent of Release into the Environment
    • 7.2 Persistence of the Vector in the Environment/Cumulative Impacts
    • 7.3 Extent of Exposure to Non-Target Species
    • 7.4 Behaviour of Parent Microorganisms and Vector in Non-Target Species
  • 8. Environmental Consequences
    • 8.1 Risks and Benefits
    • 8.2 Relative Safety Compared to Other Vaccines
  • 9. Mitigative Measures
    • 9.1 Worker Safety
    • 9.2 Handling Vaccinated or Exposed Animals
  • 10. Monitoring
    • 10.1 General
    • 10.2 Human
    • 10.3 Animal
  • 11. Consultations and Contacts
  • 12. Conclusions and Actions
  • 13. References

Summary

West Nile Virus (WNV) Vaccine, live flavivirus chimera consists of the live attenuated human yellow fever (YF) vaccine virus (Strain 17 D) modified by the introduction of a gene sequence from the WNV. This WNV Vaccine (PreveNile, manufactured by Intervet Inc., Millsboro, Delaware) was evaluated by the Veterinary Biologics Section (VBS), Canadian Food Inspection Agency (CFIA) for licensing in Canada. As part of the requirements for licensing this product in Canada, an "Environmental Assessment" was conducted and a public document which contains information on the molecular and biological characteristics of the live recombinant (chimeric) organism, target animal and non-target animal safety, human safety, environmental considerations and risk mitigation measures prepared. The product review process determined that the above product satisfactorily met the requirements for licensing veterinary biologics in Canada.

1. Introduction

1.1 Proposed Action

The VBS, Terrestrial Animal Health Division, CFIA is responsible for licensing veterinary biologics for use in Canada. The legal authority for the regulation of veterinary biologics in Canada is provided under the Health of Animals Act and Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA with regard to safety, purity, efficacy and potency of the product. Intervet Canada Ltd. has submitted the following equine WWNV Vaccine for licensing in Canada:

  • WNV Vaccine, Live Flavivirus Chimera (Trade name: PreveNile), United States Department of Agriculture (USDA) Product Code 1991.R1, VBS File 830VV/W4.0/I6.2.

The Environmental Assessment was prepared by VBS as part of the overall assessment for licensing the above vaccine in Canada, and was based on information provided by the manufacturer as well as documents from other sources.

1.2 Background

The above vaccine containing a live chimeric YF virus-WNV is manufactured by Intervet Inc., Millsboro, Delaware (US Veterinary Biologics Establishment License No 286). This vaccine is currently licensed for sale in the US. The chimeric vaccine consists of live attenuated YF vaccine virus, modified by the insertion of genetic material from the WNV. This is a stand-alone product at the present time. VBS has not previously licensed live chimeric flavivirus constructs for use in animals, but a related vaccine based on the same proprietary technology (ChimeriVax-West Nile) is currently being tested for use in humans in clinical trials by the company Acambis.

2. Purpose and Need for Proposed Action

2.1 Significance

The label indication for PreveNile is for the vaccination of healthy horses as early as five months of age to prevent viremia and as an aid in the prevention of disease and encephalitis due to WNV infection.

2.2 Rationale

The VBS evaluates veterinary biologics submissions for licensure under the Health of Animals Act and Regulations. General criteria for licensure are: (a) the product must be pure, safe, potent and efficacious, (b) the product must be licensed in the country of origin, (c) vaccine components must be relevant to Canadian disease conditions, and (d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices". This U.S.-origin vaccine meets the general criteria and presented no unacceptable importation risk, and therefore was evaluated for licensure by VBS.

3. Alternatives

The two alternative options available are: a) to issue a Permit to Import Veterinary Biologics to Intervet Canada Ltd. for the importation of PreveNile from the U.S., if all licensing requirements are satisfactory, or b) not to issue a Permit to Import Veterinary Biologics, if licensing requirements are not met.

4. Molecular and Biological Characteristics of Backbone and Recombinant Vaccine Organisms

4.1 Identification, Sources, and Strains of Backbone Organism

The proposed vaccine qualifies as a Category III Veterinary Biologic (live expression vectors that contain heterologous genes for immunizing antigens and/or other immune stimulants). The vaccine construct is a chimeric flavivirus vaccine containing gene sequences from the WNV inserted into the attenuated YF vaccine recipient. PreveNile is derived from an infectious clone from the attenuated licensed vaccine strain of YF virus (strain 17D), which is also the recipient of several experimental human vaccines currently being developed against WNV, Japanese encephalitis virus, and dengue (various ChimeriVax products, from Acambis). This proprietary ChimeriVax technology was licensed to Intervet Inc. for use in veterinary vaccines by Acambis in 2003.

The Public Health Agency of Canada surveillance report for WNV up to November 17, 2007, identified 109 presumptive or confirmed positive horses in Canada that year, in Alberta, Saskatchewan and Manitoba. Their report also indicated that there were 1682 positive WNV mosquito pools from Ontario to Alberta, and 2351 clinical cases in humans so far in 2007, indicating that there is still a significant risk of infection with this virus in Canada.

4.2 Source, Description and Function of Foreign Genetic Material

The ChimeriVax technology employs the YF vaccine capsid and nonstructural genes to deliver the premembrane (prM) and envelope (E) genes of the WNV.

The donor gene sequences are from a WNV field isolate, and have not been used previously in a veterinary biologic.

4.3 Method of Accomplishing Genetic Modification

The ChimeriVax two-plasmid technology divides the cloned YF backbone into two plasmids. The WNV prM and E genes used were cloned from a field isolate of WNV. Details of the methods used to create the recombinant virus are on file with VBS.

4.4 Genetic and Phenotypic Stability of the Vaccine Organism

The WNV vaccine, live flavivirus chimera is not recovered from the plasma or from the nasal, oral or rectal swabs of horses vaccinated by the recommended intramuscular route. Thus the vaccine would be expected to retain genetic stability in the target animal species (horses). Studies showed that the Master Seed virus is genetically and phenotypically stable and is free of extraneous agents as per 9CFR 113.300.

4.5 Horizontal Gene Transfer and Potential for Recombination

Intertypic recombination between flaviviruses has been reported for dengue virus during extended periods of high viremia in humans, but appears to be of low frequency and incidence. Recombination between different flavivirus species has not been reported in the literature. Since replication of the vaccine virus does not occur at a significant level in the equine host, and as this vaccine will not be administered in combination with other modified live viruses, the likelihood of recombination events is very low. Viremia following WNV infection in horses is also typically of low titre and short duration, so although it is theoretically possible for a recombination event between WNV and the WNV vaccine, live flavivirus chimera, the likely outcome would be replacement of the prM or E proteins in either virus, and this would likely be as attenuated as the vaccine virus.

4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities

The YF virus strain used in the construct is an attenuated vaccine strain that has been widely used around the world, including in Canada. In vivo and in vitro studies conducted by Acambis and Intervet indicate that biological properties (host range specificity and virulence properties) of ChimeriVax-WNV vaccine strains are similar to those of the backbone YF virus vaccine strain (17D). Replication and dissemination of the WNV vaccine, live flavivirus chimera is unlikely, since this vaccine has limited to no replication in cells of mosquitoes or in live mosquito vectors of WNV and YF virus.

4.7 Comparison of the Recombinant Vaccine Organisms to Backbone Organism Properties

The host range of the recombinant vaccine organism appears to be similar to the backbone vaccine strain, which is also not transmitted by mosquitoes.

4.8 Route of Administration/Transmission

Results from studies in horses, mosquitoes, birds, mice and non-human primates demonstrated that the donor WNV DNA sequences did not enhance the virulence or ability of the vaccine to survive in target and non-target animals. There is unlikely to be any virus shed or spread from vaccinates.

5. Human Safety

The ChimeriVax chimeric flavivirus vaccines have been developed for use in humans by Acambis, Inc., of Cambridge, Massachusetts, and phase II clinical trials in humans with the related ChimeriVax-WNV vaccine are currently in progress. Intervet Inc. has also provided additional safety data for the PreveNile equine WNV vaccine, live flavivirus chimera, including data on safe use in non-human primates, and data is available from humans tested in the phase I clinical trials with the related vaccine.

5.1 Previous Safe Use

The original ChimeriVax-based Japanese encephalitis vaccine has been safely tested in phase I and phase II human clinical trials, and phase I clinical trials with the ChimeriVax-WNV2 vaccine have been reported. A ChimeriVax-based dengue vaccine is also in development, to simultaneously immunize people to all four serotypes of the dengue virus. These vaccines were well tolerated in humans and there have been no significant adverse events reported to date.

The WNV vaccine, live flavivirus chimera, has a 21-day withdrawal period for vaccinated horses to ensure the safety of humans consuming horse meat, and the vaccine components are considered safe for human consumption. Other stabilizing agents in the vaccine are commonly used in food animal vaccines and are listed as GRAS (generally recognized as safe).

5.2 Probability of Human Exposure

Human exposure to the vaccine is likely to be limited to veterinarians, animal technicians, manufacturing staff and testing laboratory staff. Inadvertent injection of the vaccine into human subjects is expected to cause no serious adverse effects. Any viremia occurring in humans accidentally injected with this product is not considered sufficient to infect insect vectors, so spread by accidental injection is unlikely. Vaccinated animals do not shed the vaccine, so as a result human exposure is not expected to occur through animal excretions.

5.3 Possible Outcomes of Human Exposure

Human volunteers who have been injected with related ChimeriVax-based live flavivirus chimeric vaccines reported no severe adverse events, and the frequency and severity of events reported was comparable to the group vaccinated with placebos.

5.4 Pathogenicity of Parent Microorganisms in Humans

The parental strain of YF vaccine virus (strain 17D) was developed and attenuated in 1937, and over a 50-year period of large-scale use involving more than 500 million doses have established an acceptable safety profile. However, there have been some recent reports of rare, sometimes fatal, serious adverse events post-vaccination at an estimated frequency of three to five cases per million doses distributed. The ChimeriVax-WN vaccine for human use was demonstrated to be less neurovirulent than the commercial YF 17D vaccine in mice and non-human primates. Various ChimeriVax-derived vaccines have been tested in humans, as discussed in Section 5.1, and no serious adverse events were reported as a consequence of their use.

5.5 Effect of Gene Manipulation on Pathogenicity in Humans

WNV vaccine, live flavivirus chimera, manufactured for use in horses, is not expected to be any more pathogenic in humans than the backbone YF vaccine, which has been used safely in almost 500 million humans since 1937.

5.6 Risk Associated with Widespread Use of the Vaccine

The widespread use of the vaccine is not expected to have any public health significance. The likelihood of an adverse event occurring in the case of accidental human injection is likely to be low to negligible (USDA licensing conclusion was "Finding of No Significant Impact"), and accidentally injected humans may develop some immune protection from infection with the WNV, as well as YF virus.

6. Animal Safety

6.1 Previous Safe Use

Safety of the WNV vaccine, live flavivirus chimera, has been demonstrated in horses, mice, non-human primates, mosquitoes and birds, as part of the licensing of this veterinary biologic. The detailed data are on file at VBS. No related products have been registered for veterinary use in Canada at this time, but the YF vaccine is licensed in Canada for use in humans travelling to regions with YF virus infections.

6.2 Fate of the Vaccine in Target and Non-Target Species

The WNV vaccine, live flavivirus chimera, was found to be safe in experimental studies in horses (intended target species) as well as in several non-target species (mice, non-human primates, mosquitoes and birds). All the studies in target and non-target species indicate that the host range and tropism of the WN vaccine, live flavivirus chimera, were not altered from the backbone YF vaccine strain. Studies on the potential for neurovirulence in mice using direct inoculation indicated that the WNV vaccine, live flavivirus chimera, required more than 10,000 times more virus to achieve 50 percent mortality in a group of mice. Additional studies comparing the donor strain of WNV and the WNV vaccine, live flavivirus chimera, verified that injection of mice was not lethal at doses up to 10 million plaque-forming units (PFU), whereas between one and three PFU of a field isolate of WNV can kill 50 percent of a group of mice.

6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals

Studies in horses demonstrated that there was no indication of virus shed or spread as evidenced by the lack of vaccine virus recovery after vaccination. Studies in chickens with YF or WNV, live flavivirus chimera, also did not result in any detectable viremia (or neutralizing antibody) following injection. Similar studies with the injection of various doses of vaccine virus in crows also did not lead to detectable viremia or protection from WNV when subsequently challenged. Experimental injection of nonhuman primates resulted in a mild viremia but no clinical signs during an observation period, and postmortem analysis revealed minimal lesions with no involvement of the neurons.

6.4 Reversion to Virulence Resulting from Back Passage in Animals

Studies showed that the Master Seed virus is genetically and phenotypically stable and is free of extraneous agents as per 9CFR 113.300. The vaccine virus does not replicate significantly in horses, and back passage attempts have not shown any reversion to virulence.

6.5 Effect of Overdose in Target and Potential Non-Target Species

Studies injecting 10 times the expected minimum protective dose in horses did not demonstrate any adverse clinical signs in horses, and the vaccine virus was not isolated from plasma, nasal, oral or rectal swabs. Transmission of the vaccine virus between these vaccinated horses and sentinel horses maintained in contact also did not occur.

6.6 The Extent of the Host Range and the Degree of Mobility of the Vector

WNV, live flavivirus chimera is restricted in its host range. This vaccine virus does not result in detectable viremia and is not shed from vaccinated horses and, thus, no spread of the organism is expected. The replication and dissemination of WNV, live flavivirus chimera in mosquitoes and mosquito cell lines was compared to that of YF-17D and to a field isolate of WNV, and the equine vaccine had reduced replication in mosquito cells (from Aedes aegypti), when compared to YF-17D. Neither YF-17D or the WNV, live flavivirus chimera could be isolated from Culex species of mosquitoes when cultured after inoculation, whereas mosquitoes inoculated with a virulent field strain of WNV had high titres of virus at this time. This indicates that the vaccine virus is unlikely to be disseminated by mosquitoes, which are recognized as vectors for YF or WNV.

6.7 Safety in Pregnant Animals and to Offspring Nursing Vaccinated Animals

Since WNV, live flavivirus chimera does not replicate in and shed from vaccinated horses, the safety profile of the vaccine is not expected to be different for pregnant animals and offspring nursing vaccinated animals. The insert in the vaccine describes the field safety study, which included 302 pregnant mares, and observed no significant post vaccinal events. It is generally recommended to avoid causing stress by injecting horses with anything during pregnancy, though.

7. Affected Environment

7.1 Extent of Release into the Environment

There are no ecological concerns associated with the use of flavivirus-vectored vaccines. For example, shed-spread studies with this virus in the horse have shown that the recombinant virus is not shed or spread. In addition, studies with this WNV vaccine, live flavivirus chimera demonstrated that the vaccine is less able to replicate in mosquito cell lines and is unable to be disseminated by mosquitoes, when compared to the backbone YF vaccine virus or WNV. Potential for occasional limited environmental release through accidental spills, unintended syringe aerosols or contamination of the skin and hair around the vaccination site does exist during routine use of this vaccine, but the vaccine virus is unlikely to infect animals or insects by this route.

7.2 Persistence of the Vector in the Environment/Cumulative Impacts

The WNV vaccine, live flavivirus chimera has been reported to be viable at -70°C and 4°C for prolonged periods, but viability is greatly reduced at room temperature and above. The vaccine is susceptible to inactivation by most disinfectants, and the intramuscular injection of vaccine into horses should not release virus into the environment.

7.3 Extent of Exposure to Non-Target Species

The very limited host range WNV vaccine, live flavivirus vector, reduces the risk of spread to non-target mammalian species. The risk of using this vaccine in horses is expected to be no greater than the risk of using the YF vaccine in humans in Canada.

7.4 Behaviour of Parent Microorganisms and Vector in Non-Target Species

As noted above, the YF vaccine backbone is typically only infectious in humans or nonhuman primates, and dissemination by insect vectors has not been demonstrated. The donor DNA organism, WNV, infects a variety of mammals, birds, and mosquito species in Canada, which is the reason for needing a vaccine to protect horses. As described above, the WNV vaccine, live flavivirus chimera, is more comparable to the YF vaccine, and does not have any additional virulence due to the donor WNV.

8. Environmental Consequences

8.1 Risks and Benefits

For any vaccine, risks of vaccination can be attributed to potential adverse reactions. This risk is identified on the product label along with the recommendation that appropriate symptomatic treatment be given, which may include antihistamines, anti-inflammatory drugs, and/or epinephrine. The efficacy of the WNV vaccine, live flavivirus chimera, in protecting horses against viremia has been demonstrated in vaccination-challenge experiments, and WNV infection is a significant risk to horses in Canada.

8.2 Relative Safety Compared to Other Vaccines

The WNV, live flavivirus chimera does not have the ability to cause WNV infection, and does not require inactivation. The lack of potential for reversion to virulence and absence of adjuvants typical of killed virus vaccines are also positive safety features inherent with live vectored vaccines. To be licensed in Canada, all veterinary vaccines must be shown to be pure, potent, safe and efficacious when used according to label recommendations.

9. Mitigative Measures

9.1 Worker Safety

The vaccine is manufactured at Intervet Inc. in Millsboro, Delaware, which is a Veterinary Biologics Establishment licensed by the USDA. Individuals working with the vaccine in this production facility and veterinarians and animal technicians can be exposed to the live chimeric vaccine. As already discussed in the section above on human safety, such exposure is not considered to be a safety concern.

9.2 Handling Vaccinated or Exposed Animals

Exposure of groups such as horse owners to the live recombinant organism is likely to be very low since vaccinated animals do not shed the virus and unintended contamination of hair and skin at the vaccination site is not considered to be of public health significance.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all suspected significant adverse reactions to the CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. On VBS receipt of an adverse reaction complaint, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and CFIA. If the problem is resolved to the satisfaction of the veterinarian/client, no further action is usually requested by VBS. However, if the investigation is not satisfactory, VBS may initiate regulatory action depending on the case which may include further safety testing, temporarily stop sale or product withdrawal from the market.

10.2 Human

No special monitoring of the human safety of the product will be carried out.

10.3 Animal

Veterinarians/horse owners and manufacturers should report any suspected adverse reactions to VBS as indicated above. For reporting purposes, adverse reactions are divided into Type 1, 2, 3 and 4 reactions. Type 1 reactions are defined as any systemic adverse reaction, anaphylactic or hypersensitivity requiring veterinary treatment including: persistent fever, recumbency, persistent lethargy, decrease in activity, muscle tremors, shivering, hypersalivation, dyspnea and other respiratory problems, cyanosis, diarrhea, vomiting, colic and other gastrointestinal problems, eye problems, abortions and other reproductive problems and neurological signs. Type 2 reactions are defined as death following vaccination. Type 3 reactions are defined as local persistent reactions such as edema, abscess, granuloma, fibrosis, alopecia, hyperpigmentation and excessive pain at the injection site. Type 4 cases consist of cases of suspected lack of efficacy. Suspected adverse reactions should be reported using the form Notification of Adverse Events to Veterinary Biologics (CFIA/ACIA 2205).

11. Consultations and Contacts

Manufacturer:

Intervet Inc.
35500 West 91st Street
De Soto, Kansas, USA 66018

Importer:

Intervet Canada Ltd.
250 Water Street
Whitby, Ontario, L1N 9T5

12. Conclusions and Actions

The Permit to Import Veterinary Biologics held by Intervet Canada Ltd., Ontario, may be amended to allow importation of the following product from the manufacturer Intervet Inc., Millsboro, Delaware:

  • WNV Vaccine, Live Flavivirus Chimera (Trade name: PreveNile), United States Department of Agriculture (USDA) Product Code 1991.R1, VBS File 830VV/W4.0/I6.2.

13. References

Arroyo J., et al. ChimeriVax-WNV live-attenuated vaccine: Preclinical evaluation of safety, immunogenicity, and efficacy. 2004. Journal of Virology 78: 12497-12507.

Arya, S.C. Commentary: YF vaccine safety: a reality or a myth? 2002. Vaccine 20: 3627-3628.

Barrett, A.D.T. et al. Conference report: 17D YF vaccines: New insights. A report of a workshop held during the World Congress on Medicine and Health in the Tropics, Marseille, France, Monday 12 September 2005. 2007. Vaccine 25: 2758-2765.

CDC. Notice to Readers: Fever, jaundice, and multiple organ system failure associated with 17D-derived YF vaccination, 1996-2001. Morbidity and Mortality Weekly Report 2001: 50: 643-5.

CDC. Adverse events associated with 17D-derived YF vaccination — United States, 2001-2002. Morbidity and Mortality Weekly Report 2002: 51: 989-993.

Hall, R.A., and A.A. Khromykh. Drug evaluation: ChimeriVax-West Nile vaccine. 2007. Current Opinion in Molecular Therapeutics 9: 498-504.

Johnson, B.W., et al. Growth characteristics of the veterinary vaccine candidate ChimeriVax-West Nile (WN) virus in Aedes and Culex mosquitoes. 2003. Medican and Veterinary Entomology 17: 235-243.

Marianneau, P., M.-C. Georges-Corbot, and V. Deubel. Rarity of adverse effects after 17D yellow-fever vaccination. 2001. Lancet 358: 84-85.

Monath, T.P., et al. WNV vaccine. 2001. Current Drug Targets - Infectious Disorders. 1: 37-50. (Free Online)

Monath, T.P. YF: an update. 2001. The Lancet Infectious Diseases 1: 11-20.

Monath, T.P., et al. Recombination and flavivirus vaccines: a commentary. 2005. Vaccine 23: 2056-2058.

Monath, T.P., et al. A live, attenuated recombinant WNV vaccine. 2006. Proceeding of the National Academy of Science of the United States of America 103: 6694-6699.

Murphy, B.R., J.E. Blaney, and S.S. Whitehead. Arguments for live flavivirus vaccines. 2004. Lancet 364: 498-500.

Pollack, M.P. Moderator- ProMed-mail post: YF vaccine-associated deaths - Peru: suspected, request for information 2007/10/19.

Public Health Agency of Canada. WNV national surveillance report, November 11, 2007 to November 17, 2007 (Week 46).

Seligman, S.J., and E.A. Gould. Live flavivirus vaccines: reasons for caution. 2004. Lancet 363: 2073-2075.


Prepared and revised by:

Veterinary Biologics Section
Terrestrial Animal Health Division
Canadian Food Inspection Agency