Environmental Assessment for the Canadian Licensing of Laboratorios Hipra's Product: Bovine Rhinotracheitis Vaccine, Modified Live Virus (HIPRABOVIS IBR Marker Live)

March 15, 2017

Prepared and revised by the Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA), this environmental assessment includes information that was current at the time of its preparation. It is possible, however, that the situation may have changed since that time. Please consult CCVB, if you have any questions.

Table of contents

Summary

Laboratorios Hipra's Bovine Rhinotracheitis Vaccine, Modified Live Virus (Trade Name: HIPRABOVIS IBR Marker Live) contains a live bovine herpesvirus type 1 virus (BoHV-1) that has been genetically modified to delete large segments of two viral genes. The deletions are in the genes encoding the BoHV-1 Glycoprotein E and Thymidine Kinase proteins, and serve to attenuate the virus. The HIPRABOVIS IBR Marker Live vaccine is labelled for use in cattle, 3 months of age or older, to reduce clinical signs of infectious bovine rhinotracheitis (IBR) and BoHV-1 field virus excretion. As part of the requirements for licensing this product in Canada, the Canadian Centre for Veterinary Biologics of the Canadian Food Inspection Agency conducted an environmental assessment on the vaccine organism. This public Environmental Assessment summary document contains information on the molecular and biological characteristics of the live genetically modified vaccine organism, target animal and non-target animal safety, human safety, environmental considerations, and risk mitigating measures.

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. Laboratorios Hipra (Girona, Spain) has submitted an application to license the following vaccine in Canada:

Bovine Rhinotracheitis Vaccine, Modified Live Virus (Trade Name: HIPRABOVIS IBR Marker Live); CCVB File 810VV/B10.0/H14.

This Environmental Assessment was prepared by the CCVB as part of the overall assessment for licensing the above vaccine in Canada. It is based on information provided by the manufacturer as well as information independently obtained from the published literature.

1.2 Background

Bovine Rhinotracheitis Vaccine, Modified Live Virus (hereafter referred to as HIPRABOVIS IBR ML) is manufactured in Spain by the company, Laboratorios Hipra, which holds a veterinary medicines establishment licence (No. 4.242-E) issued by Spanish authorities. The vaccine is currently licensed for sale throughout the European Union. The vaccine contains a live bovine herpesvirus type 1 virus (BoHV-1) that has been genetically attenuated by deletion of large segments of its glycoprotein E (gE) and thymidine kinase (tk) genes. Both the gE and tk genes are considered important for BoHV-1 viral virulence[1]. Studies published by various researchers have demonstrated BoHV-1 virus attenuation by deletion of one or both of these genes[2, 3, 4].

BoHV-1 is probably most known for its role in causing respiratory disease in cattle, or infectious bovine rhinotracheitis (IBR). BoHV-1 (commonly called IBR virus) is one of the viruses implicated in Bovine Respiratory Disease (BRD), or "Shipping Fever", a respiratory disease complex of significant economic importance often seen around the time of weaning and assembly of calves into feedlots. Clinical signs of IBR and BRD include fever of over 40°C (104°F), nasal discharge, difficulty breathing, conjunctivitis, depression, and diminished appetite. BoHV-1 infections additionally pre-dispose cattle to secondary bacterial pneumonias, such as those caused by Mannheimia hemolytica and Pasteurella multocida, which can be life-threatening. BoHV-1 infections also cause abortion, infectious pustular vulvovaginitis and infectious pustular balanoposthitis.

BoHV-1 viruses are enveloped, double-stranded DNA viruses of the alpha-herpesvirus subfamily with an almost worldwide distribution. They are typically transmitted from animal to animal via nasal, eye, or genital secretions. Vertical transmission also occurs whereby a fetus becomes infected when the virus crosses the placenta.

2. Purpose and Need for Proposed Action

2.1 Significance

The labelling for HIPRABOVIS IBR ML indicates that the vaccine is intended for intramuscular administration to cattle, 3 months of age or older, to reduce clinical signs of infectious bovine rhinotracheitis and BoHV-1 field virus excretion. It is a live IBR vaccine that is labelled for use in pregnant cattle, without the need to vaccinate cows and heifers prior to breeding to ensure safety during pregnancy.

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 general criteria for licensing are as follows:

  1. the product must be pure, safe, potent and efficacious;
  2. vaccine components must be relevant to Canadian disease conditions;
  3. foreign products must be licensed in the country of origin; and
  4. the product must be produced and tested in accordance with generally accepted good manufacturing practices. This European origin vaccine meets these general criteria and thus was evaluated for licensing by the CCVB.

3. Alternatives

The two alternative options being considered are:

  1. to issue a Permit to Import Veterinary Biologics allowing the importation of HIPRABOVIS IBR ML, if all licensing requirements are met; or
  2. 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

4.1 Identification, Sources and Strains of Parental Organisms

The parental strain of BoHV-1, which was originally used to create the double gene-deleted gE- tk- BoHV-1 vaccine virus, was derived from a clinical isolate obtained from a cow during an IBR outbreak in Spain in 1988. This strain, known as the FM strain, belongs to the subtype 1 of BoHV-1 (BoHV-1.1). According to Laboratorios Hipra, this strain was only mildly pathogenic. BoHV-1.1 strains are prevalent in North America and Europe, and tend to be associated with respiratory disease as opposed to venereal disease[5, 6].

4.2 Source, Description and Function of Foreign Genetic Material

No foreign genetic material has been added. Segments of the BoHV-1 viral genome have only been deleted. The manufacturer has performed DNA sequencing, PCR and Southern blot analyses to confirm no integration of the plasmids used to introduce the deletion mutations.

4.3 Method of Accomplishing Genetic Modification

Details of the methods used to create the recombinant virus are on file with the CCVB. In brief, the deletions were introduced by homologous recombination. Homologous recombination was obtained by co-transfection of the shuttle vectors with the non-enveloped viral DNA and selection of recombinant viruses.

4.4 Genetic and Phenotypic Stability of the Vaccine Organism

The manufacturer examined the stability of the deletion mutations in the viral genome after passage in GBK cells, the cell type used to propagate the virus during vaccine production. The banding patterns obtained after digestion with three different restriction enzymes were the same for the initial master seed virus (MSV) and the virus obtained after 5 passages in GBK cells, the maximum number of passages permitted during vaccine production. The regions surrounding the gene deletions were also sequenced and no differences between the MSV and the fifth passage virus (MSV+5) were found. These data support the genetic stability of the recombinant virus in the vaccine production cells.

4.5 Horizontal Gene Transfer and Potential for Recombination

Compared to wild type BoHV-1 viruses, the vaccine strain presents a lower risk of horizontal gene transfer and potential for recombination. Deletion of the gE and tk genes reduces the capacity of the virus to infect, disseminate within, and persist within an exposed animal. The virus does not appear to be shed from intramuscularly vaccinated animals, or spread from animal to animal. Therefore, there is relatively little opportunity for the virus to exchange genetic material with other viruses. Recombination typically requires co-infection of the same cell with viruses of similar sequence. The fact that wild type BoHV-1 viruses replicate predominantly in the respiratory tract or genital tissue, while the vaccine virus does not disseminate to these regions before being cleared from the animal, diminishes the likelihood of co-infection and hence recombination. If recombination were to occur, deletion of two genes at distinct sites within the gE- tk- BoHV-1 viral genome reduces the possibility of such an event restoring the BoHV-1 genome to its full complement of genes. Even if the gE- tk- BoHV-1 were to regain functional gE and tk genes, the outcome would be a virus not unlike field strains of BoHV-1. According to the manufacturer, the parental FM BoHV-1 field virus making up the bulk of the vaccine virus was only a mildly pathogenic strain.

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

Field strains of BoHV-1 typically infect an animal via the nasal mucosa to replicate within epithelial cells of the nasal mucosa and upper respiratory tract. In some animals, the virus will subsequently migrate through nerve cells to establish latent infections in neuronal cell bodies of the trigeminal ganglia. The tonsils may also become latently infected. BoHV-1 genital infections also occur, with virus replicating in the mucous membranes of the vagina or prepuce and establishing latent infections in the sacral ganglia. Latent infections can re-activate later in life during times of stress and result in additional BoHV-1 infectious virus shedding into the environment. BoHV-1 viruses are predominantly known for infecting cattle, but they can also infect other animals of the Bovidae family, including sheep and goats.

Studies performed by the manufacturer demonstrate that when the gE- tk- BoHV-1 vaccine virus is injected intramuscularly (IM) into the neck muscles of calves, it is undetectable at 2, 4 and 6 days post-injection in:

  • typical target body organs: nasal mucosa, conjunctiva, trachea, lungs, trigeminal ganglion;
  • main body fluids: whole blood, serum, white blood cells;
  • secretions: urine, feces, and nasal, ocular, saliva, vaginal and balanal (glans penis) swabs; and
  • reproductive organs: testis, seminal vesicle, prostate, ovaries, uterine mucosa and vaginal mucosa.

Thus, although the gE- tk- BoHV-1 virus is replication competent (it will multiply in the GBK cells used for virus production), it does not appear to be able to disseminate from the intramuscular injection site to infect the normal target tissues of BoHV-1. Instead, it seems to be rapidly cleared from the body of a vaccinated animal.

The manufacturer conducted separate studies to determine whether the virus can spread from vaccinated calves to other in-contact calves. In the first, three-month-old calves were administered (IM in the neck) a 10X dose of the vaccine on days 0 and 21. Sentinel calves were housed together with the vaccinated calves. All sentinel calves remained BoHV-1 seronegative until the end of the study on day 42. Nasal swabs taken on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39 and 42 from the sentinel calves were negative for vaccine virus by PCR and virus isolation (nasal swabs from vaccinated calves were also negative at these time points). In the second study, three-month-old calves were administered (IM in the neck) a 10X dose of the vaccine on days 0, 21 and 42. Sentinel calves were housed together with the vaccinated calves. All sentinel calves remained BoHV-1 seronegative until the end of the study on day 60. Vaccine virus was not recovered from the nasal, ocular or salivary swabs taken on days 0, 1-10, 21-31, and 42-52 from the sentinel or vaccinated calves. The results of these studies show that the vaccine virus does not spread from IM vaccinated calves to in-contact calves.

4.7 Comparison of the Modified Organisms to Parental Properties

The vaccine organism differs from the parental BoHV-1 virus by deletion of selected base pairs (bp) of the gE gene and tk gene. These deletion mutations serve to attenuate the virus so that it does not cause clinical disease in vaccinated animals.

4.8 Route of Administration/Transmission

The HIPRABOVIS IBR ML vaccine is to be administered parenterally by the intramuscular route, preferentially in the neck region of cattle. No transmission from vaccinated animals to in-contact animals is expected.

5. Human Safety

5.1 Previous Safe Use

The gene deleted gE- tk- BoHV-1 virus has been used as a vaccine in Europe since 2011. Over 5 million doses of the vaccine have been sold in Europe.

5.2 Probability of Human Exposure

Individuals administering HIPRABOVIS IBR ML to cattle could on occasion become exposed to the vaccine in the event of accidental spillage or self-injection. Human exposure to the gE- tk- BoHV-1 virus is not expected for individuals handling vaccinated animals based on the manufacturer's inability to recover shed vaccine virus in nasal and other bodily secretions post-IM administration.

5.3 Possible Outcomes of Human Exposure

Human exposure to the gene deleted BoHV-1 virus is not expected to be a health concern. Field strains of BoHV-1 are not pathogenic to humans[1, 7, 8] so there is no reason to believe the attenuated, gene deleted vaccine virus would be pathogenic.

5.4 Pathogenicity of Parent Microorganisms in Humans

BoHV-1 is not thought to be pathogenic to humans. Field strains of BoHV-1, and BoHV-1 infected cattle, are prevalent in many countries including Canada and are not recognized as a zoonotic concern for the farmers and veterinarians routinely exposed to these viruses.

5.5 Effect of Gene Manipulation on Pathogenicity in Humans

The genetic manipulation has eliminated the ability of the virus to cause disease in cattle, and thus has presumably further reduced the chance of the virus being pathogenic to humans.

5.6 Risk Associated With Widespread Use of the Vaccine

No risks to human safety associated with the widespread use of the vaccine have been identified.

6. Animal Safety

6.1 Previous Safe Use

Prior to European licensing, HIPRABOVIS IBR ML was field tested on several farms in Spain with differing production practices, in cows and calves. Injection site swelling was observed in up to 30% of cows (compared to 10% of control cows administered a blank vaccine); however, none of the animals showed pain at the injection site, no nodules were felt, and all swellings resolved within 96 hours of appearance. No cow showed abnormal general clinical signs attributable to the vaccine. In calves, no local reactions at the injection site were observed, nor were any general clinical signs attributable to the vaccine. Safety studies were also conducted in three-month-old calves at Hipra to ensure safety of an accidental 10X overdose and safety of extra repeated doses (tested by administering 3 doses 14 days apart). No abnormal general clinical signs attributed to the vaccine were observed in these overdose studies.

The vaccine was additionally tested at Hipra for safety in pregnant cattle by administering a 10X dose to pregnant cows at 4 months of gestation, pregnant cows at 5 months of gestation, and pregnant cows at 6-7 months of gestation. The cows used in this study had never previously received an IBR vaccine and were sourced from certified IBR negative herds. They tested negative for BoHV-1 antigen and antibody prior to receiving the HIPRABOVIS IBR ML vaccine during pregnancy. Two cows aborted, both from the 4 months of gestation group, at 14 and 38 days post vaccination. In both cases, the fetus and placental samples available tested negative for the presence of BoHV-1 and thus were unlikely to have been caused by infection by the vaccine virus (evidence of an in utero bacterial infection was found in one case). Analysis of European post-licensing pharmacovigilance data for HIPRABOVIS IBR ML did not reveal problems with abortions. Safety of the vaccine in pregnant cows is additionally supported by the tissue distribution data showing the gene-deleted virus does not disseminate to reproductive tissues and is undetectable in the blood following vaccination.

Over 5 million doses of HIPRABOVIS IBR ML have been sold in Europe since 2011.

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

The manufacturer performed a study to examine the fate of the vaccine in calves post-intramuscular vaccination. Two-month-old calves were administered a 10X dose and then one male calf and one female calf were sacrificed on days 2, 4, and 6 post-vaccination. Samples from the following organs, body fluids and secretions were collected and tested for BoHV-1 by virus isolation and PCR:

  • typical target body organs: nasal mucosa, conjunctiva, trachea, lungs, trigeminal ganglion
  • main body fluids: whole blood, serum, white blood cells
  • secretions: urine, feces, and nasal, ocular, saliva, vaginal and balanal (glans penis) swabs
  • reproductive organs: testis, seminal vesicle, prostate, ovaries, uterine mucosa and vaginal mucosa

All samples tested negative for BoHV-1, suggesting that the intramuscularly injected vaccine virus does not disseminate throughout the body.

Field strains of BoHV-1 can establish a latent infection in nerve cells, particularly of the trigeminal ganglion, and in lymphoid cells of the tonsil[6, 9]. As mentioned above, the vaccine virus could not be recovered from the trigeminal ganglion or tonsil after 2, 4 and 6 days post-vaccination. Ganglia closer to site of IM injection (in the neck) were not checked to confirm the absence of latent virus.

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

In the tissue tropism study described above in section 6.2, no vaccine virus could be isolated from the urine, feces, nasal secretions, ocular secretions, saliva or vaginal/penis swabs post-vaccination. Another study examined milk samples at 14, 21 and 45 days post-vaccination and no evidence of vaccine virus shedding in the milk was found.

Studies were performed to determine whether the virus would spread from vaccinated calves to other in-contact calves. In one study, three-month-old calves were administered (IM in the neck) a 10X dose of the vaccine on day 0 and 21. Sentinel calves were housed together with the vaccinated calves. All sentinel calves remained BoHV-1 seronegative until the end of the study on day 42. Nasal swabs taken on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39 and 42 from the sentinel calves were negative for vaccine virus by PCR and virus isolation (nasal swabs from vaccinated calves were also negative at these time points). In another study, three-month-old calves were administered (IM in the neck) a 10X dose of the vaccine on days 0, 21 and 42. Sentinel calves were housed together with the vaccinated calves. All sentinel calves remained BoHV-1 seronegative until the end of the study on day 60. Vaccine virus was not recovered from the nasal, ocular or salivary swabs taken on days 0, 1-10, 21-31, and 42-52 from the sentinel or vaccinated calves.

Taken together, these data indicate that the vaccine virus is not shed from IM-vaccinated animals and does not spread to other calves held in contact with the vaccinates.

If the vaccine virus does not spread from vaccinated calves to other calves, the natural host of the virus, it is unlikely that the vaccine will spread from vaccinated calves to non-target animals.

6.4 Reversion to Virulence Resulting from Back Passage in Animals

The manufacturer initiated a back passage study in calves starting with the vaccine virus being administered by the route of administration indicated for vaccination. For this study, three-month-old calves were intramuscularly administered a 10X dose of the vaccine virus for the first pass. However, no virus could be recovered from the nasal swabs taken from these calves to inoculate the second group of calves. Nasal swabs were checked for virus every day until 14 days post-vaccination. All swabs tested negative for vaccine virus by virus isolation and PCR. All calves, however, seroconverted to BoHV-1 antigen, confirming virus exposure. These results, together with the shed/spread results, suggest there is a lack of opportunity for the vaccine virus to revert to virulence due to limited replication and transmission between animals.

To further confirm the stability of the virus, the manufacturer performed an additional reversion to virulence study with virus administered to the first group of calves by the intranasal route. Following intranasal inoculation, some virus can be recovered from nasal swab samples. Nasal swab samples, collected on days 2-7 post inoculation, were then used to intranasally inoculate subsequent groups of calves in a back passage study. No signs of viral reversion to virulence were evident after five passages in calves. It was noted that the amount of virus recoverable from nasal swabs declined with each passage.

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

The vaccine has been tested in three-month-old calves, the youngest age for which the vaccine is recommended, at a 10X dose, both with and without a 1X booster dose 21 days later. No clinical signs suggestive of an IBR infection were observed, and mean rectal temperatures remained below 39.5°C.

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

BoHV-1 viruses are predominantly known for infecting cattle, but they can also infect other animals of the Bovidae family, including sheep and goats. However, BoHV-1 infections do not appear to be a common cause of disease in sheep and goats. The gene deleted vaccine virus is expected to have the same host range, except it is unlikely to encounter these other host species. The mobility of the vector is impaired as it has not been found to be able to spread from vaccinated cattle to other in-contact cattle.

7. Affected Environment

7.1 Extent of Release into the Environment

Release into the environment is expected to be low. Release could occur in cases of accidental spillage. Release via shedding from vaccinated animals is not expected.

7.2 Persistence of the Vector in the Environment and Cumulative Impacts

The gE- tk- BoHV-1 gene deleted virus of the vaccine is not expected to be any more persistent in the environment than field strains of BoHV-1 which are prevalent in Canada. BoHV-1, as an enveloped virus, is susceptible to many common lipophilic disinfectants and solvents[6, 8]. Ultraviolet radiation can inactivate the virus after 8-12 hours of exposure[8].

7.3 Extent of Exposure to Non-Target Species

Little exposure to non-target animal species is expected based on manufacturer studies showing that the vaccine virus is not shed in the urine, feces, nasal secretions, ocular secretions, saliva or milk after intramuscular administration.

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

BoHV-1 can infect other species in the Bovidae family, but does not appear to be a common cause of disease in these species. The mutations in the vaccine virus which render it non-virulent for cattle are expected to render the virus non-virulent for other species.

8. Environmental Consequences

8.1 Risks and Benefits

The benefit of the vaccine is that it has been shown to help reduce clinical signs of IBR, including rhinitis and dyspnea, in calves post challenge with a virulent IBR virus. The challenge virus used was of US-origin suggesting the Spanish origin vaccine should cross-protect against North American strains of BoHV-1. The vaccine appears to be safe for use in pregnant cows, and calves nursing pregnant cows, even if the pregnant cow has not received prior vaccinations against IBR. Because of deletion of the gE gene, the vaccine can allow for the differentiation of vaccinated animals from infected animals, as animals vaccinated with HIPRABOVIS IBR ML alone will not test positive on IBR diagnostic tests designed to detect antibodies against the gE protein.

No significant risks associated with use of the vaccine in Canada have been identified.

8.2 Relative Safety Compared to other Vaccines

HIPRABOVIS IBR ML is as safe as other modified live virus IBR vaccines already approved for the Canadian market.

9. Mitigative Measures

9.1 Worker Safety

Veterinarians and workers at cattle farms responsible for vaccinating cattle might be exposed to the live genetically modified organism in situations of accidental spillage (e.g., during rehydration of the vaccine) or self-injection. As is always the case, vaccinators should be well trained to reduce the chance of accidental self-injection. HIPRABOVIS IBR ML does not contain an oil adjuvant likely to cause significate injection site lesions in humans. For the reasons mentioned in section 5, human health concerns, even in cases of accidental self-injection, are not expected from exposure to this vaccine.

9.2 Handling Vaccinated or Exposed Animals

Vaccinated animals do not appear to shed the vaccine virus thus no mitigative measures are suggested for those handing vaccinated animals.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all significant suspected 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. If an adverse reaction 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

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

10.3 Animal

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

11. Consultations and Contacts

Manufacturer:
Laboratorios Hipra, S.A.
Avda. la Selva, 135
17170 Amer (Girona)
Spain

12. Conclusions and Actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Bovine Rhinotracheitis Vaccine, Modified Live Virus (Trade Name: HIPRABOVIS IBR Marker Live) in Canada would not be expected to have any significant adverse effect on the environment, when manufactured and tested as described in the approved Outline of Production, and used according to label directions.

Following this assessment and the completion of the Canadian veterinary biologics licensing process, the Permit to Import Veterinary Biologics held by Hipra Animal Health Canada may be amended to allow the importation and distribution of the following product in Canada:

Bovine Rhinotracheitis Vaccine, Modified Live Virus (Trade Name: HIPRABOVIS IBR Marker Live); CCVB File 810VV/B10.0/H14.

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

13. References

1. The biology of bovine herpesvirus 1 (BoHV-1). 2005, Australian Government Department of Health and Ageing. www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/5DCF28AD2F3779C4CA257D4E001819B9/$File/biology-bovineherpesvirus.pdf. Accessed February 6, 2017.

2. Kalthoff, D., et al., Immunization and challenge experiments with a new modified live bovine herpesvirus type 1 marker vaccine prototype adjuvanted with a co-polymer. Vaccine, 2010. 28(36): p. 5871-7.

3. van Engelenburg, F.A., et al., A glycoprotein E deletion mutant of bovine herpesvirus 1 infects the same limited number of tissues in calves as wild-type virus, but for a shorter period. J Gen Virol, 1995. 76 (Pt 9): p. 2387-92.

4. Kaashoek, M.J., et al., Virulence and immunogenicity in calves of thymidine kinase- and glycoprotein E-negative bovine herpesvirus 1 mutants. Vet Microbiol, 1996. 48(1-2): p. 143-53.

5. Graham, D.A., Bovine herpes virus-1 (BoHV-1) in cattle-a review with emphasis on reproductive impacts and the emergence of infection in Ireland and the United Kingdom. Ir Vet J, 2013. 66(1): p. 15.

6. Biswas, S., et al., Bovine herpesvirus-1 (BHV-1) - a re-emerging concern in livestock: a revisit to its biology, epidemiology, diagnosis, and prophylaxis. Vet Q, 2013. 33(2): p. 68-81.

7. EPA staff supporting memorandum for application APP201842: to import for release a genetically modified virus contained within a veterinary vaccine. 2015. http://www.epa.govt.nz/search-databases/HSNO Application Register Documents/APP201842_APP201842_Memorandum_FINAL.pdf. Accessed February 6, 2017.

8. Hiprabovis IBR Marker Live: European Public Assessment Report - Scientific Discussion 2011. http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/veterinary/medicines/000158/vet_med_000226.jsp&mid=WC0b01ac058001fa1c. Accessed February 6, 2017.

9. Winkler, M.T., A. Doster, and C. Jones, Persistence and reactivation of bovine herpesvirus 1 in the tonsils of latently infected calves. J Virol, 2000. 74(11): p. 5337-46

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