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Background/introduction Porcine parvovirus (PPV), the causative agent of severe reproductive failures in pigs, is present worldwide. The witnessed spread of the virulent 27a type PPV strains since its recognition raised concerns about the efficacy of the available commercial vaccines. Methods To address this question, vaccinated pregnant gilts were challenged with a PPV-27a-like virus strain and parameters related to vaccine efficacy were compared. Results The K22 vaccine strain of Parvoruvax® (PVX) was characterized as "Kresse-like" based on the epitope mapping data. Vaccination of the gilts induced a low level of antibody responses. Based on foetal mortality, the number of sows which had challenge virus-affected foetuses, the percent of PPV positive piglets/litters plus their PPV genome and viral load PVX outscored the other vaccinated groups. Conclusion Stronger protection was provided by the "Kresse-like" K22 PPV strain-based vaccine than by the NADL-2 and NADL-like strain-based commercial vaccines against a PPV-27a cluster strain challenge. Vaccine-induced antibody levels as measured pre-challenge were not found to be an accurate indicator of protection.

Duck hepatitis A virus (DHAV), an avian picornavirus, causes high-mortality acute disease in ducklings. Among the three serotypes, DHAV-1 is globally distributed, whereas DHAV-2 and DHAV-3 serotypes are chiefly restricted to Southeast Asia. In this study, we analyzed the genomic evolution of DHAV-1 strains using extant GenBank records and genomic sequences of 10 DHAV-1 strains originating from a large disease outbreak in 2004–2005, in Hungary. Recombination analysis revealed intragenotype recombination within DHAV-1 as well as intergenotype recombination events involving DHAV-1 and DHAV-3 strains. The intergenotype recombination occurred in the VP0 region. Diversifying selection seems to act at sites of certain genomic regions. Calculations estimated slightly lower rates of evolution of DHAV-1 (mean rates for individual protein coding regions, 5.6286 × 10−4 to 1.1147 × 10−3 substitutions per site per year) compared to other picornaviruses. The observed evolutionary mechanisms indicate that whole-genome-based analysis of DHAV strains is needed to better understand the emergence of novel strains and their geographical dispersal.

Background: Actinobacillus pleuropneumoniae is a major economically significant bacterial respiratory pathogen of pigs, and vaccine use is considered an integral control method to prevent disease. The objective of this multi-study analysis was to evaluate the serovar independent efficacy in growing pigs of the C-vaccine (Coglapix®, Ceva, France), which comprises whole cells of A. pleuropneumoniae serovars 1 and 2 expressing ApxI, ApxII and ApxIII toxins. Efficacy was based on protection against lung lesions, since there is good correlation between the severity/extension of lung lesions and losses induced by pleuropneumonia. Vaccine efficacy was determined against challenge with the most common serovars (1, 2, 4, 5, 6, 7, 9/11 and 13) of A. pleuropneumoniae in a total of 13 studies of the same design and reproducibility was validated.Results: Protection against homologous serovars 1 and 2 significantly reduced lung lesion scores (LLS) compared to the positive controls: p = 0.00007 and p = 0.00124, respectively. The protection against heterologous serovars 4, 5, 6, 7, 9/11, and 13 also significantly reduced LLS: range p = 2.9e-10 to p = 0.00953. Reproducibility between challenge studies was excellent with the estimated random effect of study (the fraction of the total variation attributed by differences between studies) being only 2.6%, 2.2% and 4.8% for three serovar 2, two serovar 9/11 and serovar 4 challenge studies, respectively. An outlier was the 35% of variation attributable to trial between the two serovar 6 challenges, possibly explained by a Streptococcus spp. outbreak.Conclusions: A highly significant serovar independent reduction of pathological lung lesions by the C-vaccine was demonstrated for all the serovars tested (1, 2, 4, 5, 6, 7, 9/11 and 13). High levels of protection with similar significance-values were obtained for both homologous and heterologous serovar challenge. To our knowledge the aerosol chamber challenge concept based on accurate individual pig dosing and a standardized biologically weighted lung lesion scoring is the first to be validated statistically as reproducible and reliable. The C-vaccine was demonstrated to be a good candidate to fulfil the demands in the field for an A. pleuropneumoniae-vaccine i.e., high protective capability against disease caused by multiple serovars.

Fowl adenoviruses (FAdV), detected during routine diagnostic investigations from 38 countries (5 continents) over a decade, were partially sequenced and grouped by phylogenetic analysis. The partial polymerase gene nucleotide sequences of the 365 fowl adenovirus isolates resulted in the following species distribution: 11% FAdV-A; 3% FAdV-B; 2% FAdV-C; 34% FAdV-D; and 50% FAdV-E. Noticeably, only 79 of the detected strains could be associated with adenovirus-specific pathologic conditions: 62 (79%) with inclusion body hepatitis; 9 (11%) with gizzard erosion; and 8 (10%) with hepatitis hydropericardium syndrome. The remainder of the FAdV strains was detected as concomitant infection from other disease conditions almost exclusively in boilers of 27 to 42 d of age: the majority of them was FAdV-E followed by FAdV-D, and to a lesser extent of FAdV-A, B, and C, the latter ones have not been associated with any of the established adenovirus-caused syndromes in our collection. The highest ratio of coinfections was observed for FAdV-B (62%), while it was about 30% for the rest of the FAdV species. The most frequent coinfection, in connection with all FAdV species, was with the avian infectious bronchitis virus. The presented database will serve as the basis for comparative whole genome and cross-neutralization analysis of selected FAdV isolates.

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 &lt, 0.001) suppresses the local replication of the challenge virus in the upper respiratory mucosa and consequent oro-nasal shedding.

The recombinant herpesvirus of turkey (rHVT) vaccines targeting Newcastle disease (ND) and H5Nx avian influenza (AI) have been demonstrated efficient in chickens when used individually at day-old. Given the practical field constraints associated with administering two vaccines separately and in the absence of a currently available bivalent rHVT vector vaccine expressing both F(ND) and H5(AI) antigens, the aim of this study was to investigate whether interference occurs between the two vaccines when simultaneously administered in a single shot. The studies have been designed to determine (i) the ND and AI-specific protection and antibody response conferred by these vaccines inoculated alone or in combination at day-old, (ii) the influence of maternally-derived antibodies (MDA), and (iii) the potential interference between the two vaccine. Our results demonstrate that their combined administration is efficient to protect chickens against clinical signs of velogenic Newcastle disease virus (vNDV) and H5-highly pathogenic avian influenza virus (HPAIV) infections. Viral shedding following co-vaccination is also markedly reduced, while slightly lower NDV- and AIV-specific antibody responses are observed. NDV- and AIV-specific MDA show negative effects on the onset of the specific antibody responses. However, if AIV-specific MDA reduce the protection against H5-HPAIV induced by rHVT-H5(AI) vaccine, it was not observed for ND.

Among the different vaccines used to control highly pathogenic avian influenza, an HVT vector-based live recombinant avian influenza vaccine, expressing the haemagglutinin gene of an H5N1 HPAI virus, has been used by the poultry industry since 2012. The objective of the study presented in this paper was to test the efficacy of the commercially available HVT-based recombinant H5 vaccine against antigenically drifted H5N1, H5N8 and H5N2 HPAI virus circulating in Egypt recently. Groups of SPF chicks vaccinated at day-old with the HVT-based recombinant H5 vaccine were challenged, along with non-vaccinated controls, with 10

We determined the genomic sequence of a Ukrainian strain of fowl adenovirus B (FAdV-B). The isolate (D2453/1) shared 97.2% to 98.4% nucleotide sequence identity with other viruses belonging to the species Fowl aviadenovirus B. Marked genetic divergence was seen in the hexon, fiber, and ORF19 genes, and phylogenetic analysis suggested that recombination events had occurred in these regions. Our analysis revealed mosaicism in the recombination patterns, a finding that has also been described in the genomes of strains of FAdV-D and FAdV-E. The shared recombination breakpoints, affecting the same genomic regions in viruses belonging to different species, suggest that similar selection mechanisms are acting on the key neutralization antigens and epitopes in viruses of different FAdV species. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-04972-9.

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.

In the chicken bursa of Fabricius (BF), the interfollicular epithelium (IFE) consists of cylindrical- and cuboidal-shaped cells. Among the cylindrical-shaped epithelial cells, mucus-producing and caveolin-1 (Cav-1)-expressing cells can be distinguished. Occasionally, the cuboidal-shaped cells also express Cav-1, which suggests that they are precursors of both mucus-producing and Cav-1-expressing cells. Very virulent infectious bursal disease virus (IBDV) impedes the differentiation of Cav-1-expressing cells and shifts the differentiation of cuboidal cells towards mucus-producing cells. In control birds exclusively, the IFE surface shows a mucous membrane, but after IBDV infection, the surfaces of both IFE and FAE are also covered by a mucous membrane. After IBDV infection, the cells of FAE also produce mucus, providing evidence for cell transformation. In late postinfection (pi; 28 d pi), the Cav-1 expression returned in the IFE cells, whereas the follicle (the primary lymphoid organ) underwent atrophy. The appearance of the renewed Cav-1-positive cells is similar to that of the normal basal cell, but they randomly locate in different levels of IFE, suggesting the loss of epithelial polarity. Between days 2 and 7 pi, the Cav-1 expression in the endothelial cells of the cortico-medullary capillary web is variable, which may explain the hemorrhage in several infected birds. The IBDV infection stops the Cav-1 expression and subsequently the cholesterol efflux into the bursal lumen. In the infected birds, the high cholesterol level may further worsen the clinical syndrome of IBDV.

Complete genomic sequences of two orthoreovirus strains, D2533/4/1-10 and D2533/6/1-10, isolated from Pekin ducklings in Germany have been determined. Pairwise sequence comparisons and phylogenetic analyses indicated that strain D2533/4/1-10 might have acquired its genomic segments from three different origins, from classical and novel waterfowl reoviruses, and a yet unknown orthoreovirus strain. D2533/6/1-10 proved to be only distantly related to previously described orthoreoviruses. Reassortment, host species transmission events, and successful adaptation of novel variants may signify a challenge for animal health and maintenance of economic production.

Background/Introduction Porcine parvovirus (PPV), the causative agent of severe reproductive failures in pigs, is present worldwide. The witnessed spread of the virulent 27a type PPV strains since its recognition raised concerns about the efficacy of the available commercial vaccines. Methods To address this question, vaccinated pregnant gilts were challenged with a PPV-27a-like virus strain and parameters related to vaccine efficacy were compared. Results The K22 vaccine strain of Parvoruvax® (PVX) was characterized as “Kresse-like” based on the epitope mapping data. Vaccination of the gilts induced a low level of antibody responses. Based on foetal mortality, the number of sows which had challenge virus-affected foetuses, the percent of PPV positive piglets/litters plus their PPV genome and viral load PVX outscored the other vaccinated groups. Conclusion Stronger protection was provided by the “Kresse-like” K22 PPV strain-based vaccine than by the NADL-2 and NADL-like strain-based commercial vaccines against a PPV-27a cluster strain challenge. Vaccine-induced antibody levels as measured pre-challenge were not found to be an accurate indicator of protection.

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.

Avian reoviruses are well-known pathogens seriously affecting the productivity of poultry industry. Game birds represent a small segment of the agricultural sector and much remained to be learnt about factors affecting productivity. Here we show that reovirus infections might occur in pheasants and demonstrate that reoviruses of pheasants are of diverse origin. The complete or coding-complete genomic sequences of two Hungarian reovirus strains, D1996/2/1 and Reo/HUN/Pheasant/216/2015, have been determined in this study. The strain D1996/2/1 was isolated in 2012 from birds with gizzard erosion, whereas the other strain was isolated in 2015 from diarrheic pheasant poults. Phylogenetic analyses showed that none of the Hungarian isolates shared common origin with a pheasant reovirus detected recently in the United States. Additionally, we found that Reo/HUN/Pheasant/216/2015 is a multi-reassortant reovirus within the species Avian orthoreovirus that shared genetic relationship with turkey reoviruses (σC), partridge reoviruses (λA, σB), and chicken reoviruses (λB, λC, μA, σA, and σNS), in the respective gene phylogenies, whereas two genes (μB and μNS) did not reveal any possible common ancestors. The other isolate, D1996/2/1, was found to be distantly related to previously described reoviruses raising the possibility that it might represent a novel orthoreovirus species or a new genogroup within the newly accepted species, Neoavian orthoreovirus. The genetic diversity among pheasant reoviruses could raise challenges for virus classification as well as for development of molecular diagnostic tools and vaccine based prevention and control measures.

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

Avian orthoreoviruses cause various diseases in wild birds and domesticated poultry. In this study we report the detection and genomic characterization of a partridge (Perdix perdix) origin reovirus strain, D1007/2008. The virus was isolated on cell culture from acute pneumonia and infra-orbital sinusitis. The 23,497 nucleotide long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Sequence and phylogenetic analyses showed that the partridge reovirus strain was related to orthoreoviruses of gallinaceous birds. In fact, five (λB, λC, μB, σC, σNS) and one (σB) out of 10 genes clustered definitely with turkey or chicken origin orthoreoviruses, respectively, whereas in the λA, μA, μNS and σA phylogenies a more distant genetic relationship was observed. Our data indicate that the identified reovirus strain is composed of a mixture of chicken and turkey orthoreovirus related alleles. This finding implies that partridges may serve as natural reservoirs for orthoreoviruses of domesticated poultry.

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.

Porcine circovirus type 2 (PCV2), a highly prevalent, economically important swine pathogen is classified into different genotypes (PCV2a-f) based on phylogenetic analysis. Since the introduction of extensive vaccination programs, at least two major shifts have been observed in the prevalence of PCV2 genotypes. The first genotype shift from 2a towards 2b occurred around 2003, while in recent years, we are witnessing the second change in genotype prevalence from the predominant 2b towards 2d. In this study, a PCV2d-2 isolate was characterized as a potential challenge virus for the evaluation of PCV2 vaccine efficacy. Ten-week-old pigs carrying low to moderate levels of maternally derived antibodies to PCV2 were infected with the isolate by the nasal route. Over the next 4 weeks post-infection, the pigs were monitored for the presence of viremia, fecal virus excretion, and humoral immune responses. At the end of the post-infection observation period, samples were taken from the mediastinal and mesenteric lymph nodes of the animals and tested for viral load. The gradual depletion of maternally derived antibodies in the sera of piglets was demonstrated by ELISA and virus neutralization tests. Following experimental infection by PCV2d-2, specific IgM antibodies were first detected at 14 days post challenge (dpch), while IgG class antibodies were first detected at 21 dpch. Both viremia and virus shedding could be detected at 7 dpch, in 36 and 50% of the pigs, respectively. The proportion of shedders reached 100% by 14 dpch and remained at this level, while viremia was demonstrated in 86, 100, and 100% of the pigs at 14, 21, and 28 dpch, respectively. Both the mediastinal and mesenteric lymph nodes contained high levels of virus (7.6 and 8.5 log10 copies/mg tissue, respectively).

From October 2016 to July 2017, 47 countries have been affected by highly pathogenic avian influenza (HPAI) viruses of the H5N8 clade 2.3.4.4 subtype, including European and African, and it has been the most severe HPAI outbreak ever in Europe. The development of effective influenza vaccines is required to combine preventive and control measures in order to avoid similar avian influenza epidemics taking place. Here we describe a novel prototype recombinant virus-like particle (VLP) vaccine based on a clade 2.3.4.4 H5 HA derived from a French duck HPAI H5N8 isolate of the 2016-2017 epidemics. Prototype vaccines with different antigen content were formulated and the immunogenicity was examined in specific-pathogen-free chickens and in ducks. Serum samples were collected at 3 and 4 weeks postvaccination, and development of the immune response was evaluated by hemagglutination inhibition test and ELISA. The VLP vaccines induced a dose-dependent and high level of antibody response in both chickens and ducks. The results of HPAI H5N8 challenge experiments in ducks are reported separately.Construcción rápida y pruebas de inmunogenicidad de un nuevo prototipo de vacuna H5 con base en partículas similares a virus contra el clado 2.3.4.4 del virus de la influenza aviar altamente patógeno H5N8. Desde octubre del 2016 hasta julio del 2017, 47 países se han visto afectados por los virus de la influenza aviar altamente patógena del subtipo H5N8 clado 2.3.4.4, incluidos los europeos y africanos y ha sido el brote de influenza aviar de alta patogenicidad más grave en Europa. Se requiere del desarrollo de vacunas efectivas contra la influenza para combinar medidas preventivas y de control para evitar que ocurran epidemias similares de influenza aviar. En este estudio se describe un nuevo prototipo de vacuna recombinante con partículas similares a virus (VLP) basada en una hemaglutinina H5 clado 2.3.4.4 derivada de un aislamiento del virus de influenza aviar de alta patogenicidad H5N8 de patos en Francia presente en las epidemias entre los años 2016 al 2017. Se formularon prototipos de vacunas con diferente contenido de antígeno y se examinó la inmunogenicidad en pollos libres de patógenos específicos y en patos. Las muestras de suero se recolectaron a las tres y cuatro semanas después de la vacunación y el desarrollo de la respuesta inmune se evaluó mediante la prueba de inhibición de la hemaglutinación y ELISA. Las vacunas con partículas similares a virus indujeron un alto nivel de respuesta de anticuerpos dependiente de la dosis tanto en pollos como en patos. Los resultados de los experimentos de desafío con un virus de influenza aviar de alta patogenicidad H5N8 en patos se informan por separado.

Understanding the epidemiology and improving vaccinal protection against the highly variable chicken infectious bronchitis virus (IBV) requires the knowledge of circulating IBV serotypes/genotypes in defined geographic areas. Accordingly, the authors initiated a survey among the major poultry producers in Hungary in order to reveal the prevailing IBV serotypes in the country. Tracheal swabs and organ samples (caecal tonsils, kidneys, and trachea) were collected from broiler, layer, and meat-type breeder flocks, and were subjected to IBV detection by virus isolation and polymerase chain reaction (PCR). The IBV-positive samples were further characterised by nucleotide sequencing and phylogenetic analysis of a portion of the S1 IBV gene. Seventeen out of the 26 submitted samples proved to be positive for IBV. Sequence analyses revealed ten 4/91 and six QX serotypes, and a single D274 type IB virus. One sample contained a mixture of QX and Massachusetts serotype viruses. Presumably most of the 4/91 and D274 type viruses were vaccine strains. The proportion of QX type viruses and their observed variation are in good agreement with the situation in a few other European countries. The detected viruses clustered largely according to their geographic origin, with a few exceptions. If updated regularly, the preliminary ‘virus map’ will be useful for the adjustment of vaccination protocols.

Background Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. Methods To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. Results Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. Conclusions Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use. Copyright © 2015 John Wiley & Sons, Ltd.

The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle-associated epithelium (FAE). The IFE comprises (i) cylindrical-shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two-way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti-caveolin-1 (Cav-1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav-1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav-1 indicates that no caveolin-mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav-1 positive cells can be found among the SC, which are designated SC II. Cav-1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav-1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans-Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21-23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav-1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.

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.

The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout the world indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control ND. There is a need for vaccination programmes that could be initiated at 1-day-old for mass application and which would induce a long-lasting immunity, with no need for a booster vaccination at a later age. In this context, the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with a classical programme that included a conventional live and an inactivated ND vaccine at the same age in commercial layer chickens. The humoral, cell-mediated and local immunity were followed weekly and birds were challenged with a viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the conventional vaccine-based regime. This might be related to a T-helper type 1 (Th1) cellular-driven immunological response, in contrast to the T-helper type 2 (Th2) humoral-oriented immune response provided by the current conventional vaccine-based vaccination programmes.

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.

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).

In countries where avian influenza has become endemic, early vaccination of layer pullets or broilers with classical inactivated vaccines at the hatchery is no longer an option because of interference with passive immunity indirectly induced by the necessary vaccination of the breeders. On the other hand, injection of thousands of chicks from 7 to 10 days old on farms has been determined to be unreliable and, therefore, poorly efficacious. For these reasons, interest has arisen regarding a newly developed live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 highly pathogenic avian influenza virus (HPAIV) strain (rHVT-H5), which in theory is capable of breakthrough passive immunity to both the vector (HVT) and the insert (H5) and is consequently applicable at the hatchery. The objectives of this trial were to evaluate the impact of maternally derived antibodies (MDAs) specific to H5N1 on the immunity and the efficacy (protection and virus shedding) of different vaccination programs including rHVT-H5 and inactivated H5N1 and H5N2 vaccines applied alone or in combination. Therefore, broilers carrying MDAs against both HVT and Asian H5N1 HPAIV were vaccinated on the first day of age with rHVT-H5, with or without boosting vaccination by an inactivated vaccine after 10 days. The different groups were challenged with two antigenically highly divergent Egyptian dade 2.2.1 H5N1 HPAIVs at 4 wk of age. Protection against challenge was compared with unvaccinated birds or vaccinated birds without MDAs. Between 70% and 90% clinical protection could be observed in the vaccinated groups possessing MDAs, indicating no or very low interference of MDAs with vaccination. Results regarding clinical protection, humoral, cell-mediated, and mucosal immunity, as well as re-excretion of challenge virus are presented and discussed.

1. CVI-ChNL 74·3, a dendritic cell-specific monoclonal antibody (mAb) also identifies chicken lung granular pneumocytes (type II pneumocytes), which produce surfactant. 2. The 74·3 mAb does not cross-react with any other avian or mammalian granular pneumocyte, and provides a convenient tool for monitoring the status of type II pneumocytes in the chicken lung.

The effects of infectious bursal disease virus (IBDV) (strain F52/70) infection were studied by immunohistochemical methods on the splenic extracellular matrix (ECM). The major fibrillar components of the ECM, the type I and type III collagens and the main ECM organizing glycoproteins (laminin, tenascin and fibronectin) were monitored up to 11 days post-infection (d.p.i.). By 3 d.p.i., the collagens that form the basic scaffold of the antigen-trapping region of the spleen are destroyed, which is followed by deterioration of the glycoproteins. The ECM in the red pulp and the other regions of the white pulp (periarteriolar lymphatic sheath and germinal centre) seem to be normal. The reason for the significantly different pathological alterations in the ECM between the two regions of the spleen may be explained by the origin of the reticular cells. The reticular cells in the antigen-trapping zone and other splenic regions are of haemopoietic and mesenchymal origins, respectively. Possibly, the reticular cells of the haemopoietic origin are more susceptible for the IBDV infection than the mesenchymal ones. Development of the antigen-trapping, B-cell-dependent zone of the splenic white pulp precedes that of the periarteriolar lymphatic sheath and germinal centre, which suggests that this region may contribute to B-cell maturation. Damage of the ECM in the antigen-trapping zones results in impairment of tissue organization, which may contribute to the permanent immunosuppression.

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.

Avian orthoreoviruses (ARVs) are responsible for considerable economic losses in broiler chickens; yet, the genetic characterization of most ARV strains is limited to a few genes, and the full coding region has been determined for only S1133 and 138, two ARV strains associated with tenosynovitis. Recently, in parts of the United States, ARVs with novel neutralization antigen type were isolated from chickens afflicted with runting-stunting syndrome. One such strain, AVS-B, was selected for full genome sequencing and phylogenetic analysis. The complete genome was 23,494 bp in size and included 12 open reading frames. The lengths of the coding regions, as well as those of the 5' and 3' ends, were fairly well conserved between AVS-B and other reference strains. In pairwise comparisons to the S1133 and 138 strains, the AVS-B strain shared a wide range of sequence identities along each genome segment, i.e., a range of 54-55% for the σC coding region of S1 genome segment and 91-93% for the S2 genome segment. Phylogenetic analyses of individual genes of AVS-B did not identify any single common ancestor among more completely characterized ARV strains for which sequence data are available. One exception to this lack of identity was strain 138, which shared 90-93% nt identity with AVS-B along seven of ten genome segments; only M2, M3, and S1 segments of these strains shared lower sequence identities. Collectively, our analyses indicated that multiple reassortment events and strong divergence caused by the accumulation of point mutations could have led to the observed assortment and genetic heterogeneity of the AVS-B genome.