Search

Infectious bursal disease virus (IBDV) is a ubiquitous immunosuppressive pathogen which is a severe burden to the poultry industry. Due to frequent mutation and reassortment events, its double-stranded, bi-segmented RNA genome displays a considerable heterogeneity, which in turn produces profound differences in antigenicity and pathogenicity. In recent years, the investigation of IBDV epidemiology has greatly benefited from the proposal of multiple phylogenetic classification systems which prompted the execution of molecular surveys all over the world, producing a wealth of standardized and easily sharable data. The present epidemiological survey was conducted in six Near East and Persian Gulf countries to characterize the field IBDVs circulating in the region. Ninety-three broiler flocks, immunized with different vaccination protocols, were investigated over a one-year period by collecting eight individual bursal samples at a time. Using molecular assays targeting both genome segments, field IBDVs belonging to four genotypes were detected from 42 flocks across all investigated countries. While traditional very virulent IBDVs (genotype A3B2) represented a minority, multiple atypical IBDV genotypes (A3B1, A4B1 and A6B1) were found to circulate in most of the region. Many of the detected strains were related to sequences from other continents, supporting the involvement of the Near and Middle East in complex networks of viral spreading. The obtained results, combined with the available literature, allowed us to update the epidemiological picture, hopefully favouring the identification of epidemiological threats and the planning of effective monitoring and control strategies. Different field IBDVs were found to circulate in the Near and Middle East. Multiple atypical genotypes (A3B1, A4B1, A6B1) were found to circulate extensively. Traditional very virulent IBDVs (A3B2) were a minority of the detected strains. Viral exchanges can be hypothesized between the region and different continents. [ABSTRACT FROM AUTHOR]

Infectious bursal disease virus (IBDV) is a highly contagious birnavirus causing a burdensome immunosuppressive disease in chickens. IBDV features a remarkable antigenic, pathogenic and genetic heterogeneity, with significant implications on disease manifestation, control measures and diagnostic approaches. The recent proposals of comprehensive phylogenetic classification systems offered the ideal platform for large-scale molecular surveys, which are crucial to gather epidemiological data and inform control efforts. In this study, the IBDV scenario was investigated in most of Western Europe by considering the results of diagnostic activities performed internationally throughout 2021. In total, 470 bursal samples from nine different countries were analysed by RT–PCR targeting the VP2. When a field virus was identified, the VP1 was also characterized. Most of the 132 detected field viruses were highly homologous reassortants featuring a very virulent-like VP2 and a classical-like VP1 (genotype A3B1). Despite emerging recently, these reassortants were already signalled in several countries in North-Western Europe and associated with subclinical infections. Here, we report their further spread in the region, where they currently represent the dominant field threat. Two other IBDV types were found, one in Italy, where all the identified viruses clustered in a clade of the A3B1 genotype previously reported only in Russia and the Middle East, and the other in Portugal, where the recently characterized A9B1 genotype was confirmed to circulate. The obtained data suggest the recent occurrence of a major shift in the Western European epidemiological landscape of IBDV, stressing the importance of steady monitoring and sharing of information among different countries and laboratories. RESEARCH HIGHLIGHTS The IBDV scenario in Western Europe seems to have radically changed in recent years. IBDV reassortants were found to be the dominant field type in the region. Local circulation of two other IBDV types was detected in Italy and Portugal. [ABSTRACT FROM AUTHOR]

Avian metapneumovirus (aMPV) economically affects the global poultry industry causing respiratory and reproductive disorders. Considering the paucity of data on aMPV occurrence in European free-ranging avifauna, a molecular survey was conducted on wild birds of 23 species belonging to the orders Anseriformes, Charadriiformes or Passeriformes, captured alive and sampled in Northeast Italy as part of the national avian influenza virus (AIV) surveillance activities. A total of 492 oropharyngeal swabs, collected from 2007–2010, all AIV-negative, were screened from aMPV by subtype-specific qRT-PCR. An aMPV-C strain, named aMPV/C/IT/Wigeon/758/07, was found in a wintering young Eurasian wigeon (Mareca penelope) sampled in November 2007. The matrix, fusion, and attachment glycoprotein genes of the detected strain were subsequently amplified by specific independent RT-PCRs, then sequenced, and compared in a phylogenetic framework with known aMPV homologous sequences retrieved from GenBank. Close genetic relationships were found between the aMPV/C/IT/Wigeon/758/07 strain and subtype C Eurasian lineage strains isolated in the late 1990s in French domestic ducks, suggesting epidemiological links. Eurasian wigeons are medium/long-range migrant dabbling ducks that move along the Black Sea/Mediterranean flyway; our finding might, therefore, be related to migratory bridges between countries. To our knowledge, this is the first molecular evidence of the occurrence of aMPV subtype C in Italy and backdates the aMPV-C circulation to 2007. Moreover, the results suggest the susceptibility of Eurasian wigeons to aMPV. Broader investigations are needed to assess the role of wild ducks and the significance of the wildfowl/poultry interface in aMPV-C epidemiology. Wild birds live-captured in Italy were tested for aMPV detection and characterization. aMPV-C Eurasian lineage was found for the first time in a wintering Eurasian wigeon. Migratory birds could be involved in the aMPV epidemiology. [ABSTRACT FROM AUTHOR]

Infectious bursal disease virus (IBDV) is an immunosuppressive pathogen with a bi-segmented, double-stranded RNA genome. Two serotypes, named 1 and 2, are recognized, and serotype 1 is traditionally divided into classical, variant and very virulent strains. Despite having great practical relevance, this classification lacks standardization and may therefore generate confusion. The widespread adoption of molecular techniques, which allow direct assessment of either pathogenicity or antigenicity, further complicates this scenario. Recently, two phylogeny-based classification methods have been proposed to obtain informative and standardized results. The first is based on the analysis of the capsid protein VP2 gene, considered as the main determinant of virulence, while the other one considers both VP2 and RNA polymerase VP1, located in different genome segments. The latter approach is therefore better suited to detect reassortment events, which play a significant role in IBDV evolution. This work reports the detection of eight strains in Portuguese broiler farms that diverged from all available IBDV sequences. Subsequent analyses of VP1 and VP2 sequences confirmed that these strains form a new genotype having segment A of a new genogroup and segment B of classical-like genogroup. No recombination events were identified, while several unique amino acid changes were found in key positions within the VP2. This discovery emphasizes the importance of steady and attentive monitoring of IBDV epidemiology. Further research efforts will be devoted to characterizing the novel genotype, by isolating new strains, assessing their pathogenicity and determining the level of protection ensured by commonly administered IBDV vaccines. Eight IBDV strains with unique VP2 features were detected in Portugal. Based on two distinct classification methods, the strains belong to a new genotype. [ABSTRACT FROM AUTHOR]

Despite the efforts to achieve a consistent classification scheme based on the complete S1 gene, the genetic characterization of infectious bronchitis virus (IBV) is often performed on partial S1 regions due to economic and time constraints in the diagnostic routine. Sanger sequencing remains the most common and cost-effective option even if the analysis of samples where multiple field and vaccine strain populations coexist can lead to partial or misleading results. The present study aimed to evaluate the different diagnostic outcomes of three commonly used RT–PCR methods targeting two regions of the S1 gene. A possible bias in IBV detection and characterization was investigated in relation to the adopted method, the strain concentration as well as their ratio in mixed samples. Thirty samples were prepared by artificially mixing two vaccine strains, combined at different ratios and selected among four different IBV lineages, i.e. GI-1 (Mass), GI-13 (793/B), GI-19 (QX), GI-23 (Israeli Variant 2). Sequence analysis was conducted both manually and with bioinformatic methods. The result agreement among methods, replicates and analysis approaches was statistically evaluated. Consistent results emerged among the three assays, with a few discrepancies likely caused by primer affinity and target amount. This study confirms the complexity of IBV strain identification and highlights the importance of evaluating and updating the available diagnostic assays for a reliable detection of all circulating IBV strains. [ABSTRACT FROM AUTHOR]

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogenfree eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype. [ABSTRACT FROM AUTHOR]

Live vaccines predominantly control avian metapneumovirus (aMPV) infection in poultry flocks, but vaccine virus can be found for extended periods after application. The most frequently used aMPV vaccine in Italy, VCO3 subtype B, was shown to contain a uniqueTru9Irestriction endonuclease site within the amplicons produced by a commonly used aMPV diagnostic reverse transcriptase (RT)-nested polymerase chain reaction (PCR). Analysis of European and database logged subtype B aMPV sequences confirmed that the sequence occurred only in the VC03 vaccine. A subsequent RT-PCR restriction endonuclease study of field samples, collected from turkeys between 2007 and 2012, detected subtype B vaccine-derived strains in 12 of 90 samples tested that were collected from birds under 12 weeks of age. [ABSTRACT FROM AUTHOR]

In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10−0.41median infectious dose/ml and 101.15median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were <0.2. Standard curves, generated either using the serial dilution of an RNA suspension or RNA extracted from the serial dilution of titrated viral suspensions as templates, exhibited good linearity (R2>0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity. [ABSTRACT FROM PUBLISHER]

Avian metapneumovirus (aMPV) subtype A was isolated from 7-week-old turkeys showing respiratory disease typical of turkey rhinotracheitis. Comparison of the virus sequence with previously determined vaccine marker sequences showed that the virulent virus had originated from a licensed live subtype A aMPV vaccine. The vaccine had neither been in use on the farm within a period of at least 6 months nor had it been used on farms within a distance of approximately 5 km. Isolation of the virus and exposure to naive turkeys caused disease typical of a virulent aMPV field strain. The study shows that disease was caused by exposure to aMPV vaccine-derived virus that was present in the environment, and indicates that such virus is able to circulate for longer than was previously envisaged. [ABSTRACT FROM AUTHOR]