A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI ("pigeon variant" of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

Recombinant turkey herpesvirus expressing H9 hemagglutinin providing protection against H9N2 avian influenza.

H9N2 avian influenza viruses (AIVs) were prevailing in chickens, causing great economic losses and public health threats. In this study, turkey herpesviruses (HVT) was cloned as an infectious bacterial artificial chromosomes (BAC). Recombinant HVT (rHVT-H9) containing hemagglutinin (HA) gene from H9N2 virus were constructed via galactokinase (galK) selection and clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. The recombinant rHVT-H9 showed no difference with parent HVT in plague morphology and virus replication kinetics. H9 protein expression of rHVT-H9 could be detected by western blot and indirect immunofluorescence assay (IFA) in vitro and in vivo. Immunization with rHVT-H9 could induce robust humoral and cellular immunity in chickens. In the challenge study, no chicken shed H9N2 virus from oropharynx and cloaca, and no H9N2 virus was found in viscera in vaccination groups. The result suggests that rHVT-H9 provides effective protection against H9N2 AIV in chickens.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Recombinant Turkey Herpesvirus-AI Vaccine Virus Replication in Different Species of Waterfowl.

Waterfowl play a key role in the epidemiology of the H5N1 subtype of highly pathogenic avian influenza (HPAI) virus; therefore, efficient immunization of domesticated ducks and geese to maximize the impact of other control measures is of great importance. A recombinant (r)HVT-AI, expressing the HA gene of a clade 2.2 H5N1 HPAI strain had been developed and proved to be efficient against different clades of H5N1 HPAI virus in chickens after a single vaccination at 1 day old and could provide long-term immunity. We investigated whether rHVT-AI applied at 1 day old is able to replicate in different species and crossbreeds of ducks and in geese with the aim of collecting data on the possible application of rHVT-AI vaccine in different species of waterfowl for the control of H5N1 HPAI. We tested the possible differences among different waterfowl species, i.e., between geese (Anser anser, domesticated greylag goose), Muscovy ducks (Cairina moschata forma domestica), Pekin ducks (Anas platyrhynchos forma domestica), and mule ducks (Muscovy duck × Pekin duck), in their susceptibility to support the replication of rHVT-AI. Vaccine virus replication was followed by real-time PCR in spleen, bursa, and feather tip samples. Humoral immune response to vaccination was tested using the hemagglutination inhibition (HI) test and H5-specific commercial ELISA. Significant differences among the different waterfowl species regarding the rate of rHVT-AI replication was detected that were not reflected by the same difference in the immune response to vaccination. Replication of the rHVT-AI vaccine was very limited in Pekin ducks, somewhat better in mule ducks, and the vaccine virus was replicating significantly better in Muscovy ducks and geese, reaching 100% detectability at certain time points after administration at 1 day old. Results indicated that the vaccine virus could establish different levels of persistent infection in these species of waterfowl. No humoral immune response could be detected either by HI test or ELISA during the tested postvaccination period (5 wk).

Efficacy of a Recombinant Turkey Herpesvirus H5 Vaccine Against Challenge With H5N1 Clades 1.1.2 and 2.3.2.1 Highly Pathogenic Avian Influenza Viruses in Domestic Ducks (Anas platyrhynchos domesticus).

Domestic ducks are the second most abundant poultry species in many Asian countries and have played a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI).In this study, the protective efficacy of a live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAI strain (A/Swan/Hungary/4999/ 2006) (rHVT-H5/2.2), given at 3 days of age, was examined in Pekin ducks (Anas platyrhynchos domesticus). The vaccine was given alone or in combination with an inactivated H5N1 clade 2.3.2.1 reverse genetic (rgGD/2.3.2.1) vaccine given at 16 days of age, either as a single vaccination or in a prime-boost regime. At 30 days of age, ducks were challenged with one of two H5N1 HPAI viruses: A/duck/Vietnam/NCVD-2721/2013 (clade 1.1.2) or A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1.C). These viruses produced 100% mortality in less than 5 days in nonvaccinated control ducks. Ducks vaccinated with the rgGD/2.3.2.1 vaccine, with or without the rHVT-H5/2.2 vaccine, were 90%-100% protected against mortality after challenge with either of the two H5N1 HPAI viruses. The rHVT-H5/2.2 vaccine alone, however, conferred only 30% protection against mortality after challenge with either H5N1 HPAI virus; the surviving ducks from these groups shed higher amount of virus and for longer than the single-vaccinated rgGD/2.3.2.1 group. Despite low protection, ducks vaccinated with the rHVT-H5/2.2 vaccine and challenged with the clade 1.1.2 Vietnam virus had a longer mean death time than nonvaccinated controls (P = 0.02). A booster effect was found on reduction of virus shedding when using both vaccines, with lower oropharyngeal viral titers at 4 days after challenge with either HPAI virus (P < 0.05). Neither rHVT-H5/2.2 nor standard HVT vaccine could be detected in samples collected from multiple tissues at different time points, indicting minimal levels of viral replication. In conclusion, although a minor effect on survival was observed, this study demonstrates the suboptimal protection with the rHVT-H5/2.2 vaccine given alone in Pekin ducks against H5N1 HPAI viruses and only a minor additive effect on virus shedding reduction when used with an inactivated vaccine in a prime-boost regime.

The interplay between MDV and HVT affects viral miRNa expression.

It is well established that herpesviruses encode numerous microRNAs (miRNAs) and that these virally encoded small RNAs play multiple roles in infection. The present study was undertaken to determine how co-infection of a pathogenic MDV serotype one (MDV1) strain (MD5) and a vaccine strain (herpesvirus of turkeys [HVT]) alters viral miRNA expression in vivo. We first used small RNA deep sequencing to identify MDV1-encoded miRNAs that are expressed in tumorigenic spleens of MDV1-infected birds. The expression patterns of these miRNAs were then further assessed at an early time point (7 days postinfection [dpi]) and a late time point (42 dpi) in birds with and without HVT vaccination using real-time PCR (RT-PCR). Additionally, the effect of MDV1 co-infection on HVT-encoded miRNAs was determined using RT-PCR. A diverse population of miRNAs was expressed in MDV-induced tumorigenic spleens at 42 dpi, with 18 of the 26 known mature miRNAs represented. Of these, both mdv1-miR-M4-5p and mdv1-miR-M2-3p were the most highly expressed miRNAs. RT-PCR analysis further revealed that nine MDV miRNAs were differentially expressed between 7 dpi and 42 dpi infected spleens. At 7 dpi, three miRNAs were differentially expressed between the spleens of birds co-infected with HVT and MD5 compared with birds singly infected with MD5, whereas at 42 dpi, nine miRNAs were differentially expressed. At 7 dpi, the expression of seven HVT-encoded miRNAs was affected in the spleens of co-infected birds compared with birds only receiving the HVT vaccine. At 42 dpi, six HVT-encoded miRNAs were differentially expressed between the two groups. Target prediction analysis suggests that these differentially expressed viral miRNAs are involved in regulating several cellular processes, including cell proliferation and the adaptive immune response.

The Meleagrid herpesvirus 1 genome is partially resistant to transposition.

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.

Inhibition of toll-like receptor 2-mediated NF-kappaB activation in Vero cells with herpesvirus of turkeys.

In a previous study, vaccination with a live bivalent vaccine consisting of herpesvirus of turkeys (HVT) and SB-1 was found to be associated with distinct cytokine expression patterns and the modulation of cytokine responses in the spleen. This vaccine could play a role in mediating protection against infection with the RB1B strain of Marek's disease virus. In the present study, vectors for chicken Toll-like receptor 1 (chTLR1) and 2 (chTLR2) expression were constructed and transfected into Vero cells. Nuclear factor kappa light-chain enhancer of activated B cell (NF-kappaB) activation was detected after HVT infection. Compared with normal Vero cells, NF-kappaB activation was significantly inhibited by HVT in Vero cells transfected with chTLR1-1, chTLR1-2, or both. The results demonstrate the significant characteristics of HVT in activating TLR2 signaling. chTLR1 plays a key role in TLR2 subfamily-mediated NF-kappaB inhibition after HVT infection.

Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotypes 1, 2, and 3.

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.

Targeting Marek's disease virus by RNA interference delivered from a herpesvirus vaccine.

Live attenuated herpesvirus vaccines such as herpesvirus of turkey (HVT) have been used since 1970 for the control of Marek's disease (MD), a highly infectious lymphoproliferative disease of poultry. Despite the success of these vaccines in reducing losses from the disease, Marek's disease virus (MDV) strains have shown a continuing increase in virulence, presumably due to the inability of the current vaccines in preventing MDV replication. The highly specific and effective nature of RNA interference (RNAi) makes this technology particularly attractive for new antiviral strategies. In order to exploit the power of RNAi-mediated suppression of MDV replication in vivo delivered through existing vaccines, we engineered recombinant HVT expressing short hairpin RNA (shRNA) against MDV genes gB and UL29. The levels of protection induced by the RNAi-expressing HVT against virulent virus challenge were similar to the parent pHVT3 virus. However, chickens vaccinated with recombinant HVT expressing shRNA showed moderate reduction of challenge virus replication in blood and feather samples. Delivery of RNAi-based gene silencing through live attenuated vaccines for reducing replication of pathogenic viruses is a novel approach for the control of infectious diseases.

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES.

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.