Avian influenza (AI) remains a major threat to public health as well as to the poultry industry. AI vaccines are considered a suitable tool to support AI control programs in combination with other control measures such as good biosecurity and monitoring programs. We constructed recombinant turkey herpesvirus (HVT) vector vaccines expressing the hemagglutinin gene of AI virus H5 subtype (rHVT‐H5) and evaluated their characteristics and efficacy against AI. We found that the cytomegalovirus (CMV) promoter is the most suitable for expression of the hemagglutinin gene among three promoters we evaluated. The rHVT‐H5 vaccine did not cause any adverse reactions and did not revert to virulence after passages in chicken. Finally, efficacy of the rHVT‐H5 vaccine was evaluated. We demonstrated that it provided protection against diverse AI H5 viruses belonging to different clades and reduced virus shedding from the challenged chicken. We also proved that efficacy provided by the rHVT‐H5 vaccine was not significantly affected by presence of maternally derived antibodies (MDA) against AI virus. Furthermore, the rHVT‐H5 vaccine could be applicable to the differentiating infected from vaccinated animals (DIVA) strategy. In summary, we successfully developed a HVT vector AI vaccine that possesses features that could be beneficial to AI control.
- avian influenza
- turkey herpesvirus
- vector vaccines
- hemagglutinin gene
Avian influenza (AI) is an important zoonotic disease and it remains a major threat to public health and to the poultry industry. The highly pathogenic (HP) H5N1 outbreaks were first reported in China in 1996 and then spread to other parts of the world. These HP avian influenza viruses (AIV) have become endemic in several countries including China, Indonesia, Vietnam, and Egypt . Between 2003 and 2015, 846 confirmed human cases of AI (H5N1) have been reported and 449 of those human patients have died (WHO, 2016). Most recently, HP H5N2 and H5N8 viruses caused outbreaks in the United States from December 2014 to June 2015, which resulted in depopulation of more than 48 million chickens and turkeys. These outbreaks cost farmers, the government, and consumers in the United States billions of dollars. France has also been hit by HP H5 viruses since November 2015 and the outbreaks have caused significant damages to its poultry industry. These recent HP H5 AIV continued to evolve into various clades as defined by the World Health Organization (WHO)/World Organisation for Animal Health (OIE)/Food and Agriculture Organization (FAO) H5N1 Evolution Working Group according to phylogenetic topology based on
Control of HP AI in poultry has been achieved traditionally through (1) education, (2) biosecurity, (3) diagnostics and surveillance, and (4) elimination of infected poultry (stamping‐out) . Viruses were successfully eradicated through a combination of those measures in many countries affected by HP AIV. However, in endemically infected countries, viruses had spread widely before executing effective measures, and therefore, it was impossible to identify and eliminate all of infected birds. In such endemic situations, vaccines are considered suitable and powerful tools to support AI eradication or control programs in combination with other control measures such as good biosecurity and monitoring programs [4–6]. When used properly, vaccines for AI have been demonstrated to protect poultry against clinical signs and mortality, increase resistance to infection, and reduce virus shedding markedly, thus decreasing the possibility of virus spreading among birds . Most frequently used AI vaccines have been oil‐adjuvanted, inactivated whole‐virus vaccines and fowlpox virus or Newcastle disease virus (NDV)‐vectored vaccines are also available. However, efficacy of these vaccines is limited especially against antigenically distant viruses [8–10]. Also, efficacy of these vaccines is known to be severely impaired by maternally derived antibodies (MDA) [11, 12]. Furthermore, oil‐adjuvanted, inactivated whole‐virus vaccines impede virus surveillance programs based on serology because serological responses elicited by the inactivated whole‐virus vaccines are indistinguishable to those elicited by live field viruses. Therefore, development of novel vaccines which are efficacious in the face of MDA and are compatible with so‐called differentiate infected from vaccinated animals (DIVA) strategy is necessary.
Turkey herpesvirus (HVT), or the meleagrid herpesvirus 1, belongs to the family of
2. Construction of rHVT‐H5 vaccines
2.1. Construction of recombinant viruses
A number of HVT vector vaccines have been constructed and evaluated for their characteristics and efficacy against avian pathogens [21–26]. There are three important elements in HVT vector vaccines that can impact efficacy of these vaccines in chicken: insertion sites, antigen genes, and promoters. Several insertion sites including US2, US10, UL39 , and an intergenic region between UL45 and UL46 (UL45/46) [24–26] have been evaluated. Out of these potential insertion sites, we demonstrated that insertion of extraneous genes at the UL45/46 site did not alter their capacity to replicate . Furthermore, using the UL45/46 as the insertion site, we were successful in constructing HVT vector vaccines having antigen genes of NDV, IBDV, or ILTV that are highly efficacious against these pathogens [24–26]. Therefore, we decided to use the UL45/46 site for insertion of antigen genes of AIV.
Eight segments of genomic RNA of influenza A virus encode three membrane‐associated proteins, HA, neuraminidase (NA), and Matrix (M) 2, five internal proteins, nucleoprotein (NP), PB1, PB2, PA, and M1, and two nonstructural (NS) proteins, NS1 and NS2. Out of these viral proteins, the HA surface glycoprotein is known to be the major antigen and elicits neutralizing antibodies that provide protection against the disease . We used the
Selection of promoters that control the expression of antigen genes is also an important factor. Tsukamoto et al. compared the cytomegalovirus (CMV) promoter and CMV/chicken β‐actin chimera (Pec) promoter for expression of IBDV
To prepare for construction of recombinant HVT, HVT insertion site sequences (
2.2. In vitro characterization of rHVT‐H5
Figure 1 shows genomic structures of the constructed rHVT‐H5 vaccines. The genomic structures of the rHVT‐H5 vaccines were confirmed by PCR assays with one primer binding to the insert sequence and the other primer binding to the HVT insertion site sequences (data not shown). The genomic structures were further confirmed by Southern blot analysis using digoxigenin‐labeled probes specific to the
In order to assess genetic and phenotypic stability of the rHVT‐H5 vaccines, the viruses were passed in CEF 20 times. Viruses after the passages were characterized by PCR, Southern blot, BPA, and western blot. Results obtained with the passed viruses were identical to those obtained with the viruses before the passage (data not shown), and therefore, it was concluded that these viruses were genetically and phenotypically stable.
2.3. Evaluation of promoters for expression of HA gene
In order to identify the most suitable promoter among the CMV promoter, the Bac promoter, and the Pec promoter for expression of the
One‐day‐old specific pathogen free (SPF) White Leghorn chicks were vaccinated subcutaneously with one of the rHVT‐H5 vaccines. A group of chickens was held as a non‐inoculated negative control group and another group of chickens was vaccinated subcutaneously with inactivated A/turkey/Wisconsin/68 (H5N9) vaccine at 3 weeks of age as an inactivated vaccine control group. Chickens in each group were bled each week between 3 and 7 weeks of age (6 and 7 weeks of age for the inactivated vaccine control group) and obtained sera were evaluated by the AIV hemagglutination inhibition (HI) test and AIV enzyme‐linked immunosorbent assay (ELISA). The AIV HI tests were conducted using four hemagglutination units of an inactivated AIV homologous antigen of the A/turkey/Wisconsin/68 (H5N9) strain according to the standard procedure .
As shown in Figure 5, the rHVT‐H5 vaccines induced increased HI titers as early as 3 weeks of age and the increased titers were maintained through 7 weeks of age. Chickens vaccinated with HVT‐CMV‐H5Wis68 had higher mean HI titers than HVT‐Pec‐H5Wis68 and HVT‐Bac‐H5Wis68 vaccinated groups had between 4 and 7 weeks of age, with the differences statistically significant at 5 and 6 weeks of age. As expected, the inactivated A/turkey/Wisconsin/68 (H5N9) vaccine induced high HI titers at 6 and 7 weeks of age (3 and 4 weeks post‐vaccination), confirming validity of the assay. When tested with the commercial AIV ELISA kits, Flock‐Chek™ AIV Ab kit (Idexx Laboratories) and ProFLOK® AIV Ab test kit (Zoetis), sera collected from the rHVT‐H5 vaccinated chickens were negative between 3 and 7 weeks of age, whereas sera collected from the inactivated vaccine control chickens showed highly positive ELISA titers with both kits (Figure 6).
This result demonstrated that the CMV promoter was the most suitable for expression of the
3. Safety of rHVT‐H5 vaccine
Based on the results described above, the CMV promoter was selected for the expression of the
Safety of the rHVT‐H5h vaccine was evaluated by an overdose study and a backpassage study. For the overdose study, SPF embryos at 18 days of incubation or day‐of‐age SPF chicks received rHVT‐H5h at 10 times the typical field dose. In ovo application of rHVT‐H5h at overdose did not affect hatchability and the vaccinated chickens remained free from any clinical signs or adverse reactions until 18 weeks of age. Chickens were necropsied at 18 weeks of age and no gross lesions were observed in any of the vaccinated chickens. Similarly, rHVT‐H5h did not cause any clinical signs, adverse reactions, or gross lesions in other avian species such as turkeys, quail, pheasants, and pigeons.
For the backpassage study, to confirm that the rHVT‐H5h vaccine will not revert to virulence, rHVT‐H5h was passed five times in SPF chickens by using heparinized blood from chickens in the previous passage to inoculate a new set of SPF chicks. Chickens inoculated with the virus at the fifth passage, that is, inoculated with the heparinized blood from the fourth passage, were observed closely for any clinical signs for 45 days. Chickens were then necropsied and observed for grossly observable lesions. No chickens had any clinical signs, adverse reactions, or gross lesions. Therefore, it was concluded that rHVT‐H5h did not revert to virulence after backpassages in chickens.
We conducted another study to evaluate the ability of the rHVT‐H5h vaccine to transmit from vaccinated chickens to non‐vaccinated chickens in contact. Day‐of‐age SPF chickens vaccinated in ovo with rHVT‐H5h or parental HVT were commingled in isolators with non‐vaccinated contact chickens. Virus isolation was attempted from the peripheral mononuclear blood lymphocytes collected from chickens at 10, 14, and 21 days of age. No virus was isolated from any of the contact chickens at any time points while the viruses were isolated from the vaccinated chickens, indicating that neither rHVT‐H5h nor parental HVT spread from vaccinated chickens to non‐vaccinated contact chickens. It appears that transmissibility of HVT parent and the rHVT‐H5h vaccine between chickens is negligible.
In summary, similar to parent HVT vaccines and other HVT vector vaccines, the rHVT‐H5h vaccine is extremely safe causing no adverse effects and does not revert to virulence after passages in chickens. The lack of transmission of the rHVT‐H5h vaccine further strengthens the safety profile of this vaccine in terms of containment of the genetically modified organism.
4. Efficacy of rHVT‐H5 vaccine
4.1. Efficacy against homologous challenge
Finally, efficacy of the rHVT‐H5h vaccine was evaluated. After vaccination, humoral immune response was evaluated by the AIV HI tests. After challenge with HP AIV, vaccine efficacy was evaluated for (1) protection against mortality and clinical signs and (2) reduction of challenge virus shedding from challenged birds. Both the criteria are important for AI vaccines because these features will ensure that the vaccine will be beneficial as a measure to contain spread of field AI viruses as well as to reduce economical burdens in both endemic and emergent situations [4, 31].
Initial evaluation of the rHVT‐H5h vaccine efficacy was conducted using homologous AIV as a challenge virus . Day‐of‐age SPF chicks were vaccinated subcutaneously with either a frozen, cell‐associated (ca) form or a lyophilized, cell‐free (cf) form of the rHVT‐H5h vaccine. At 5 weeks of age before challenge, increased HI titers were observed in all the rHVT‐H5h vaccinated chicken with mean titers of 26.3 for the ca group and 25.1 for the cf group (Figure 7) when using homologous antigen.
Chickens were challenged with 106 mean embryo infectious dose (EID50) HP H5N1 AIV A/Whooper Swan/Mongolia/3/2005 clade 2.2 strain at 6 weeks of age. The
We observed 3–6 log10 reduction of challenge virus shedding in the rHVT‐H5h vaccinated chickens. From oropharyngeal swabs at 2 days post‐challenge (dpc), only 4/30 (13%) chickens in the ca rHVT‐H5h group and 3/20 (15%) chickens in the cf rHVT‐H5h group shed virus with minimal virus titers (101–103 EID50/ml), while all the challenge controls shed significant amounts (105–107EID50/ml) of virus (Figure 8A). From cloacal swabs, no virus was isolated from any of the rHVT‐H5h vaccinated chickens at 2 dpc, while all the challenge controls shed virus at 102–105EID50/ml (Figure 8B).
These results demonstrated high potential of the rHVT‐H5h vaccine as an excellent tool to support control of AI. Therefore, we proceeded to further evaluation of the rHVT‐H5h, especially for protection against heterologous virus challenge and effects of MDA. Since the ca form appeared to provide slightly better immunogenicity than the cf form, the following evaluation was conducted using the ca form of the vaccine.
4.2. Efficacy against heterologous challenge
Since HP H5 viruses have become diverse and have evolved into various different clades, it is highly important that AI vaccines exert broad “cross‐clade” efficacy against diverse AIV strains. Indeed, one of the limitations of conventional oil‐adjuvanted, inactivated vaccines is that they are not as effective against heterologous viruses as homologous viruses [8–10]. Therefore, we tested efficacy of the rHVT‐H5h vaccine against various AIV HP H5 strains.
Day‐of‐age SPF broiler chicks were vaccinated with the rHVT‐H5h vaccine. A commercially available inactivated vaccine based on H5N2 Mexican strain (iH5N2) was used as a control and injected into birds at 10 days of age. Challenge was conducted at 4 weeks of age using the Indonesian A/chicken/WestJava Sbg/29/2007 H5N1 strain (CW07). The CW07 virus belongs to clade 2.1.3 and sequence similarity of the HA gene between the rHVT‐H5h insert and the CW07 virus was 93%. After challenge, all chickens in the sham‐vaccinated, challenge control group died within 2 days. In the group vaccinated with rHVT‐H5h, 80% (16/20) of the chickens survived the challenge, while only one bird (5%) vaccinated with iH5N2 survived the challenge. When rHVT‐H5h vaccinated chickens received boost with iH5N2 vaccine at 10 days of age, protection increased to 90% (18/20). Reduction in virus shedding up to 3 log10 was observed in rHVT‐H5h vaccinated chickens compared to the challenge control. When the homologous antigen was used, rHVT‐H5h, either with or without boost with iH5N2 vaccine, it induced average HI titers of 25–26 prior to challenge. HI titers were much lower with the heterologous CW07 antigen with average between 21 and 22. These results indicate that cell‐mediated immunity as well as mucosal immunity provided by HVT vector vaccines might be involved in protective efficacy against heterologous CW07 strain.
In the next trial, another AIV HP H5 strain, A/Viet Nam/1203/04 (H5N1) (VN04), was used for challenge. The VN04 virus belongs to clade 1 and shares 96.5% HA gene similarity with the rHVT‐H5h insert. Vaccination was conducted at the day of age to SPF layer chicks and challenge was conducted at 4 weeks of age. Protection was 85% (17/20) while all challenge control chickens died and reduction of virus shedding of 3 log10 was observed in the rHVT‐H5h group.
Since these two trials using heterologous HP H5 strains demonstrated that the rHVT‐H5h provided “cross‐clade” efficacy, we went further and conducted another efficacy trial using Mexican H5N2 HP strain which shares only 82% HA gene similarity with the rHVT‐H5h insert. Day‐of‐age chicks vaccinated with rHVT‐H5h were challenged with A/chicken/Queretaro/14588‐19/95 (H5N2) strain at 4 weeks of age. Nineteen out of 20 (95%) chickens survived the challenge, while all the challenge control died. This result demonstrated very broad cross‐protective efficacy provided by rHVT‐H5h.
We further evaluated efficacy of the rHVT‐H5h vaccine against various HP AIV H5 isolates including several Egyptian isolates and a recent 2014 H5N8 isolate from Germany, as summarized in Figure 9. Protection in rHVT‐H5h vaccinated chickens ranged between 60 and 100% with significant reduction in virus shedding, further strengthening evidence of very broad “cross‐clade” efficacy provided by the rHVT‐H5h vaccine. In one study where layer chickens were vaccinated and raised under field conditions in Egypt, a single rHVT‐H5h vaccination at day of age conferred a high level of protection (60–73%) for a relatively extended period (up to 19 weeks of age) against an Egyptian isolate .
To see if cellular immunity indeed is involved in this broad protective efficacy, we conducted in vitro cytotoxicity assay. Splenic lymphocytes collected from chickens vaccinated with the rHVT‐H5h vaccine were incubated with chicken lung cells infected with either H5N9, H6N2, H7N2, or H9N2 low pathogenic AIV. The highest level of lysis by splenic cytotoxic T cells was observed with H5‐infected target cells. Lysis was also observed with other heterologous AIV (H6, H7, or H9), although to a lower degree. Negligible lysis was observed with naïve uninfected lung cells. These results indicated that the rHVT‐H5h vaccine induced AIV‐specific cytotoxic T‐cell activity and it may contribute in part to broad protective immunity induced by the rHVT‐H5h.
4.3. Efficacy of rHVT‐H5 in chicken with maternally derived antibodies
In endemic countries, most breeders are vaccinated with AI vaccines and/or exposed to field AIV challenge and therefore, their progeny possess MDA against AIV. Efficacy of oil‐adjuvanted, inactivated AIV vaccines and fowlpox‐vectored AI vaccines has been shown to be significantly impaired in the presence of MDA. On the other hands, HVT vector vaccines have been shown not to be excessively affected by the presence of MDA against inserted antigens. Indeed, with the rHVT‐H5h vaccine, several studies demonstrated lack of significant interference on its protective efficacy by the presence of MDA when administered to day‐of‐age chicks [10, 34].
AI surveillance is conducted through serological assays including ELISA and HI. Except in endemic countries, positive serological response in those assays will lead to immediate and extreme actions including “stamping‐out.” Also, there are trade implications because many countries ban importation of poultry from AI‐positive countries. Therefore, when AI vaccination is introduced, it is critical that AI vaccines do not interfere with the AI surveillance. It is highly favorable that serological responses elicited by AI vaccines may be distinguished from those elicited by infection of field AIV. Since conventional inactivated vaccines elicit humoral immune responses that lead to positive titers in both ELISA and HI tests, these vaccines do interfere with the surveillance .
The rHVT‐H5h vaccine elicits antibodies against the HA protein and the antibody responses can be detected by the HI tests. However, those sera from vaccinated chickens were negative in commercial ELISA kits because the ELISA kits are designed to detect antibodies against more conserved internal protein (NP) in order to offer coverage over different subtypes of AIV. When we examined serological responses in chickens that were vaccinated with the rHVT‐H5h and then challenged with HP AIV, we found positive ELISA titers in chickens that excreted challenge viruses. These results demonstrated that the rHVT‐H5h vaccines may be applied to the DIVA strategy and do not interfere with AI surveillance.
Our studies demonstrated that the rHVT‐H5h vaccine possesses characteristics that could be beneficial to control of AI in endemic countries and in emergency situations. Those characteristics are (1) broad “cross‐clade” protective efficacy against diverse AIV H5 isolates, (2) lack of interference by MDA, (3) applicability to hatchery vaccination, and (4) applicability to DIVA. The rHVT‐H5h vaccine has been approved by authorities in Egypt, Mexico, and Bangladesh and is in use in the field. An independent survey conducted by FAO, General Organization for Veterinary Services in Egypt, and Centre International de Recherche en Agriculture pour le Développement in France concluded that day‐of‐age vaccination utilizing the rHVT‐H5h vaccine at hatcheries is more efficient than the program using the inactivated vaccines at farms and it would have a positive impact for disease control in Egypt .
It is clear that vaccines alone cannot solve all the problems associated with AI. However, we believe that in conjunction with active and efficient surveillance and strict biosecurity measures, the rHVT‐H5h vaccine can contribute to disease control by increasing resistance against infection and decreasing the amount of virus shed to the environment. It would also remove economical burdens from farmers and consumers and improve animal welfare by protecting chickens from mortality and clinical signs. In conclusion, we successfully developed a HVT vector AI vaccine that possesses many features that could be beneficial to AI control. It remains to be seen whether this vaccine is truly useful in the field.
Food and Agriculture Organization (FAO). Approaches to controlling, preventing and eliminating H5N1 highly pathogenic avian influenza in endemic countries. Rome; 2011.
WHO/OIE/FAO H5N1 Evolution Working Group. Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. Influenza and Other Respiratory Viruses. 2012; 6:1–5.
Swayne D.E., Suarez D.L., Sims L.D. Influenza. In: Swayne DE, Glisson JR, McDougald LR, Nair V, Nolan LK, Suarez DL, editors. Diseases of Poultry. 13th ed. Ames, IA: Wiley‐Blackwell; 2013. pp. 181–218.
Swayne D.E. The role of vaccines and vaccination in high pathogenicity avian influenza control and eradication. Expert Review of Vaccines. 2012; 11:877–880.
Capua I., Alexander D.J. Avian influenza vaccines and vaccination in birds. Vaccine. 2008; 26:D70–D73.
Sims L.D. Lessons learned from Asian H5N1 outbreak control. Avian Diseases. 2007; 51:174–181.
Swayne D.E. Vaccines for list A poultry diseases: Emphasis on avian influenza. Developmental Biology (Basel). 2003; 114:201–212.
Abdelwhab E.M., Grund.C, Aly M.M., Beer M., Harder T.C., Hafez H.M. Multiple dose vaccination with heterologous H5N2 vaccine: immune response and protection against variant clade 2.2.1 highly pathogenic avian influenza H5N1 in broiler breeder chickens. Vaccine. 2011; 29:6219–6225.
Swayne D.E., Kapczynski D. Strategies and challenges for eliciting immunity against avian influenza virus in birds. Immunolgical Reviews. 2008; 225:314–331.
Rauw F., Palya V., Gardin Y., Tatar‐Kis T., Dorsey K.M., Lambrecht B. et al. Efficacy of rHVT‐AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains. Avian Diseases 2012; 56:913–922.
Abdelwhab E.M., Grund C., Aly M.M., Beer M., Harder T.C., Hafez H.M. Influence of maternal immunity on vaccine efficacy and susceptibility of one day old chicks against Egyptian highly pathogenic avian influenza H5N1. Veterinary Microbiology. 2012; 155:13–20.
De Vriese J., Steensels M., Palya V., Gardin Y., Dorsey K.M., Lambrecht B. et al. Passive protection afforded by maternally‐derived antibodies in chickens and the antibodies’ interference with the protection elicited by avian influenza‐inactivated vaccines in progeny. Avian Diseases. 2010; 54:246–252.
Schat K.A., Nair V. Marek's disease. In: Swayne D.E., Glisson J.R., McDougald L.R., Nolan L.K., Suarez D.L., Nair V, editors. Diseases of Poultry. 13th ed. Hoboken, NJ: Wiley‐Blackwell; 2013. pp. 515–552.
Okazaki W., Purchase H.G., Burmester B.R. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Diseases. 1970; 14:413–429.
Heller E. D., Schat K.A. Enhancement of natural killer cell activity by Marek's disease vaccines. Avian Pathology. 1987; 16:51–60.
Kitamoto N., Ikuta K., Kato S., Yamaguchi S. Cell‐mediated cytotoxicity of lymphocytes from chickens inoculated with herpesvirus of turkey against a Marek's disease lymphoma cell line (MSB‐1). Biken Journal. 1979; 22:11–20.
Rauw F., Gardin Y., Palya V., Anbari S., Lemaire S., Boschmans M. et al. Improved vaccination against Newcastle disease by an in ovo recombinant HVT‐ND combined with an adjuvanted live vaccine at day‐old. Vaccine. 2010; 28:823–833.
Witter R.L., Offenbecker L. Duration of vaccinal immunity against Marek's disease. Avian Diseases. 1978; 22:396–407.
Palya V., Tatár‐Kis T., Mató T., Felföldi B., Kovács E., Gardin Y. Onset and long‐term duration of immunity provided by asingle vaccination with a turkey herpesvirus vector ND vaccine in commercial layers. Veterinary Immunology and Immunopathology. 2014; 158:105–115.
Afonso C.L., Tulman E.R., Lu Z., Zsak L., Rock D.L., Kutish G.F. The genome of turkey herpesvirus. Journal of Virology. 2001; 75:971–978.
Darteil R., Bublot M., Laplace E., Bouquet J.F., Audonnet J.C., Rivière M. Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens. Virology. 1995; 211:481–490.
Morgan R.W., Gelb J. Jr, Schreurs C.S., Lütticken D., Rosenberger J.K., Sondermeijer P.J.. Protection of chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of Turkeys vaccine expressing the Newcastle disease virus fusion protein. Avian Diseases. 1992; 36:858–870.
Ross L.J., Binns M.M., Tyers P., Pastorek J., Zelnik V., Scott S. Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus. Journal of General Virology. 1993; 74:371–377.
Tsukamoto K., Saito S., Saeki S., Sato T., Tanimura N., Isobe T. et al. Complete, long‐lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. Journal of Virology. 2002; 76:5637–5645.
Esaki M., Godoy A., Rosenberger J.K., Rosenberger S.C., Gardin Y., Yasuda A. et al. Protection and antibody response caused by turkey herpesvirus vector Newcastle disease vaccine. Avian Diseases. 2013; 57:750–755.
Esaki M., Noland L., Eddins T., Godoy A., Saeki S., Saitoh S. et al. Safety and efficacy of a turkey herpesvirus vector laryngotracheitis vaccine for chickens. Avian Diseases. 2013; 57:192–198.
Swayne D.E., Suarez D.L., Sims L.D. Influenza. In: Swayne D.E., Glisson J.R., McDougald L.R., Nolan L.K., Suarez D.L. and Nair V, editors. Diseases of Poultry. 13th ed. Hoboken, NJ: Wiley‐Blackwell; 2013. pp. 181–218.
Sonoda K., Sakaguchi M., Okamura H., Yokogawa K., Tokunaga E., Tokiyoshi S. et al. Development of an effective polyvalent vaccine against both Marek's and Newcastle diseases based on recombinant Marek's disease virus type 1 in commercial chickens with maternal antibodies. Journal of Virology. 2000; 74:3217–3226.
Ma C., Zhang Z., Zhao P., Duan L., Zhang Y., Zhang F. et al. Comparative transcriptional activity of five promoters in BAC‐cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus. Journal of Virological Methods. 2014; 206:119–127.
Pedersen J.C. Hemagglutination‐Inhibition Test for Avian Influenza Virus Subtype Identification and the Detection and Quantitation of Serum Antibodies to the Avian Influenza Virus. Methods in Molecular Biology. 2008; 436:53–66.
Gardin Y., Palya V., Dorsey K.M., El‐Attrache J., Bonfante F., de Wit S. et al. Experimental and field results regarding immunity induced by a rHVT‐H5 vector vaccine against H5N1 and other H5 type highly pathogenic avian influenza viruses. Avian Diseases. Forthcoming. DOI: 10.1637/11144‐050815‐ResNote.1
Kapczynski DR, Esaki M, Dorsey KM, Jiang H, Jackwood M, Moraes M, Gardin Y. Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus. Vaccine. 2015; 33:1197–1205.
Kilany W.H., Dauphin G., Selim A., Tripodi A., Samy M., Sobhy H. et al. Protection conferred by recombinant turkey herpesvirus avian influenza (rHVT‐H5) vaccine in the rearing period in two commercial layer chicken breeds in Egypt. Avian Pathology. 2014; 43:514–523.
Kilany W.H., Hassan M.K., Safwat M., Mohammed S., Selim A., VonDobschuetz S. et al. Comparison of the effectiveness of rHVT‐H5, inactivated H5 and rHVT‐H5 with inactivated H5 prime/boost vaccination regimes in commercial broiler chickens carrying MDAs against HPAI H5N1 clade 2.2.1 virus. Avian Pathology. 2015; 44:333–341.
Peyre M., Choisy M., Sobhy H., Kilany W.H., Gély M., Tripodi A. et al. Added value of avian influenza (H5) day old chick vaccination for disease control in Egypt. Avian Diseases. Forthcoming. DOI: 10.1637/11131‐050715‐ResNote.1