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Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.

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Vaccines are an essential tool for the control of viral infections in domestic animals. We generated recombinant vector herpesvirus of turkeys (vHVT) vaccines expressing computationally optimized broadly reactive antigen (COBRA) H5 of avian influenza virus (AIV) alone (vHVT-AI) or in combination with virus protein 2 (VP2) of infectious bursal disease virus (IBDV) (vHVT-IBD-AI) or fusion (F) protein of Newcastle disease virus (NDV) (vHVT-ND-AI). In vaccinated chickens, all three vHVT vaccines provided 90-100% clinical protection against three divergent clades of high pathogenicity avian influenza viruses (HPAIVs), and significantly decreased number of birds and oral viral shedding titers at 2 days post-challenge compared to shams. Four weeks after vaccination, most vaccinated birds had H5 hemagglutination inhibition antibody titers, which significantly increased post-challenge. The vHVT-IBD-AI and vHVT-ND-AI vaccines provided 100% clinical protection against IBDVs and NDV, respectively. Our findings demonstrate that multivalent HVT vector vaccines were efficacious for simultaneous control of HPAIV and other viral infections.

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H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.

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Turkey herpesvirus (HVT) is widely used as an effective recombinant vaccine vector for expressing protective antigens of multiple avian pathogens from different loci of the HVT genome. These include the HVT029/031 (UL22-23) locus for the insertion of IBDV VP2 and the recently identified HVT005/006 locus as a novel site for expressing heterologous proteins. In order to compare the efficacy of recombinant vaccines with the HA gene at different sites, the growth curves and the HA expression levels of HVT-005/006-hCMV-HA, HVT-005/006-MLV-HA, and HVT-029/031-MLV-HA were first examined in vitro. While the growth kinetics of three recombinant viruses were not significantly different from those of parent HVT, higher expression of the HA gene was achieved from the HVT005/006 site than that from the HVT029/031 site. The efficacy of the three recombinant viruses against avian influenza H9N2 virus was also evaluated using one-day-old SPF chickens. Chickens immunized with HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA displayed reduced virus shedding compared to HVT-029/031-MLV-HA vaccinated chickens. Moreover, the overall hemagglutination inhibition (HI) antibody titers of HVT-005/006-HA-vaccinated chickens were higher than that of HVT-029/031-HA-vaccinated chickens. However, HVT-005/006-MLV-HA and HVT-005/006-hCMV-HA did not result in a significant difference in the level of HA expression in vitro and provided the same protective efficacy (100%) at 5 days after challenge. In the current study, the results suggested that recombinant HVT005/006 vaccines caused better expression of HA than recombinant HVT029/031 vaccine, and that HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA could be a candidate vaccine for the protection of chickens against H9N2 influenza.

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Turkey herpesvirus (HVT) has been widely used as a successful live virus vaccine against Marek's disease (MD) in chickens for more than five decades. Increasingly, HVT is also used as a highly effective recombinant vaccine vector against multiple avian pathogens. Conventional recombination, or recombineering, techniques that involve the cloning of viral genomes and, more recently, gene editing methods have been used for the generation of recombinant HVT-based vaccines. In this study, we used NHEJ-dependent CRISPR/Cas9-based approaches to insert the mCherry cassette for the screening of the HVT genome and identifying new potential sites for the insertion of foreign genes. A novel intergenic site HVT-005/006 in the unique long (UL) region of the HVT genome was identified, and mCherry was found to be stably expressed when inserted at this site. To confirm whether this site was suitable for the insertion of other exogenous genes, haemagglutinin (HA) of the H9N2 virus was inserted into this site, and a recombinant HVT-005/006-HA was rescued. The recombinant HVT-HA can grow well and express HA protein stably, which demonstrated that HVT-005/006 is a promising site for the insertion of foreign genes.

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Herpesvirus of turkeys (HVT), a commonly used live vaccine against Marek's disease, has proven to be a highly effective viral vector for the generation of recombinant vaccines that deliver protective antigens of other avian pathogens. In this study, a vaccine designated rHVT-NDV-opti F was constructed by inserting a codon-optimized genotype Ⅶ Newcastle disease virus (NDV) fusion (F) gene into the US2 gene of HVT Fc126 vaccine strain using CRISPR/Cas9 gene-editing technology coupled with two single-guide RNAs (sgRNA). The F protein expression of rHVT-NDV-opti F was detectable by western blotting and an indirect immunofluorescence assay. Compared with wildtype HVT, rHVT-NDV-opti F has similar plaque morphology but lower in vitro replication capacity. The F protein of rHVT-NDV-opti F is genetically stable and predominantly expressed in the cell plasma. Immunization of one-day-old specific pathogen-free chickens with 4000 plaque-forming units of rHVT-NDV-opti F induced NDV-specific antibodies and provided 70% protection against a homologous NDV challenge, effectively reducing virus shedding, clinical signs, tissue viral load, and mortality. These results suggest that rHVT-NDV-opti F could be a potential vaccine candidate against Newcastle disease in chickens and that HDR-CRISPR/Cas9 combined with dual sgRNA can rapidly and efficiently construct HVT-vectored vaccine candidates.

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Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens.

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The advent of turkey herpesvirus (HVT) vector vaccine technology (vHVT) has made a huge improvement in the prevention and control of several poultry diseases. The objective of this study was to compare, under experimental conditions, the protection conferred by different vaccination programs based on an HVT double-insert (infectious bursal disease {IBD] and Newcastle disease [ND]) vector vaccine (vHVT-IBD-ND) and an HVT single-insert (vHVT-ND) vector vaccine followed by a vaccination with a live ND vaccine at Day 1 only or at Days 1 and 14. Commercial broilers were vaccinated by the recombinant ND virus vaccines subcutaneously at 1 day old, in the hatchery, and challenged at 30 days of age using the Moroccan ND virus velogenic viscerotropic JEL strain. The results showed that the tested vaccine induced 95% to 100% clinical protection against mortality and clinical signs. The humoral immune response to vaccination was detected from 3 wk of age using enzyme-linked immunosorbent assay and hemagglutination inhibition tests. ND challenge virus shedding was significantly reduced in the vaccinated birds as compared to controls. Significant reduction of the cloacal shedding suggests that the vHVT-IBD-ND vaccine stimulates actively the immunity against the tested ND challenge virus. No significant differences were found between the vaccination programs based on vHVT-IBD-ND or on vHVT-ND.

Evaluación de la eficacia de las vacunas recombinantes contra el virus de la enfermedad de Newcastle (vHVT-IBD-ND de doble inserto y vHVT-ND de inserto único) seguidas de una vacunación con una vacuna viva para la enfermedad de Newcastle contra un desafío de la enfermedad de Newcastle velogénico marroquí en pollos de engorde comerciales. El advenimiento de la tecnología de vacunas recombinantes (vHVT) del virus herpes del pavo (HVT) ha provocado una mejora en la prevención y el control de varias enfermedades avícolas. El objetivo de este estudio fue comparar, en condiciones experimentales, la protección conferida por diferentes programas vacunales basados en una vacuna recombinante HVT con doble inserto (bursitis infecciosa [EII] y enfermedad de Newcastle [ND]) (vHVT-IBD-ND) y una vacuna recombinante HVT con inserto única (vHVT-ND) seguida de una vacunación con una vacuna para Newcastle viva aplicada en el día 1 o en los días 1 y 14. Pollos de engorde comerciales se vacunaron con las vacunas recombinantes del virus de la enfermedad de Newcastle por vía subcutánea al día de edad, en la incubadora y se expusieron a los 30 días de edad utilizando la cepa JEL viscerotrópica velogénica del virus de la enfermedad de Newcastle de Marruecos. Los resultados mostraron que la vacuna evaluada indujo una protección clínica del 95% al 100% contra la mortalidad y los signos clínicos. La respuesta inmune humoral a la vacunación se detectó a partir de las 3 semanas de edad mediante ensayo inmunoabsorbente ligado a enzimas y pruebas de inhibición de la hemaglutinación. La excreción del virus de Newcastle de desafío se redujo significativamente en las aves vacunadas en comparación con los controles. La reducción significativa de la eliminación cloacal sugiere que la vacuna vHVT-IBD-ND estimula activamente la inmunidad contra el virus de Newcastle de desafío analizado. No se encontraron diferencias significativas entre los programas de vacunación basados en vHVT-IBD-ND o en vHVT-ND.

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The control of poultry diseases has relied heavily on the use of many live and inactivated vaccines. However, over the last 30 yr, recombinant DNA technology has been used to generate many novel poultry vaccines. Fowlpox virus and turkey herpesvirus are the two main vectors currently used to construct recombinant vaccines for poultry. With the use of these two vectors, more than 15 recombinant viral vector vaccines against Newcastle disease, infectious laryngotracheitis, infectious bursal disease, avian influenza, and Mycoplasma gallisepticum have been developed and are commercially available. This review focuses on current knowledge about the safety and efficacy of recombinant viral vectored vaccines and the mechanisms by which they facilitate the control of multiple diseases. Additionally, the development of new recombinant vaccines with novel vectors will be briefly discussed.

Estudio Recapitulativo- Revisión acerca de las vacunas con vectores recombinantes para la avicultura. El control de las enfermedades en la avicultura se ha basado en gran medida en el uso de varias vacunas vivas e inactivadas. Sin embargo, durante los últimos 30 años, la tecnología de ADN recombinante se ha utilizado para generar nuevas vacunas avícolas. El virus de la viruela aviar y el virus del herpes del pavo son los dos vectores principales que se utilizan actualmente para construir vacunas recombinantes para la avicultura. Con el uso de estos dos vectores, se han desarrollado y están disponibles comercialmente más de 15 vacunas con vectores virales recombinantes contra la enfermedad de Newcastle, la laringotraqueítis infecciosa, enfermedad infecciosa de la bolsa, influenza aviar y Mycoplasma gallisepticum. Esta revisión se enfoca en el conocimiento actual sobre la seguridad y eficacia de las vacunas con vectores virales recombinantes y los mecanismos por los cuales facilitan el control de múltiples enfermedades. Además, se discutirá brevemente el desarrollo de nuevas vacunas recombinantes con nuevos vectores.

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The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8ß+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8ß+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

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Marek's disease virus (MDV) is an important poultry pathogen that is controlled through widespread vaccination with avirulent and attenuated strains. However, continued evolution of field viruses to higher virulence has required ongoing improvement of available vaccine strains, and these vaccine strains offer an attractive platform for designing recombinant vector vaccines with cross-protection against MDV and additional pathogens. Recent reports of failures in vaccine licensing trials of positive controls to reach appropriately high levels of Marek's disease incidence prompted us to evaluate possible combinations of outbred specific-pathogen-free layer lines and alternative virulent challenge strains that could provide more consistent models for serotype 3 vectored vaccine development. Choice of layer line and virulent MDV challenge strain each contributed to the ability of a challenge model to reach 80% virulence in unvaccinated positive control groups in the majority of trials, without overwhelming serotype 3 vectored vaccine protection in vaccinated groups. Conversely, reducing challenge virus dose by a factor of four, or vaccine dose by half, had no consistent effect across these models. Although MDV strain 617A had the most potential as an alternative to strains that are currently approved for licensing trials, no combination of layer line and challenge virus consistently met the goals for a successful challenge model in all study replicates, indicating that high variability is an inherent difficulty in MDV challenge studies, at least when outbred birds are used.

Artículo regular­Comparación de las cepas de desafío del virus de la enfermedad de Marek y los tipos de aves para la obtención de licencias de vacunas. El virus de la enfermedad de Marek (MDV) es un patógeno importante en la avicultura que se controla mediante la vacunación generalizada con cepas avirulentas y atenuadas. Sin embargo, la evolución continua de los virus de campo hacia una mayor virulencia ha requerido una mejora continua de las cepas vacunales disponibles y estas cepas vacunales ofrecen una plataforma atractiva para diseñar vacunas con vectores recombinantes que induzcan protección cruzada contra el virus de la enfermedad de Marek y patógenos adicionales. Los reportes recientes de fallas en los controles positivos para alcanzar niveles apropiadamente altos de incidencia de la enfermedad de Marek en los ensayos para obtener la licencia de vacunas llevaron a evaluar posibles combinaciones de líneas de postura híbridas libres de patógenos específicos y cepas de desafío virulentas alternativas que podrían proporcionar modelos más consistentes para el desarrollo de vacunas con vectores de serotipo 3. Tanto la elección de la línea de postura como de la cepa de desafío virulenta de Marek contribuyeron a obtener un modelo de desafío con capacidad para alcanzar el 80% de virulencia en grupos controles positivo no vacunados en la mayoría de los ensayos, sin una protección abrumadora de la vacuna con vector de serotipo 3 en los grupos vacunados. Por el contrario, la reducción de la dosis del virus de desafío en un factor de cuatro, o la dosis de vacuna a la mitad, no tuvieron un efecto constante en estos modelos. Aunque la cepa 617A de Marek mostró el mayor potencial como alternativa a las cepas que actualmente están aprobadas para ensayos de licenciar vacunas, ninguna combinación de línea de postura y virus de desafío cumplió consistentemente los objetivos de un modelo de desafío exitoso en todas las réplicas del estudio, lo que indica que la alta variabilidad es una dificultad inherente en los estudios de desafío para la enfermedad de Marek, al menos cuando se utilizan aves híbridas.

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The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.

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Understanding the pathogenesis of herpesvirus of turkeys (HVT) in its natural host is necessary before recombinant HVT (rHVT) can be used efficiently in turkey flocks. The objectives of this study were to evaluate when commercial turkey flocks get infected with wild type HVT, to study replication of HVT (conventional and recombinant rHVT-Newcastle disease, rHVT-ND) and other Marek's disease (MD) vaccines (SB-1 and CVI988) in turkey embryonic tissues, and to evaluate the expression of TLR-3 and IFN-γ in the lung and spleen of one-day-old turkeys after in ovo vaccination with MD vaccines. Our results demonstrated that commercial turkeys got exposed to wild type HVT within the first days of life; therefore, there is a potential of interaction between wild type HVT and rHVT when administered at day of age. On the other hand, all evaluated vaccines (especially HVT and rHVT-ND) replicated very well in turkey embryonic tissues. In ovo vaccination with HVT and CVI988 increased transcription of TLR-3 in the spleen of one-day-old turkeys. However, no effect on the transcription of TLR-3 or IFN-γ in the lungs and IFN-γ in the spleen in newly hatched turkeys was detected in the present study. Because of the limitations of evaluated genes, timepoints, and studied tissues, future studies are warranted to better understand the effect of MD vaccines on the turkey embryo immune responses.RESEARCH HIGHLIGHTS Commercial turkey flocks get infected with wild type HVT within the first days of life.HVT and rHVT replicates readily in turkey embryonic tissues.SB-1 and CVI988 also replicate in turkey embryonic tissues, but at lower rates than HVT and rHVT.HVT and CVI988 increase transcription of TLR-3 in the spleen.

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The use of novel vector vaccines (viral, bacterial and apicomplexan) can have a significant impact on the control of poultry disease. They offer a cost effective, convenient and effective means of mass vaccine delivery combined with the ability to switch on both antibody and cell-mediated immunity. In addition, recent viral vector constructs have enabled farmers to vaccinate against up to three important pathogens with a single in ovo administration. As the technology develops, it is likely that this means of vaccine administration will be utilized further and it will play a key role in the control of both existing and new emerging diseases of poultry in the future.

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Current methods to combat highly pathogenic avian influenza (HPAI) outbreaks in poultry rely on stamping out and preventive culling, which can lead to high economic losses and invoke ethical resistance. Emergency vaccination could be an alternative as vaccination is one of the most efficient and cost-effective measures to protect poultry from HPAI infection, preventing spreading to other poultry and greatly reducing the potential transmission to humans. Current conventional inactivated AI vaccines may be useful for combating AI outbreaks, but do not fulfil all targets of an ideal AI vaccine, including mass applicability and rapid onset of immunity. We aimed to further investigate the potential of Herpesvirus of Turkeys (HVT) as a vector containing a recombinant H5 hemagglutinin of HPAI H5N1. This HVT-H5 vector was analysed in vitro, tested for onset of immunity against AI challenge, breadth of protection, reduction of virus shedding, and induction of both antibody and cellular responses in SPF layers or broiler chicks containing maternal derived antibodies (MDA+). In SPF layers HVT-H5 provided full protection to lethal challenges with 4 antigenically diverse HPAI H5N1 strains from 2 weeks post vaccination (w.p.v.), while in MDA+ birds full protection was provided from 3 w.p.v. to homologous challenge. Also shedding of challenge virus was reduced in both SPF and MDA+ birds. HVT-H5 induced a protective HI titre (≥4) to 11 HPAI H5N1 strains at 3 w.p.v. in 3-week-old SPF layers and to HPAI H5N8 A/ch/Neth/14015531/2014. Besides inducing a protective antibody response HVT-H5 also induced an influenza-specific T cell response. This data demonstrates that HVT-H5 vaccine appears to fulfil many of the criteria for an ideal AI vaccine including early onset of immunity, a broad protection, reduced virus shedding, protection in presence of AI-MDA and could be a useful tool in the combat of AI outbreaks worldwide.

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A double construct vaccine of turkey herpesvirus (HVT) was prepared that contains the fusion (F) gene from Newcastle disease virus (NDV) and the viral protein 2 (VP2) gene from infectious bursal disease virus (IBDV). Safety of the vaccine (HVT-ND-IBD) was confirmed and efficacy was evaluated after subcutaneous (SC) vaccination at 1 day of age or the in ovo route of vaccination. Challenges were performed with velogenic NDV strains (Texas GB and Herts Weybridge 33/56), with different strains of IBDV (classical strain STC; very virulent strain CS89 and variant E strain) and with Marek's disease virus (MDV) strain RB1B. Vaccination with HVT-ND-IBD induced a high level of protection against these challenges. Vaccination with HVT is often combined with Rispens CVI988 vaccine and live ND vaccines for higher and earlier, MD and ND protection, respectively. HVT-ND-IBD vaccination in combination with these vaccines showed MD protection as early as 4 days post vaccination and ND protection as early as 2 weeks post vaccination. The long protection as seen with HVT vaccination was confirmed by demonstrating protection against NDV up to 60 weeks. Finally, to evaluate the performance of the vaccine in commercial birds with maternally-derived antibodies, two field trials were performed, using in ovo vaccination in broilers and SC vaccination in combination with Rispens CVI988 vaccine in layer-type birds. The efficacy was confirmed for all components by challenges. These results demonstrate that HVT-ND-IBD is a safe and highly efficacious vaccine for simultaneous control of ND, IBD and MD. RESEARCH HIGHLIGHTS A double construct HVT vaccine with the NDV F and the IBDV VP2 genes was prepared. The vaccine protects against three important diseases: MDV, NDV and IBDV. In ovo and sub-cutaneous vaccination was evaluated in the field in commercial chickens.

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Venous thrombosis within the hepatic vasculature is associated with a distinct array of risk factors, characteristics, and potential complication. As such, it entails unique management considerations and strategies relative to the more common categories of venous thromboembolic disease. Although broadly divided into thrombosis of the afferent vasculature (the portal venous system) and efferent vasculature (the hepatic venous system), presentations and management strategies within these groupings are heterogeneous. Management decisions are influenced by a variety of factors including the chronicity, extent, and etiology of thrombosis. In this review we examine both portal vein thrombosis and hepatic vein thrombosis (and the associated Budd-Chiari Syndrome). We consider those factors which most impact presentation and most influence treatment. In so doing, we see how the particulars of specific cases introduce nuance into clinical decisions. At the same time we attempt to organize our understanding of such cases to help facilitate a more systematic approach. Critically, we must recognize that although increasing evidence is emerging to help guide our management strategies, the available data remain limited and largely retrospective. Indeed, current paradigms are based largely on observational experiences and expert consensus. As new and more rigorous studies emerge, treatment strategies are likely to be continually refined, and paradigm shifts are sure to occur.

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The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells.IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek's disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

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Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek's disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.

RESUMEN

Avian influenza viruses (AIVs) are highly contagious and have caused huge economical loss to the poultry industry. AIV vaccines remain one of the most effective methods of controlling this disease. Turkey herpesvirus (HVT) is a commonly used live attenuated vaccine against Marek's disease; it has also been used as a viral vector for recombinant AIV vaccine development. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a gene editing tool which, in vaccinology, has facilitated the development of recombinant DNA viral-vectored vaccines. Here, we utilize homology-directed repair (HDR) for the generation of a HVT-H7N9 HA bivalent vaccine; a H7N9 HA expression cassette was inserted into the intergenic region between UL45 and UL46 of HVT. To optimize the selection efficiency of our bivalent vaccine, we combined CRISPR/Cas9 with erythrocyte binding to rapidly generate recombinant HVT-H7HA candidate vaccines.