Environmental Assessment for the Canadian licensing of Boehringer Ingelheim Vetmedica Inc.'s Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus

December 31, 2018

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 Canadian Centre for Veterinary Biologics if you have any questions.

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 parental and recombinant organisms
5 Human safety
6 Animal safety
7 Affected environment
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
11 Conclusions and actions
12 References

Summary

1. Introduction

1.1 Proposed action

The Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (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 the Health of Animals Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA regarding the safety, purity, potency, and efficacy of the product. Boehringer Ingelheim (Canada) has submitted an application to license the following novel vaccine in Canada:

Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus, Trade name: Ingelvac Provenza, CCVB file number: 880VV/S2.5/B8.2, USDA Product Code: 19A5.R2.

This Environmental Assessment was prepared by the CCVB as part of the overall assessment for licensing the above vaccine in Canada.

1.2 Background

Swine influenza virus (SIV) disease is one of the most important infectious diseases in North America's swine population, contributing to high morbidity and economic losses. A highly variable, segmented negative sense RNA virus, its genome segments have the potential for re-assortment events in a co-infection. This strategy presents challenges to developing an effective and currently relevant vaccine as one tool in an effective herd management plan. As a zoonotic pathogen, SIV can be transmitted to agricultural workers and other in contact individuals thereby raising public health concerns. Influenza in mammalian and avian species is an immediately notifiable disease and positive identification requires notification of the provincial or territorial animal health authorities.

The Canadian Swine Health Intelligence Network reports that the number of confirmed SIV cases has steadily increased in recent years, with clear divisions in the SIV lineages detected in western versus eastern Canada. H1N1 was the predominant subtype in Canada until the emergence of H3N2 subtype in 2004. H1N2 – 1A.1 (α-H1) is prevalent in western Canada, while H1N1-1A.2 (β-H1) is common in eastern Canada with H1N2 starting to increase. For H3N2, the majority of cases lie within the IV-B, IV-C and IV-D clusters.

SIV vaccines licensed for use and distribution in Canada contain inactivated viruses of H1 or H3 subtypes, which may or may not be similar to today's circulating strains. Additionally, an inactivated vaccine will only elicit a humoral antibody response without stimulating an adaptive, cell-mediated response. Vaccine efficacy is determined by homology with circulating strains, the amount of antigenic material included in the vaccine and the type of adjuvant. To address the diversity of the evolving strains, the use of autogenous vaccines for swine influenza has also seen increased demands. For a current review of the state of SIV disease, diagnostic and vaccination options refer to Sandbulte et al. (2015).

2. Purpose and need for proposed action

2.1 Significance

A live, attenuated vaccine comprising two SIV strains, H1N1 & H3N2, has completed licensing requirements in the U.S. and is undergoing the review process in Canada. This is the first instance of a live influenza vaccine being used for swine. Both strains have a partial deletion of the NS1 sequences which serves to attenuate the virus and provides a means of detection and differentiation in a surveillance program. However, as these are genetically modified strains, a risk assessment is required as part of the veterinary biologic licensing process in Canada.

2.2 Rationale

The CCVB evaluates veterinary biologic product submissions for licensure under the Health of Animals Act and the Health of Animals Regulations. The criteria for considering a licensing application are as follows: a) the product must be pure, safe, potent and efficacious; b) vaccine components must be relevant to Canadian disease conditions; c) foreign products must be licensed in the country of origin; and d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices." This US origin vaccine meets these criteria and thus was evaluated for licensing by the CCVB.

3. Alternatives

The two options being considered are: a) to issue a Permit to Import Veterinary Biologics to Boehringer Ingelheim (Canada) allowing the importation of Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus Vaccine, 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 parental and recombinant organisms

The vaccine contains two strains of swine influenza virus. Both strains were generated through a reverse transcription process to produce genetically reassorted, attenuated viruses. The process for rescuing the influenza A virus from cloned cDNA was as described by Hoffmann et al. (2000). In this system cloned cDNA for the influenza genome is distributed onto eight plasmids, with each plasmid containing the viral cDNA for one of the eight influenza RNA segments inserted between the pol I promoter and the pol I terminator sequence (known as the pol I transcription unit). The pol I transcription unit I is flanked by the human cytomegalovirus pol II promoter and a polyadenylation signal. After transfection into mammalian cells, the pol I promoter is used to synthesize negative-sense vRNA while the pol II promoter is used to transcribe mRNA from each segment; in turn these are translated into viral proteins. In this manner, the viral replication cycle is initiated and infectious, modified influenza A virus is produced.

In this vaccine, both strains of the swine influenza virus (H1N1 and H3N2) contain a deletion in the NS1 gene segment resulting in a carboxy-truncated NS1 non-structural protein. The H1N1 reassorted vaccine strain virus consists of the HA (haemagglutinin) and NA (neuraminidase) proteins from an A/swine H1N1 strain and the PB2 (RNA polymerase), PB1 (RNA polymerase), PA (RNA polymerase), NP (nucleoprotein), M (matrix) and truncated NS1 (non-structural) proteins from an A/swine/ H3N2 strain. The H3N2 reassorted vaccine strain virus consists of HA, NA, PB2, PB1, PA, NP, M and truncated NS1 proteins from an A/swine/ H3N2 strain.

4.1 Identification, sources and strains of parental organisms

The parental organisms used to generate the reassorted viruses are US-based isolates of swine influenza type A. The identification, sources and strains of the parental influenza virus are considered to be confidential business information.

4.2 Source, description and function of foreign genetic material

The source and description of the reassorted viruses are considered to be confidential business information. Both reassorted influenza vaccine strains contain truncated NS1 protein sequences. The truncation is directly responsible for the attenuation of the vaccine virus. The full length NS1 protein functions prominently in influenza virus replication and down regulation of the host innate immune response by suppressing the interferon response. Cells infected with a truncated NS1 swine influenza virus were shown to express higher levels of interferon α and β and TNF α - all immune modulators with demonstrated antiviral activity against influenza A viruses. The net result is an attenuation of the wild-type virus' virulence. The phenotypic properties of each master seed are described in Solorzano et al. (2005). No additional genetic material was added in the construction process.

4.3 Method of accomplishing genetic modification

The process for reassorting the influenza A virus from cloned cDNA was as described by Hoffmann et al. (2000) and Solorzano et al. (2005).

4.4 Genetic and phenotypic stability of the vaccine organism

Full genome sequencing of the master seed virus (n), master seed virus + 1 (n+1) and working seed virus (n+6) demonstrated the genetic stability of the reassorted viruses. Vaccine strains exhibit poor growth in MDCK cells and in older than eight-day-old embryonated SPF chicken eggs, i.e. in cells with competent interferon pathways. In these cells, truncated NS protein expression is very low as detected by Western blot. In contrast, vaccine strains demonstrate efficient growth in cell systems, such as Vero cells, which lack competent interferon pathways.

In laboratory-based back-passage studies of the master seed virus in piglets, no reversion to virulence was observed.

4.5 Horizontal gene transfer and potential for recombination

Influenza viruses are noted for the exchange of genomic RNA segments resulting in reassorted viruses. There is a potential for recombination between a vaccine strain with a wild type SIV strain in a co-infection scenario within an animal host. Should this occur, the new reassorted virus will not be more virulent than the wild type SIV. The vaccine strains do not contain any non-influenza sequences or genetic material that may posed an additional risk. As the vaccine strains show decreased virulence due to the truncated NS1, it is expected that the risk of a recombination event would be further decreased due to the reduced virulence and resulting reduced viral replication, shed and spread.

Reassortment events are driven by influenza virus internal genes which are similar regardless of the surface H and N glycoprotein subtype. The risk of reassortment is not affected by the NS1 sequence. Reassortment events involving the vaccine strains have been reported in U.S. swine farms; it is theorized that the population size of U.S. swine farms may be one contributing factor for influenza reassortment event. Canadian swine farms are smaller and further apart and thus a lower frequency of reassortment events is theorized to occur.

4.6 Host range/specificity, tissue tropism and shed/spread capabilities

By their nature, influenza viruses have a broad mammalian host range and may move between species, either in a direct or adaptive manner. Influenza viruses adapted to swine and humans target upper respiratory tract tissues at sialic acid receptors with α(2,6) linked carbohydrates.

The manufacturer confirmed in efficacy and safety studies that limited shedding of the vaccine viruses occurs. In a laboratory-based safety study, one-day-old pigs were administered vaccine strain virus by the intranasal route. Animals were monitored for 14 days. Within first 3 days of treatment, most vaccinated pigs had shed virus in nasal swabs at least once. No shedding was detected after day 7. Several animals had detectable vaccine strain virus in respiratory lavage fluid. Non-vaccinated sentinel animals remained negative for virus isolation.

4.7 Comparison of the modified organisms to parental properties

In the parental wildtype virus the full length NS1 protein is responsible for virus replication and down regulation of the host innate immune response by suppressing the host organism's interferon response. Both influenza vaccine strains express a truncated NS1 protein directly responsible for the attenuation of the vaccine virus. SIV mutants expressing the truncated NS1 protein were shown to induce higher levels of interferon α and β and TNF α - all with demonstrated antiviral activity against influenza A viruses.

4.8 Route of administration/transmission

The vaccine strains are administered to animals by direct installation into the nasal passages, to target the mucosal immune response pathway.

5. Human safety

5.1 Previous safe use

The vaccine strains present in the Swine Influenza Vaccine, H1N1 & H3N2, have not been assessed in humans. Similar H1N1 strains with a deletion of the NS1 region have undergone clinical phase I trials in humans; the vaccine was well tolerated and adverse events were mild and of a short duration. Wacheck et al. (2010) reported that human volunteers inoculated with 7.7 log₁₀ TCID50 of a similar construct would shed vaccine virus in nasal samples for up to 12 hrs post-vaccination.

5.2 Probability of human exposure

Human exposure to the influenza vaccine viruses may occur during manufacturing, QC testing or administration of the vaccine. Biocontainment procedures during production, filling and QC testing will prevent human exposure to the vaccine strains.

Veterinarians and farm workers administering the vaccine product in swine operations may be exposed to aerosolized droplets of the vaccine. Swine husbandry specialists and farm workers may be exposed to the virus vaccine as a result of a shedding event in a vaccinated animal.

5.3 Possible outcomes of human exposure

As the vaccine strains are attenuated with reduced virulence, a possible outcome of infection is a mild cold, nasal congestion and headache. These symptoms will be milder than those expected with a naturally circulating strain of SIV in humans: cough, headaches, fatigue, fever, laboured breathing and muscle stiffness for several days. Similar replication-deficient strains of NS1-H1N1 have undergone clinical phase I trials in humans; the vaccine was well tolerated and adverse events were mild (rhinitis-like symptoms and headaches). Wacheck et al. (2010) reported that human volunteers inoculated with 7.7 log₁₀ TCID50 of a similar construct would shed vaccine virus in nasal samples for up to 12 hrs post-vaccination.

A live, attenuated influenza vaccine for humans is licensed for use in Canada and the U.S. This vaccine (for 2018-2019, consisting of four strains: A/H3N2, A/H1N1, B/Victoria, B/Yamagata) is recommended for ages 2 to 49, and is sprayed into the nose, in a manner similar to this proposed veterinary vaccine. The U.S. Center for Disease Control has published a list of possible outcomes after administration of a live, attenuated influenza vaccine through the nasal passages as including headache, runny nose, sore throat, chills, cough, and fatigue.

There is a higher risk to an immunocompromised individual or an individual with respiratory disease or asthma should contact with the vaccine strains occur. A possible outcome in an immunocompromised individual is the development of moderate to severe influenza symptoms. It is probably that immunocompromised individuals would likely not be present within the manufacturing or QC areas of a vaccine facility, nor in contact with animals at a swine farm.

5.4 Pathogenicity of parent microorganisms in humans

The parent swine influenza strains would be expected to cause moderate flu symptoms in infected humans. Investigational studies with the two parent H1N1 and H3N2 swine influenza strains in humans have not been done.

5.5 Effect of gene manipulation on pathogenicity in humans

The truncated NS1 protein reduces the virulence of the vaccine strains in swine as the virus is unable to downregulate the host antiviral interferon pathway. This effect was also observed with similar replication-deficient strains of NS1-H1N1 in clinical phase I trials in humans; the vaccine was well tolerated, and an innate host immune response against the virus strain was activated. Observed adverse events in individuals receiving the vaccine were mild. Wacheck et al. (2010) reported that human volunteers inoculated with 7.7 log₁₀ TCID50 of a similar construct would shed vaccine virus in nasal samples for up to 12 hrs post-vaccination.

5.6 Risk associated with widespread use of the vaccine

The risk associated with widespread use of this vaccine is expected to be significantly less than with the wild-type isolates. This was demonstrated by the safety and efficacy data submitted to support product licensure.

6. Animal safety

6.1 Previous safe use

The following field study data was submitted for review: 997 newborn pigs (1 – 5 days of age) at three geographical sites in the U.S. were each administered one dose of vaccine (1 mL, intranasal route) then monitored for 14 days. Only minor clinical observations were recorded; these may or may not be attributable to the vaccine. At one site the clinical observations of depression and loss of condition were attributed to the extreme heat and humidity during the study, and not to the vaccine regimen. The most frequent clinical observations in decreasing order were: depression, loss of condition, conjunctivitis, panting, diarrhea, lameness, and lacerations.

6.2 Fate of the vaccine in target and non-target species

Individual influenza virus strains exhibit a limited host range for infection and replication. The manufacturer evaluated safety of this vaccine in rats, chickens and ferrets. The ferret influenza model closely resembles the course of influenza infection humans and other primates susceptible to influenza. Chickens and rats were included as representative of non-target species that may be in close contact with swine. Non-target animals were vaccinated via the intranasal route and observed daily for clinical signs for 14 days. At 14 days, animals were euthanized and necropsied. No clinical signs or gross lung lesions were observed in the three non-target animal species. It is concluded that there is negligible to low risk by the vaccine should it be taken up by non-target species.

6.3 Potential of shed and/or spread from vaccinate to contact target and non-target animals

A shed and spread study was submitted for review. In this study 24 1-5-day old piglets were administered the vaccine intranasally. 24 sentinel piglets, same age, were introduced approximately four hours after vaccination. All animals were observed for clinical signs and virus shed for 14 days. Minimal clinical signs were observed in both groups. Only vaccinated animals had lung lesions at necropsy. Vaccine virus was not detected in sentinel animals. The study demonstrates the low probability of a shed and/or spread event.

6.4 Reversion to virulence resulting from back passage in animals

The manufacturer submitted a back-passage study in swine to demonstrate a lack of reversion to virulence after five passages of the vaccine strains.

6.5 Effect of overdose in target and potential non-target species

Administration of a 10X dose to 2-3-day old piglets demonstrated the safety of the vaccine in an overdose situation with no untoward symptoms. An overdose in non-target species was not assessed.

6.6 The extent of the host range and the degree of mobility of the vector

Individual influenza virus strains exhibit a limited host range for infection and replication. The vaccine was evaluated in rats, chickens and ferrets as representative of non-target animal species. The ferret influenza model most closely resembles the course of influenza infection humans and other primates susceptible to influenza. Chickens and rats were included as representative of non-target species that may be in close contact with swine. No clinical signs or gross lung lesions indicative of an influenza infection were observed in the three non-target animal species. Vaccine virus was isolated from ferrets in swabs up to day 6. This confirms the limited host range of the vaccine strains.

7. Affected environment

7.1 Extent of release into the environment

The vaccine strains will be inoculated into swine at a swine operation. Influenza, as an enveloped virus, has low survivability in the environment and is susceptible to inactivation by disinfectants and ultraviolet light. The survivability of the modified vaccine strains is predicted to be similar or not greater than the survivability of the circulating, prevalent strains. The vaccine strains could also be released into the environment if shed in swine manure used to fertilize crops. Studies demonstrated that the vaccine strain is not shed and released into the environment. Current swine husbandry requirements for a thorough disinfection of premises would further minimize the risk of release into the environment.

7.2 Persistence of the vector in the environment and cumulative impacts

Influenza virus, as an enveloped virus, has low survivability in the environment and is susceptible to inactivation by disinfectants and ultraviolet light. The vaccine strains are of negligible risk to persist in the environment.

7.3 Extent of exposure to non-target species

Rats, chickens and ferrets were evaluated as representative of non-target animal species that may be exposed to the vaccine strains. The ferret was representative of the typical course of influenza infection in veterinarians and farm workers in close contact with the vaccinated animals. Chickens and rats were included as representative of non-target species that may be in close contact with swine.

7.4 Behaviour of parent microorganisms and vector in non-target species

A study in non-target species demonstrated that the vaccine strains cannot productively infect and replicate. No clinical signs or gross lung lesions were observed in ferrets, chickens and rats, confirming that non-target species are at low to negligible risk when exposed to the vaccine strains. The limited host range along with the antiviral action of host species interferon will further decrease the risk of effect on the environment.

8. Environmental consequences

8.1 Risks and benefits

The risk associated with use of the Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus is:

in animals either permissively infected with a circulating strain of swine influenza virus or vaccinated with this vaccine, there is a risk of a reassortment event between the circulating SIV wild type strain and the vaccine strain

The benefits associated with use of the Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus are the following:

the presence of the two genetically modified strains may offer a broader cross protective immunity against different subtypes and strains
live virus strains induce both humoral and cellular immune responses against variant viruses
the use of live influenza virus strains (rather than killed strains) and their induction of cellular immune responses is expected to provide a higher degree of protection against infection in the presence of maternal derived antibodies
intranasal administration of this vaccine induces the mucosal immune response pathways
the use of modified live influenza virus strains (rather than killed strains) may decrease the risk of vaccine-associated enhanced respiratory disease (VAERD)
the use of a truncated NS1 vaccine strain allows a means of detection and differentiation in a surveillance program, e.g. by an RT-PCR based restriction endonuclease test

8.2 Relative safety compared to other vaccines

Use of this vaccine by the intranasal route decreases the prevalence of injection site reactions that may be observed with an adjuvanted, killed SIV vaccine. The vaccine's attenuation properties may contribute to a low level SIV infection within the host animal. The lack of inactivation increases the risk of a recombination event with a circulating, wild type SIV strain in the swine host, in a non-target animal, in a farm worker or in the environment. The virulence of the new strain will not exceed the virulence of the wild type SIV strain.

9. Mitigative measures

9.1 Worker safety

The vaccine is produced in a USDA licensed facility according to validated procedures and under generally accepted good manufacturing practices and biocontainment protocols. Manufacturing personnel are trained in safety procedures and procedures to prevent the dissemination of the viral strains during production and testing.

9.2 Handling vaccinated or exposed animals

Veterinarians and personnel handling the vaccinated animals are trained in animal husbandry and animal vaccination. This vaccine is administered by the intranasal route without a needle; there is no risk of self-injection to the animal handler.

This vaccine is administered to animals by direct inoculation into the nasal passages; there is a risk of the modified live virus vaccine being aerosolized outside of the animal's nasal passages and infecting personnel handling the animals. The company has developed a training program on intranasal administration of vaccines to pigs, on user safety and on the correct use of personal protective equipment. The company's technical sales force provided on farm training at customers' facilities in the U.S. The company has reported several unusual events of personnel handling the vaccinated animals during the vaccination process developing mild flu-like symptoms post-administration. In all instances, individuals were not wearing the appropriate personal protective equipment as per label recommendations. The vial label contains detailed guidance concerning use of personal protective equipment to avoid inhalation and contact with eyes and skin while vaccinating animals with this vaccine as follows:

"Wear appropriate personal protective equipment to avoid inhalation and contact with eyes and skin. Wash hands thoroughly after handling. Accidental inhalation or contact may result in eye irritation, cough, fever, fatigue, chills, headache, runny nose and/or congestion. If these symptoms persist for more than 24 hours, seek medical attention. Measures to prevent accidental exposure of immunocompromised or vulnerable individuals should be taken. Pregnant women should consult their medical practitioner before working with this product."

The manufacturer has reported the detection of influenza reassortment events in animals administered the vaccine strains. A reassortment event requires that both the vaccine strain virus and the circulating wild type swine influenza virus be present in an animal at the same time. The label directions specify the use of this vaccine only in healthy animals. Animals or premise populations that are not healthy, especially those that are exhibiting signs of a respiratory or flu-like infection, should not be vaccinated with this vaccine to minimize or negate the risk of a reassortment event between the modified live virus vaccine strains and naturally circulating swine influenza strains.

Swine farm operators and workers can prevent, manage and reduce the incidence of SIV infection through biosecurity protocols, quarantine procedures for incoming animals, use of vaccines, investigating and reporting production losses that exceed expected values and other practices. Swine farm operators and workers that are themselves affected by a respiratory cold or flu illness should avoid contact with swine until their symptoms have resolved. Swine farm operators and workers should help prevent the risk of transmission of SIV by wearing gloves and a respirator mask, and by washing their hands after handling swine.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all significant suspected adverse events to the CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse events directly to the CFIA. If an adverse event complaint is received by the CCVB, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and the CFIA. If the problem is resolved to the satisfaction of the veterinarian or client, usually, no further action is requested by the CCVB. However, if the outcome of the investigation is unsatisfactory, the CCVB may initiate regulatory action, depending on the case, which may include further safety testing, temporary stoppage of product sales, or product withdrawal from the market.

10.2 Human

Any suspected adverse events or unusual flu-like events arising in personnel handling animals vaccinated with the live SIV vaccine should be reported to the CCVB as indicated in 10.1. Suspected adverse events should be reported using Form CFIA/ACIA 2205 – Notification of Suspected Adverse Events to Veterinary Biologics. Unusual flu-like events arising in personnel handling animals vaccinated with live SIV vaccine should also be reported to the local, municipal, provincial or territorial health authority.

10.3 Animal

Veterinarians, vaccinators, and producers should report any suspected adverse events to the CCVB as indicated in 10.1. Suspected adverse events should be reported using Form CFIA/ACIA 2205 – Notification of Suspected Adverse Events to Veterinary Biologics.

11. Conclusions and actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus (CCVB File No.: 880VV/S2.5/B8.2) in Canada may pose a low to negligible risk to the environment or to human health, if used in a manner that is not in agreement with the approved label directions.

Following this assessment and the completion of the Canadian Veterinary Biologics licensing process, the Permit to Import Veterinary Biologics held by Boehringer Ingelheim Canada Ltd. may be amended to allow the importation and distribution of the following product in Canada:

Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus
Trade Name: Ingelvac Provenza
CCVB File No. 880VV/S2.5/B8.2
USDA Code 19A5.R2

All serials of this product must be released by the USDA prior to importation into Canada. All conditions described in the Permit to Import Veterinary Biologics must be followed with respect to the importation and sale of this product.

12. References

Boehringer Ingelheim Vetmedica, Inc. Risk Analysis: Swine Influenza Vaccine, H1N1 & H3N2, Modified Live Virus, Product Code 19A5.R2. Version: July 10, 2014. Confidential document (complete) and confidential document (redacted for confidential business information) on file with CCVB. Risk analysis was prepared by Boehringer Ingelheim Vetmedica Inc. for the USDA-APHIS-CVB licensing and review process to support proposed field safety studies.

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Nogales A, and Martinez-Sobrido L. 2017. Reverse genetics approaches for the development of influenza vaccines. Int J Mol Sci 18(1): 20-52.

Pasick J, and Berhane Y. 2017. Swine Influenza Virus Surveillance – Canada. Reported at the OFFLU Swine Influenza Virus Meeting, 27-28 March, 2017. FAO Headquarters, Rome, Italy.

Province of Ontario, Ministry of Agriculture, Food and Rural Affairs. Animal Health and Welfare Branch. Industry Update: Influenza A – Swine. October 25, 2018.

Richt JA, Lekcharoensuk P, Lager KM, Vincent AL, Loiacono CM, Janke BH, Wu W-H, Yoon K-J, Webby RJ, Solorzano A, and Garcia-Sastre A. 2006. Vaccination of Pigs against Swine Influenza Viruses by Using an NS1-Truncated Modified Live-Virus Vaccine. J Virol 80(22): 11009-11018.

Sandbulte MR, Spickler AR, Zaabel PK, and Roth JA. 2015. Optimal Use of Vaccines for Control of Influenza A Virus in Swine. Vaccines 3:22-73.

Solorzano A, Webby RJ, Lager KM, Janke BH, Garcia-Sastre A, and Richt JA. 2005. Mutations in the NSq protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs.

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U.S. Center for Disease Control. 2015. Live, Intranasal Influenza VIS. Current edition date: 8/7/2015. https://www.cdc.gov/vaccines/hcp/vis/vis-statements/flulive.html

Wacheck V, Egorov A, Groiss F, Pfeiffer A, Fuereder T, Hoeffmayer D, Kundi M, Popow-Kraupp T, Redlberger-Fritz M, Mueller CA, Cinatl J, Michaelis M, Geiler J, Bergmann M, Romanova J, Roethl E, Morokutti A, Wolschek M, Ferko B, Seipelt J, Dick-Gudenus R, and Muster T. 2010. A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagoni

Date modified:: 2019-02-28