Grass carp peroxiredoxin 5 and 6-mediated autophagy inhibit grass carp reovirus replication and mitigate oxidative stress.

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H2O2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H2O2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.

Gga-miR-30c-5p Suppresses Avian Reovirus (ARV) Replication by Inhibition of ARV-Induced Autophagy via Targeting ATG5.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.

Recombinant Lactococcus lactis Expressing Grass Carp Reovirus VP6 Induces Mucosal Immunity Against Grass Carp Reovirus Infection.

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery.

Evaluation of Pathogenicity and Antigenicity of Avian Reoviruses and Disease Control Through Vaccination.

Avian reoviruses are ubiquitous in poultry production worldwide and can be transmitted vertically or horizontally among chickens. The pathogenicity of reoviruses can range from very pathogenic viruses that affect multiple tissues and organs to apathogenic. Avian reoviruses have been associated with many disease presentations, and two of the most economically significant diseases are viral arthritis/tenosynovitis and viral enteritis. Viral arthritis/tenosynovitis has been recognized since the 1950s and essentially disappeared after development of attenuated live and inactivated vaccines in the 1980s but re-emerged in 2011 due to the emergence of antigenic variants. Viral enteritis was first recognized in the 1970s and became the predominant reovirus-associated disease between 2006 and 2011 due to the emergence of pathogenic enterotropic reoviruses. Pathogenicity of reovirus isolates can be evaluated in several ways, including inoculation of day-old broiler chicks with low maternal reovirus antibody titers via the foot pad route or the oral and intratracheal route. Pathogenic reoviruses induce foot pad inflammation within 3 days of inoculation, and more pathogenic reoviruses are able to disseminate to and damage visceral organs. Only reovirus infections in young chickens result in disease due to age-related resistance to disease development. Reoviruses exist as many serotypes and subtypes with various degrees of interrelatedness. The earliest reovirus strains in the United States were antigenically related to each other and are referred to as S1133-like viruses, but in the 2000s, reoviruses emerged that were antigenically different from the S1133-like viruses. Virus neutralization assay using polyclonal antisera has been used to classify the emerging variant reoviruses into serogroups. The first reovirus vaccines were developed in the 1970s, and by the 1980s breeder vaccination programs were established that protected breeders, prevented vertical transmission of reovirus, and provided maternal immunity to the progeny during the crucial first 3 wk of life. With the emergence of antigenic variant reoviruses in the 2000s, vaccination programs using S1133-like vaccines became ineffective. The poultry industry has relied on vaccination with autogenous inactivated reovirus vaccines to alleviate losses due to viral arthritis/tenosynovitis and viral enteritis. Virus isolates used for autogenous vaccines must be updated regularly and are selected based on pathotype, serotype, or Sigma C (σC) genotype. Live attenuated S1133 vaccines are still used in breeder chickens for the priming effect, followed by one or more injections of the inactivated licensed and/or autogenous vaccines. The route of vaccination and the number of doses received by breeder chickens are very important for a sufficient antibody response. Intramuscular vaccination with inactivated vaccines elicits the highest antibody response, while subcutaneous vaccination with inactivated vaccines elicits a low antibody response. More recently, research has focused on development of alternative vaccines and vaccination strategies. An inactivated variant reovirus vaccine was developed that elicits protection against multiple variant serotypes, and experimental recombinant and subunit vaccines have been described and show potential. More research needs to be done to develop better vaccines, vaccination programs, and other control measures for preventing reovirus infection, transmission, and losses due to disease.

Estudio recapitulativo- Evaluación de patogenicidad y antigenicidad de reovirus aviares y control de enfermedades mediante vacunación Los reovirus aviares son ubicuos en la producción avícola en todo el mundo y pueden transmitirse por vías vertical u horizontal entre los pollos. La patogenicidad de los reovirus puede variar desde virus muy patógenos que afectan múltiples tejidos y órganos hasta virus apatógenos. Los reovirus aviares se han asociado con muchas presentaciones de enfermedades, y dos de las enfermedades más significativas desde el punto de vista económico son la artritis/tenosinovitis viral y la enteritis viral. La artritis/tenosinovitis viral se ha reconocido desde la década de 1950 y esencialmente desapareció después del desarrollo de vacunas vivas atenuadas e inactivadas en la década de 1980, pero resurgió en 2011 debido a la aparición de variantes antigénicas. La enteritis viral se reconoció por primera vez en la década de 1970 y se convirtió en la enfermedad predominante asociada a reovirus entre 2006 y 2011 debido a la aparición de reovirus enterotrópicos patógenos. La patogenicidad de los aislados de reovirus se puede evaluar de varias maneras, incluida la inoculación de pollos de engorde de un día con títulos bajos de anticuerpos maternos contra el reovirus a través de la vía del cojinete almohadilla plantar o la vía oral e intratraqueal. Los reovirus patógenos inducen la inflamación de las almohadillas de las patas dentro de los tres días posteriores a la inoculación, y más reovirus patógenos pueden diseminarse y dañar los órganos viscerales. Solo las infecciones por reovirus en pollos jóvenes resultan en enfermedades debido a la resistencia relacionada con la edad al desarrollo de la enfermedad. Los reovirus existen como muchos serotipos y subtipos con varios grados de interrelación. Las primeras cepas de reovirus en los Estados Unidos estaban relacionadas antigénicamente entre sí y se conocen como virus relacionados a la cepa S1133, pero en la década de 2000 surgieron reovirus que eran antigénicamente diferentes de los virus relacionados con S1133. Se ha utilizado el ensayo de neutralización de virus con antisueros policlonales para clasificar las variantes de reovirus emergentes en serogrupos. Las primeras vacunas contra el reovirus se desarrollaron en la década de 1970, y en la década de 1980 se establecieron programas de vacunación para reproductores que protegían a los reproductores, prevenían la transmisión vertical de reovirus y proporcionaban inmunidad materna a la progenie durante las cruciales primeras tres semanas de vida. Con la aparición de variantes antigénicas de reovirus en la década de 2000, los programas de vacunación con vacunas relacionadas con la cepa S1133 se volvieron ineficaces. La industria avícola se ha basado en la vacunación con vacunas autógenas de reovirus inactivado para aliviar las pérdidas debidas a artritis/tenosinovitis viral y enteritis viral. Los aislados de virus utilizados para las vacunas autógenas deben actualizarse con regularidad y se seleccionan según el patotipo, el serotipo o el genotipo Sigma C (σC). Las vacunas vivas atenuadas S1133 todavía se usan en pollos reproductores para el efecto de preparación, seguidas de una o más inyecciones de las vacunas inactivadas autorizadas y/o autógenas. La vía de vacunación y el número de dosis recibidas por los pollos reproductores son muy importantes para una respuesta de anticuerpos suficiente. La vacunación intramuscular con vacunas inactivadas provoca la mayor respuesta de anticuerpos, mientras que la vacunación subcutánea con vacunas inactivadas provoca una baja respuesta de anticuerpos. Más recientemente, la investigación se ha centrado en el desarrollo de vacunas alternativas y estrategias de vacunación. Se desarrolló una vacuna de reovirus variante inactivada que provoca protección contra múltiples serotipos variantes, y se han descrito vacunas experimentales recombinantes y de subunidades que muestran potencial. Se necesita más investigación para desarrollar mejores vacunas, programas de vacunación y otras medidas de control para prevenir la infección, transmisión y pérdidas por reovirus debido a la enfermedad.

Avian Reovirus in Israel, Variants and Vaccines-A Review.

Avian reovirus (ARV) has been determined to be the etiologic agent of viral arthritis/tenosynovitis. In Israel, meat-type chickens, including broilers and breeders, are the most affected. Severe disease symptoms can appear in broiler flocks at a very young age because of early exposure and vertical transmission, causing significant welfare problems. Jewish laws define birds with inflamed, damaged, or torn gastrocnemius and digital flexor tendons as religious condemnations (non-kosher), resulting in severe economic losses for the poultry industry. Vaccination of breeders is a strategy to control the disease by reducing vertical transmission and providing maternal-derived antibodies to the progeny. This review describes Israel's ARV variants and the various vaccines developed over the years. Identification of co-circulating variants triggered the development of multivalent autogenous inactivated vaccines. However, the genotype-matched vaccines failed to provide protection, resulting in an increased prevalence of Cluster II ARV (classified as genotyping cluster 5 in the ARV common world classification). Since 2014, ARV Cluster II has been dominant in Israel. In 2015, the dominant variant s7585 tropism changed the virus pathogenesis and affected broilers with severe clinical signs between 12 and 15 days of age. A new vaccine approach developed in Israel used controlled exposure of the breeding flock to virulent ARV at the age when they are resistant to infection. This approach significantly reduced clinical field cases and reovirus isolations of breeding and broiler flocks between 2020 and 2022.

Estudio recapitulativo- Reovirus aviares en Israel, variantes y vacunas: Una revisión. Se ha determinado que el reovirus aviar (ARV) es el agente etiológico de la artritis/tenosinovitis viral. En Israel, los pollos de carne, incluidos los pollos de engorde y reproductores, son los más afectados. Los signos severos de la enfermedad pueden aparecer en parvadas de pollos de engorde a una edad muy temprana debido a la exposición temprana y la transmisión vertical, lo que causa problemas significativos de bienestar. Las leyes judías definen a las aves con gastrocnemio inflamado, dañado o desgarrado y tendones flexores digitales como decomiso religiosas (no kosher), lo que resulta en graves pérdidas económicas para la industria avícola. La vacunación de reproductoras es una estrategia para controlar la enfermedad al reducir la transmisión vertical y proporcionar anticuerpos derivados de las reproductoras a la progenie. Esta revisión describe las variantes de reovirus aviares de Israel y las diversas vacunas desarrolladas a lo largo de los años. La identificación de variantes co-circulantes desencadenó el desarrollo de vacunas inactivadas autógenas multivalentes. Sin embargo, las vacunas elaboradas con genotipos compatibles no brindaron protección, lo que resultó en una mayor prevalencia de reovirus aviares del grupo II. Desde 2014, el grupo II de los reovirus aviares ha sido dominante en Israel. En 2015, el tropismo de la variante dominante s7585 cambió la patogenia del virus y afectó a los pollos de engorde con signos clínicos severos entre los 12 y los 15 días de edad. Un nuevo enfoque de vacuna desarrollado en Israel utilizó la exposición controlada de la parvada reproductora a reovirus aviares virulentos a la edad en que son resistentes a la infección. Este enfoque redujo significativamente los casos clínicos de campo y los aislamientos de reovirus de parvadas de reproductores y pollos de engorde entre 2020 y 2022.

Perspectives on the Changing Landscape of Epizootic Hemorrhagic Disease Virus Control.

Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer (Odocoileus virginianus), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.

Modeling Abundance of Culicoides stellifer, a Candidate Orbivirus Vector, Indicates Nonrandom Hemorrhagic Disease Risk for White-Tailed Deer (Odocoileus virginianus).

(1) Background: Hemorrhagic diseases in white-tailed deer (Odocoileus virginianus) are caused by orbiviruses and have significant economic impact on the deer ranching industry in the United States. Culicoides stellifer is a suspected vector of epizootic hemorrhagic disease virus (EHDV), with recent field evidence from Florida, but its natural history is poorly understood. Studying the distribution and abundance of C. stellifer across the landscape can inform our knowledge of how virus transmission can occur locally. We may then target vector management strategies in areas where viral transmission can occur. (2) Methods: Here, we used an occupancy modeling approach to estimate abundance of adult C. stellifer females at various physiological states to determine habitat preferences. We then mapped midge abundance during the orbiviral disease transmission period (May-October) in Florida. (3) Results: We found that overall, midge abundance was positively associated with sites in closer proximity to large-animal feeders. Additionally, midges generally preferred mixed bottomland hardwood and agricultural/sand/water habitats. Female C. stellifer with different physiological states preferred different habitats. (4) Conclusions: The differences in habitat preferences between midges across states indicate that disease risk for deer is heterogeneous across this landscape. This can inform how effective vector management strategies should be implemented.

Isolation and characterization of a naturally attenuated novel duck reovirus strain as a live vaccine candidate.

Novel duck reovirus (NDRV) causes high morbidity in ducklings, and recovered ducklings are often remarkably stunted in growth. In this study, four NDRV strains were isolated from the NDRV outbreaks that occurred in different regions of Shandong province, China. The biological characteristics and pathogenicity of the four NDRV strains were elucidated, and the N20 was identified as a naturally attenuated strain. Three-day-old ducklings were immunized with live N20 strain (100 ELD50/duck), and challenged with 104.52 ELD50 of virulent N19 strain at 7 days post immunization. The vaccinated ducklings showed no evidence of clinical signs, gross and histopathological lesions, or loss of body weight, and 100 % protection against the virulent NDRV N19 infection. The NDRV-specific antibodies were generated in the immunized ducklings and could neutralize different NDRV strains. These results indicated that the N20 strain was a promising live attenuated vaccine candidate against highly pathogenic NDRV infection.

Establishment of a rare minnow (Gobiocypris rarus) model for evaluation of experimental vaccines against a disease induced by grass carp reovirus genotype II.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD.

Oral Administration of Bacillus subtilis Subunit Vaccine Significantly Enhances the Immune Protection of Grass Carp against GCRV-II Infection.

Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s-CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes (TNF-α, IL-1ß, IFN1 and MHC-II) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases.

Assessment of a natural grass carp reovirus genotype II avirulent strain GD1108 shows great potential as an avirulent live vaccine.

Vaccine immunization is currently the only effective way to prevent and control the grass carp haemorrhagic disease, and the primary pathogen in these infections is grass carp reovirus genotype II (GCRV-II) for which there is no commercial vaccine. In this study, we evaluated the safety of the GCRV-II avirulent strain GD1108 which isolated in the early stage of the laboratory through continuously passed in grass carp. The immunogenicity and protective effects were evaluated after immunization by injection and immersion. The avirulent strain GD1108 could infect and replicate in the fish which did not revert to virulence after continuous passage. No adverse side effects were observed and the vaccine strain did not spread horizontally among fish. Two routes of immunization induced high serum antibody titers of OD450nm value were 0.79 and 0.76 and neutralization titers of 320 and 320 for the injection and immersion routes of inoculation, respectively. The expression of immune-related genes increased after immunization and the levels were statistically significant. Challenge of immunized fish with a virulent GCRV-II strain resulted in protection rates of 93.88% and 76.00% for the injection and immersion routes, respectively. The avirulent strain GD1108 revealed good safety and immunogenicity via two different inoculation routes. Although the injection route provided the best immune effect, two pathways provided protection against infection with virulent GCRV-II strains in various degrees. These results indicated that the avirulent strain GD1108 can be used for the development and application as live vaccine.

Highly efficient vaccines for Bluetongue virus and a related Orbivirus based on reverse genetics.

Bluetongue virus (BTV) reverse genetics (RG), available since 2007, has allowed the dissection of the virus replication cycle, including discovery of a primary replication stage. This information has allowed the generation of Entry-Competent-Replication-Abortive (ECRA) vaccines, which enter cells and complete primary replication but fail to complete the later stage. A series of vaccine trials in sheep and cattle either with a single ECRA serotype or a cocktail of multiple ECRA serotypes have demonstrated that these vaccines provide complete protection against virulent virus challenge without cross-serotype interference. Similarly, an RG system developed for the related African Horse Sickness virus, which causes high mortality in equids has provided AHSV ECRA vaccines that are protective in horses. ECRA vaccines were incapable of productive replication in animals despite being competent for cell entry. This technology allows rapid generation of emerging Orbivirus vaccines and offers immunogenicity and safety levels that surpass attenuated or recombinant routes.

A developed subunit vaccine based on fiber protein VP56 of grass carp reovirus providing immune protection against grass carp hemorrhagic disease.

Grass carp reovirus (GCRV) is the main viral pathogen that endangers grass carp seriously. Application of vaccine has been considered to be the most effective way to prevent virus infection. VP56 is a protein encoded by gene segment 7 of grass carp reovirus, and is predicted to share homology with fiber protein of mammalian reovirus (MRV). In our study, the immunogenicity of VP56 was evaluated by neutralization test. GCRV was incubated with mouse anti-VP56 antibody, and then was injected into grass carp. Results showed that disease progress and death occurrence was hindered in the experimental group compared with the control group. For further study, the recombinant VP56 protein (rVP56) expressed by pET-32a (+) vector was purified, and was used as subunit vaccine to immunize grass carp. After each fish (15 ± 1.5 g) was injected with 30 µg purified rVP56 intraperitoneally, the immune protective efficacy of recombinant VP56 protein was assessed by a series of immune parameters. The population of red blood cells in immunized fish increased significantly after 5 d post injection (dpi), and reached a peak with (2.98 ± 0.17) × 109/ml at 7 dpi (p < 0.05). The numbers of white blood cells peaked with (8.42 ±â€¯1.01) × 107/ml at 7 dpi (p < 0.05). Additionally, the percentage of monocytes and neutrophils rose to a peak with (9.05 ±â€¯0.92)% and (25.93 ±â€¯2.60)% respectively at 5 dpi (p < 0.05 or p < 0.01), whereas lymphocytes reached the highest value of (85.81 ±â€¯2.73) % at 14 dpi (p < 0.01). Serum antibody titer in the vaccinated fish increased significantly and reached a peak at 21 dpi (p < 0.01). The mRNA expression levels of type I interferon (IFN1), major histocompatibility complex class I (MHC I), Toll-like receptor 22 (TLR22), and immunoglobulin M (IgM) were significantly up-regulated in head kidney and spleen (p < 0.05 or p < 0.01). The GCRV challenge test showed that the relative survival rate in immunized group was 71%-75%. Collectively, the results indicated that rVP56 protein can induce immune protection in grass carp, and can be consider as a candidate vaccine against GCRV infection.

Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China.

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China. RESEARCH HIGHLIGHTS A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China. The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains. The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection. These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ±â€¯0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.

Oral delivery of Bacillus subtilis spores expressing grass carp reovirus VP4 protein produces protection against grass carp reovirus infection.

Grass carp (Ctenopharyngodon idellus) hemorrhagic disease (GCHD), caused by grass carp reovirus (GCRV), has given rise to an enormous loss in grass carp industry during the past years. Up to date, vaccination remained to be the most effective way to protect grass carp from GCHD. Oral vaccination is of major interest due to its advantages of noninvasive, time-saving, and easily-operated. The introduction of oral vaccination has profound impact on aquaculture industry because of its feasibility of extensive application for fish in various size and age. However, the main challenge in developing oral vaccine is that antigens are easily degraded and are easy to induce tolerance. Bacillus subtilis (B. subtilis) spores would be an ideal oral vaccine delivery system for their robust specialty, gene operability, safety and adjuvant property. VP4 protein is the major outer capsid protein encoded by GCRV segment 6 (S6), which plays an important role in viral invasion and replication. In this study, we used B. subtilis spores as the oral delivery system and successfully constructed the B. subtilis CotC-VP4 recombinant spores (CotC-VP4 spores) to evaluate its protective efficacy in grass carp. Grass carp orally immunized with CotC-VP4 spores showed a survival rate of 57% and the relative percent survival (RPS) of 47% after the viral challenge. Further, the specific IgM levels in serum and the specific IgZ levels in intestinal mucus were significantly higher in the CotC-VP4 group than those in the Naive group. The immune-related genes including three innate immune-related genes (IL-4/13A, IL-4/13B, CSF1R), four adaptive immune-related genes (BAFF, CD4L, MHC-II, CD8), three inflammation-related genes (IL-1ß, TNF-α, TGF-ß) and interferon type I (IFN-I) related signaling pathway genes were significantly up-regulated in the CotC-VP4 group. The study demonstrated that the CotC-VP4 spores produced protection in grass carp against GCRV infection, and triggered both innate and adaptive immunity post oral immunization. This work highlighted that Bacillus subtilis spores were powerful platforms for oral vaccine delivery, and the combination of Bacillus subtilis spores with GCRV VP4 protein was a promising oral vaccine.

[Assessment of immunogenic activity of the cloned human rotavirus A WA strain.]

INTRODUCTION: Rotovirus infection (RVI) caused by the dsRNA-containing virus from genus Rotavirus, Reoviridae family, belonging to group A (RVA), is the cause of severe diarrhea in human and other mammalian species. Vaccination is the most effective way to reduce the incidence of RVI. At present, the effectiveness of using gnotobiotic piglets as a universal model for reproducing human rotavirus infection and assessing the quality of RVI vaccine preparations has been experimentally proven. OBJECTIVES: Evaluation of immunogenic activity of the cloned RVA Wa strain in the new-born Vietnamese potbellied piglets trial. MATERIAL AND METHODS: Development of viral preparations of the cloned human Wa strain PBA, development of human RVA rVP6, ELISA, polymerase chain reaction with reverse transcription, immunization and experimental infection of newborn piglets. RESULTS: The article presents the results of the experiment on double immunization of newborn piglets with native virus preparations with the infection activity 5.5 lg TCID50/ml, 3 cm3 per dose, HRV with adjuvant 500 µg per dose and mock preparation (control group) followed with experimental inoculation of all animals with virulent virus strain Wa G1P[8] human RVA with infectious activity of 5.5 lg TCID50/ml in 5 cm3 dose. Development of clinical signs of disease and animal death were observed only in control group. RT-PCR system to detect RVA RNA in rectal swabs, samples of small intestine and peripheral lymph nodes was developed. ELISA based on obtained human RVA rVP6 was developed and results on RVA-specific IgG-antibodies in serum samples of experimental piglets are presented. CONCLUSION: In the course of the research, a high immunogenic activity of the native and purified virus of the cloned Wa RVA strain Wa was established and the possibility of its use as the main component of the RVI vaccine was confirmed. The possibility of using conventional newborn pigs instead of gnotobiotic piglets as an experimental model was demonstrated.

Development of a live attenuated vaccine against Muscovy duck reovirus infection.

The Muscovy duck reovirus (MDRV) is a highly pathogenic virus that causes substantial economic losses in the Muscovy duck industry. While MDRV poses a significant threat to Muscovy ducklings, no vaccine candidates are available to date to alleviate MDRV infection throughout the world. The present study presents efforts toward establishing an attenuated vaccine for MDRV. For this purpose, a live attenuated vaccine strain named CA was obtained via alternate propagation of the MDRV isolate MW9710 in both Muscovy duck embryo fibroblasts (MDEFs) and chicken embryo fibroblasts (CEFs) for 90 passages. The CA strain achieved an adaptive growth capacity in CEFs with a viral titer that ranged between 105.0-105.5 TCID50/100 µL and lost its pathogenicity in 1-day-old Muscovy ducklings. Compared to the parent strain MW9710, the CA strain has 42 scattered amino acid substitutions, most of which are located in the λB, λC, µB, σB, and σC protein. The CA strain maintained its attenuation and showed no gene mutation or virulence reversion after back propagation into 1-day-old ducklings for five rounds. The minimum protective dose was calculated to be 300 TCID50 of the CA strain. Furthermore, a single dose of CA vaccine protected immunized ducklings against lethal challenge by the virulent MDRV strain MW9710 and significantly decreased viral loads. In summary, the CA strain exhibited striking genetic stability, excellent safety, and effective immunogenicity. This CA strain of MDRV is a promising vaccine candidate for the prevention and control of MDRV infection.