A vaccine program comprising GA08 (GI-27) and Mass (GI-1) strains prevents DMV1639 (GI-17) infectious bronchitis virus transmission among broiler chickens.

SummaryEffective control of infectious bronchitis is a challenge in commercial poultry operations due to the high transmissibility of the virus. Although multiple IBV lineages are circulating in the United States, the DMV1639-type IBV strain (GI-17) is currently the major circulating variant, creating production losses in the poultry industry. This study aimed to test whether the combination of a GA08 (GI-27) and a Mass-type (GI-1) IB vaccines could significantly reduce the transmission of a DMV1639-type (GI-17) field IBV strain in 4-week-old commercial broilers. Half of them were directly challenged, whereas the other half of the group mates were put in contact twenty-four hours later. Two replicates of the same study set up, including ten directly challenged and ten contact birds per group, were run. Transmission of the challenge virus was significantly reduced in vaccinates (R=0.0), whereas all unvaccinated birds became infected (R=9.6). Reduced transmission of the DMV1639 IB challenge virus by the combined vaccination program in broiler chickens was also accompanied by clinical protection. This data is important because prevention of IBV transmission by vaccination will result in overall reduced viral replication and consequently in reduced likelihood of genetic changes that can lead to new variants. This is the first published evidence of the successful transmission control of a DMV1639 IBV strain in chickens.

High antigenic diversity of serotype 1 infectious bursal disease virus revealed by antigenic cartography.

The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

Identification of the main genetic clusters of avian reoviruses from a global strain collection.

Introduction: Avian reoviruses (ARV), an important pathogen of poultry, have received increasing interest lately due to their widespread occurrence, recognized genetic diversity, and association to defined disease conditions or being present as co-infecting agents. The efficient control measures require the characterization of the available virus strains. Methods: The present study describes an ARV collection comprising over 200 isolates from diagnostic samples collected over a decade from 34 countries worldwide. One hundred and thirty-six ARV isolates were characterized based on σC sequences. Results and discussion: The samples represented not only arthritis/tenosynovitis and runting-stunting syndrome, but also respiratory symptoms, egg production problems, and undefined disease conditions accompanied with increased mortality, and were obtained from broiler, layer or breeder flocks. In 31 percent of the cases other viral or bacterial agents were demonstrated besides ARV. The most frequent co-infectious agent was infectious bronchitis virus followed by infectious bursal disease virus and adenoviruses. All isolates could be classified in one of the major genetic clusters, although we observed marked discrepancies in the genotyping systems currently in use, a finding that made genotype assignment challenging. Reovirus related clinical symptoms could not be unequivocally connected to any particular virus strains belonging to a specific genetic group, suggesting the lack of strict association between disease forms of ARV infection and the investigated genetic features of ARV strains. Also, large genetic differences were seen between field and vaccine strains. The presented findings reinforce the need to establish a uniform, widely accepted molecular classification scheme for ARV and further, highlight the need for ARV strain identification to support more efficient control measures.

Efficacy of a Turkey Herpesvirus Vectored Newcastle Disease Vaccine against Genotype VII.1.1 Virus: Challenge Route Affects Shedding Pattern.

The control of Newcastle disease (ND) highly relies on vaccination. Immunity provided by a ND vaccine can be characterized by measuring the level of clinical protection and reduction in challenge virus shedding. The extent of shedding depends a lot on the characteristics of vaccine used and the quality of vaccination, but influenced also by the genotype of the challenge virus. We demonstrated that vaccination of SPF chicks with recombinant herpesvirus of turkey expressing the F-gene of genotype I ND virus (rHVT-ND) provided complete clinical protection against heterologous genotype VII.1.1 ND virus strain and reduced challenge virus shedding significantly. 100% of clinical protection was achieved already by 3 weeks of age, irrespective of the challenge route (intra-muscular or intra-nasal) and vaccination blocked cloacal shedding almost completely. Interestingly, oro-nasal shedding was different in the two challenge routes: less efficiently controlled following intra-nasal than intra-muscular challenge. Differences in the shedding pattern between the two challenge routes indicate that rHVT-ND vaccine induces strong systemic immunity, that is capable to control challenge virus dissemination in the body (no cloacal shedding), even when it is a heterologous strain, but less efficiently, although highly significantly (p < 0.001) suppresses the local replication of the challenge virus in the upper respiratory mucosa and consequent oro-nasal shedding.

A Herpesvirus of Turkey-Based Vector Vaccine Reduces Transmission of Newcastle Disease Virus in Commercial Broiler Chickens with Maternally Derived Antibodies.

Newcastle Disease is one of the most important infectious poultry diseases worldwide and is associated with high morbidity, mortality, and economic loss. In several countries, vaccination is applied to prevent and control outbreaks; however, information on the ability of vaccines to reduce transmission of ND virus (NDV) is sparse. Here we quantified the transmission of velogenic NDV among 42-day-old broilers. Chickens were either vaccinated with a single dose of a vector vaccine expressing the F protein (rHVT-ND) at day-old in the presence of maternally derived antibodies or kept unvaccinated. Seeders were challenged 8 h before the co-mingling with the corresponding contacts from the same group. Infection was monitored by daily testing of cloacal and oro-nasal swabs with reverse transcription-real-time PCR and by serology. Vaccinated birds were completely protected against clinical disease and virus excretion was significantly reduced compared to the unvaccinated controls that all died during the experiment. The reproduction ratio, which is the average number of secondary infections caused by an infectious bird, was significantly lower in the vaccinated group (0.82 (95% CI 0.38-1.75)) than in the unvaccinated group (3.2 (95% CI 2.06-4.96)). Results of this study demonstrate the potential of rHVT-ND vaccine in prevention and control of ND outbreaks.

Occurrence and spread of a reassortant very virulent genotype of infectious bursal disease virus with altered VP2 amino acid profile and pathogenicity in some European countries.

Reassortant strains of Infectious Bursal Disease Virus (IBDV) were detected in commercial broiler flocks in the Netherlands, Belgium, Denmark, Czech Republic and Germany and in layers and organic broilers in Sweden in the period of 2017-19. Genetic analysis, based on hypervariable region of VP2 gene showed grouping together with very virulent IBDV strains (vvIBDV, Genogroup 3), but these recent viruses formed a separate cluster, which was most closely related to Latvian IBDV strains from 2010-13. VP1 gene of these isolates was most closely related to D78 attenuated IBDV strain. The recently described reassortant IBDV strain (Bpop/03/PL) from Poland with similar genomic constellation (segment A from vvIBDV, segment B from attenuated strain) retained its pathogenicity (80 % mortality in SPF chickens). Infection with the North-West European reassortant IBDVs described in this study showed subclinical feature in the field (without complicating agents) and when tested under standardized pathogenicity test in SPF layer chickens (no mortality or clinical signs, but marked bursa atrophy was observed). Although these recent North-West European reassortant strains had all amino acid residues in their VP2 gene which are considered as markers of vvIBDV strains, they exhibited typical amino acid changes compared to vvIBDV reference strains that should contribute to the determination of pathogenicity. Diagnostic investigations indicated that co-infection with fowl adenovirus or chicken infectious anaemia virus exaggerated the outcome of the IBDV infection (10-20 % mortality). Widespread presence of this reassortant IBDV group in clinically healthy flocks draws attention to the importance of active surveillance.

Novel Orthobunyavirus Causing Severe Kidney Disease in Broiler Chickens, Malaysia, 2014-2017.

During 2014-2017, we isolated a novel orthobunyavirus from broiler chickens with severe kidney lesions in the state of Kedah, Malaysia; we named the virus Kedah fatal kidney syndrome virus (KFKSV). Affected chickens became listless and diarrheic before dying suddenly. Necropsies detected pale and swollen kidneys with signs of gout, enlarged and fragile livers, and pale hearts. Experimental infection of broiler chickens with KFKSV reproduced the disease and pathologic conditions observed in the field, fulfilling the Koch's postulates. Gene sequencing indicated high nucleotide identities between KFKSV isolates (99%) and moderate nucleotide identities with the orthobunyavirus Umbre virus in the large (78%), medium (77%), and small (86%) genomic segments. KFKSV may be pathogenic for other host species, including humans.

Virus-Like Particle Based Vaccine Provides High Level of Protection Against Homologous H5N8 HPAIV Challenge in Mule and Pekin Duck, Including Prevention of Transmission.

The most recent pandemic clade of highly pathogenic avian influenza (HPAI) H5, clade 2.3.4.4, spread widely, with the involvement of wild birds, most importantly wild waterfowl, carrying the virus (even asymptomatically) from Asia to North America, Europe, and Africa. Domestic waterfowl being in regular contact with wild birds played a significant role in the H5Nx epizootics. Therefore, protection of domestic waterfowl from H5Nx avian influenza infection would likely cut the transmission chain of these viruses and greatly enhance efforts to control and prevent disease outbreak in other poultry and animal species, as well as infection of humans. The expectation for such a vaccine is not only to provide clinical protection, but also to control challenge virus transmission efficiently and ensure that the ability to differentiate infected from vaccinated animals is retained. A water-in-oil emulsion virus-like particle vaccine, containing homologous hemagglutinin antigen to the current European H5N8 field strains, has been developed to meet these requirements. The vaccine was tested in commercial Pekin and mule ducks by vaccinating them either once, at 3 wk of age, or twice (at 1 day and at 3 wk of age). Challenge was performed at 6 wk of age with a Hungarian HPAIV H5N8 isolate (2.3.4.4 Group B). Efficacy of vaccination was evaluated on the basis of clinical signs, amount of virus shedding, and transmission. Vaccination resulted in complete clinical protection and prevention of challenge virus transmission from the directly challenged vaccinated ducks to the vaccinated contact animals.

Una vacuna basada en partículas similares a virus proporciona un alto nivel de protección contra el desafío con un virus homólogo de influenza aviar de alta patogenicidad H5N8 en patos mula y Pekin, incluida la prevención de la transmisión. El clado pandémico más reciente de influenza aviar altamente patógena H5, clado 2.3.4.4, se diseminó ampliamente, con la participación de aves silvestres, siendo las aves acuáticas más importantes, portando el virus (incluso asintomáticamente) de Asia a América del Norte, Europa, y África. Las aves acuáticas domésticas en contacto regular con aves silvestres desempeñaron un papel importante en las epizootias H5Nx. Por lo tanto, la protección de las aves acuáticas domésticas contra la infección por influenza aviar H5Nx probablemente cortaría la cadena de transmisión de estos virus y aumentaría en gran medida los esfuerzos para controlar y prevenir brotes de enfermedades en otras aves comerciales y especies animales, así como la infección en humanos. La expectativa de una vacuna de este tipo es no solo brindar protección clínica, sino también controlar la transmisión del virus de desafío de manera eficiente y garantizar que se mantenga la capacidad de diferenciar a los animales vacunados. Se ha desarrollado una vacuna emulsionada en aceite con partículas similares al virus, que contiene el antígeno de hemaglutinina homóloga a las cepas de campo H5N8 europeas actuales, para cumplir con estos requisitos. La vacuna se probó en patos de Pekín y mulas comerciales, vacunándolos una vez, a las tres semanas de edad, o dos veces (al primer día y a las tres semanas de edad). El desafío se realizó a las seis semanas de edad con un aislado de alta patogenicidad H5N8 húngaro (2.3.4.4 Grupo B). La eficacia de la vacunación se evaluó en función de los signos clínicos, la eliminación viral y la transmisión. La vacunación dio como resultado una protección clínica completa y la prevención de la transmisión del virus de desafío de los patos vacunados.

Efficacy of a Recombinant Turkey Herpesvirus AI (H5) Vaccine in Preventing Transmission of Heterologous Highly Pathogenic H5N8 Clade 2.3.4.4b Challenge Virus in Commercial Broilers and Layer Pullets.

Outbreaks caused by the highly pathogenic avian influenza virus (HPAIV) H5N8 subtype clade 2.3.4.4 were first reported in 2014 in South Korea then spread very rapidly in Asia, to Europe, and for the first time, to North America. Efficacy of a recombinant HVT-AI (H5) vaccine (rHVT-H5) to provide clinical protection as well as to significantly reduce the shedding of an H5N8 challenge virus has already been demonstrated in SPF chickens. The aim of our studies was to test the efficacy of the same rHVT-H5 vaccine in controlling the transmission of a recent Hungarian HPAIV H5N8 challenge virus in commercial chickens. Broilers and layers were vaccinated at day old according to the manufacturer's recommendation and then challenged with a 2017 Hungarian HPAIV H5N8 (2.3.4.4b) isolate at 5 or 7 weeks of age, respectively. Evaluation of clinical protection, reduction of challenge virus shedding, and transmission to vaccinated contact birds was done on the basis of clinical signs/mortality, detection, and quantitation of challenge virus in oronasal and cloacal swabs (regularly between 1 and 14 days postchallenge). Measurement of seroconversion to AIV nucleoprotein was used as an indicator of infection and replication of challenge virus. Our results demonstrated that rHVT-H5 vaccination could prevent the development of clinical disease and suppress shedding very efficiently, resulting in the lack of challenge virus transmission to vaccinated contact chickens, regardless the type of birds. Single immunization with the tested rHVT-H5 vaccine proved to be effective to stop HPAIV H5N8 (2.3.4.4b) transmission within vaccinated poultry population under experimental conditions.

Immunity Elicited by a Turkey Herpesvirus-Vectored Newcastle Disease Vaccine in Turkey Against Challenge With a Recent Genotype IV Newcastle Disease Virus Field Strain.

Newcastle disease (ND) is still a major poultry disease worldwide. Vaccination remains the principal method of controlling ND in endemic countries. Various vaccination strategies, including the use of recently developed recombinant vaccines, have been used to control it. Recombinant vaccines that use the herpesvirus of turkey (HVT) as a vector to express one of the key antigens of Newcastle disease virus (NDV) have been developed to overcome some of the drawbacks related to the use of conventional vaccines. HVT as a vector appears to have unique beneficial characteristics: it is extremely safe, it is not affected by the presence of maternally derived antibodies, and therefore can be applied in the hatchery either in ovo or to day-old chicks. Due to its persistence in the bird, the HVT vector can be expected to induce life-long immune stimulation. In the present study, the efficacy of an HVT-based vector vaccine expressing the F gene of NDV (rHVT-F) was tested against a velogenic genotype IV NDV challenge in commercial turkeys with high levels of maternal antibodies (8.7 ± 0.8 log2 hemagglutination inhibition titer). The birds were vaccinated on the day of hatch by the subcutaneous route. Development of a humoral immune response to vaccination was detectable from 4 weeks of age by ELISA. The challenge strain used represents recent NDV genotype IV field strains from Morocco. Challenge with this strain induced ND-specific clinical signs and stunting without subsequent mortality in the non-vaccinated birds, whereas the vaccinated turkey poults showed protection as early as 3 weeks of age based on lack of clinical signs, better body weight gain, and reduction of challenge virus shedding. This is the first reported efficacy study of an HVT-vectored ND vaccine against a velogenic NDV challenge in commercial turkeys.

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).

Successive occurrence of recombinant infectious bronchitis virus strains in restricted area of Middle East.

Routine molecular diagnostic testing by our laboratory, based on using a primer pair with conservative binding sites on the spike glycoprotein coding sequence, has indicated the recurring of a unique phylogenetic cluster of chicken infectious bronchitis viruses (IBV) in the Middle East since 2010. The nearly full-length S1 subunit of the spike gene phylogeny of selected strains, however, split up this grouping, suggesting potential recombination in the S1 gene. In order to clarify this, various bioinformatic analyses of the strains were carried out, which confirmed this supposition. Two patterns of recombination were found among the strains, one of which could also be identified in GenBank-deposited IBV sequences from the region. These findings demonstrate that IBV strains of different recombinant patterns occur simultaneously in the same geographic region and could circulate for an extended period of time, thus contributing to the knowledge on IBV evolution.

Tembusu-like flavivirus (Perak virus) as the cause of neurological disease outbreaks in young Pekin ducks.

A neurological disease of young Pekin ducks characterized by ataxia, lameness, and paralysis was observed at several duck farms in Malaysia in 2012. Gross pathological lesions were absent or inconsistent in most of the cases, but severe and consistent microscopic lesions were found in the brain and spinal cord, characterized by non-purulent panencephalomyelitis. Several virus isolates were obtained in embryonated duck eggs and in cell cultures (Vero and DF-1) inoculated with the brain homogenates of affected ducks. After exclusion of other viruses, the isolates were identified as a flavivirus by flavivirus-specific reverse transcription-polymerase chain reaction (RT-PCR) assays. Inoculation of 2-week-old Pekin ducks with a flavivirus isolate by the subcutaneous or intramuscular route resulted in typical clinical signs and histological lesions in the brain and spinal cord. The inoculated virus was detected by RT-PCR from organ samples of ducks with clinical signs and histological lesions. With a few days delay, the disease was also observed among co-mingled contact control birds. Phylogenetic analysis of NS5 and E gene sequences proved that the isolates were representatives of a novel phylogenetic group within clade XI (Ntaya virus group) of the Flavivirus genus. This Malaysian Duck Tembusu Virus (DTMUV), named Perak virus, has moderate genomic RNA sequence similarity to a related DTMUV identified in China. In our experiment the Malaysian strain of DTMUV could be transmitted in the absence of mosquito vectors. These findings may have implications for the control and prevention of this emerging group of flaviviruses.

Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers.

The onset and duration of immunity provided by a recombinant ND vaccine using HVT virus as vector (rHVT-ND) was followed up to 72 weeks of age in commercial layer chickens after single application or as part of two different vaccination regimes including conventional live and killed ND vaccines. Efficacy of the different vaccination programmes was checked, from 3 to 72 weeks of age, by serology as well as by challenges with a recent velogenic NDV isolate belonging to genotype VII. Assessment of protection was done based on the prevention of clinical signs and reduction of challenge virus shedding via the oro-nasal and cloacal routes. Single vaccination with the rHVT-ND vaccine at one day of age provided complete or almost complete (95-100%) clinical protection against NDV challenges from 4 weeks of age up to 72 weeks of age when the latest challenge was done. Shedding of challenge virus both by the oro-nasal and cloacal route was significantly reduced compared to the controls. Booster vaccination of rHVT-ND vaccinated birds with conventional ND vaccines significantly increased the level of anti-NDV serum antibodies and further reduced the oro-nasal excretion of challenge virus.

Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains.

Polymerase chain reaction and sequencing were used to analyse goose parvovirus field isolates and vaccine strains. Two fragments of the genome were amplified. Fragment "A" represents a region of VP3 gene, while fragment "B" represents a region upstream of the VP3 gene, encompassing part of the VP1 gene. In the region of fragment "A" the deduced amino acid sequence of the strains was identical, therefore differentiation among strains could be done only at the nucleotide level, which resulted in the formation of three groups: Hungarian, West-European and Asian strains. In the region of fragment "B", separation of groups could be done by both nucleotide and deduced amino acid sequence level. The nucleotide sequences resulted in the same groups as for fragment "A" but with a different clustering pattern among the Hungarian strains. Within the "Hungarian" group most of the recent field isolates fell into one cluster, very closely related or identical to each other, indicating a very slow evolutionary change. The attenuated strains and field isolates from 1979/80 formed a separate cluster. When vaccine strains and field isolates were compared, two specific amino acid differences were found that can be considered as possible markers for vaccinal strains. Sequence analysis of fragment "B" seems to be a suitable method for differentiation of attenuated vaccine strains from virulent strains.