Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Turkey adenovirus 3: ORF1 gene sequence comparison between vaccine-like and field strains.

Haemorrhagic enteritis is an economically significant disease reported in the majority of the countries where turkeys are raised intensively; it is caused by Turkey adenovirus 3 (TAdV-3). The aim of this study was to analyse and compare the ORF1 gene 3' region from turkey haemorrhagic enteritis virus (THEV) vaccine-like and field strains in order to develop a molecular diagnostic method to differentiate the strains from each other. Eighty samples were analysed by sequencing and phylogenetic analyses using a new set of polymerase chain reaction (PCR) primers targeting a genomic region spanning the partial ORF1, hyd and partial IVa2 gene sequences. A commercial live vaccine was also included in the analysis. The results showed that 56 of the 80 sequences obtained in this study showed ≥99.8% nucleotide identity with the homologous vaccine strain sequence. Three non-synonymous mutations - ntA1274G (aaI425V), ntA1420C (aaQ473H) and ntG1485A (aaR495Q) - were detected in the THEV field strains but not in the vaccine strain. Phylogenetic analysis confirmed the clustering of the field and vaccine-like strains in different phylogenetic branches. In conclusion, the method employed in this study could be a useful tool towards making a correct diagnosis. The data could contribute to the knowledge of field distribution of THEV strains and increase the limited existing information available on native isolates around the world.

Molecular epidemiology of fowl adenoviruses in Greece.

Outbreaks of inclusion body hepatitis (IBH) and adenoviral gizzard erosion have been anecdotally reported in Greece since approximately 2011. However, a relevant increase in clinical outbreaks compatible with IBH has been described since 2014. Unfortunately, with limited exceptions, only serological assays were performed, and involved strains were not properly characterized. In the present study, 35 outbreaks were investigated in the period between July 2017 and February 2018 in Greece. In addition to clinical and histopathological diagnosis, fowl adenovirus (FAdV) presence was investigated by PCR and sequencing. Thirty-four out of 35 samples tested FAdV positive. Twenty-nine (85.29%) and 5 (14.71%) strains were classified as FAdV-E and FAdV-D, respectively. Fowl adenovirus-E strains were genetically homogeneous and formed an independent cluster of Greek-only sequences, including the sole previously available sequence, suggesting the prolonged circulation of this species in Greece. On the contrary, FAdV-D strains were more heterogeneous and closely related to strains sampled in other European countries, testifying the occurrence of multiple introduction events. The evaluation of phylogenetic relationships, geographic clustering, age of infection, and origin of the broiler breeder flocks suggests that both vertical and horizontal transmission are important in FAdV epidemiology in Greece and highlights the limited efficacy of currently implemented control measures. Of note, a significantly higher mortality was observed in precociously infected flocks, likely because of the higher susceptibility of younger animals. This evidence stresses the need of preventing vertical and/or early infection to limit the economic impact of adenovirus-induced diseases.

Marek's disease viruses circulating in commercial poultry in Italy in the years 2015-2018 are closely related by their meq gene phylogeny.

Marek's disease (MD) is a lymphoproliferative disease important to the poultry industry worldwide; it is caused by Gallid alphaherpesvirus 2 (GaHV-2). The virulence of GaHV-2 isolates has shifted over the years from mild to virulent, very virulent and very virulent +. Nowadays the disease is controlled by vaccination, but field strains of increased virulence are emerging worldwide. Economic losses due to MD are mostly associated with its acute form, characterized by visceral lymphomas. The present study aimed to molecularly classify a group of 13 GaHV-2 strains detected in vaccinated Italian commercial chicken flocks during acute MD outbreaks, and to scrutinize the ability of predicting GaHV-2 virulence, according to the meq gene sequence. The full-length meq genes were amplified, and the obtained amino acid (aa) sequences were analysed, focusing mainly on the number of stretches of four proline molecules (PPPP) within the transactivation domain. Phylogenetic analysis was carried out with the Maximum Likelihood method using the obtained aa sequences, and the sequences of Italian strains detected in backyard flocks and of selected strains retrieved from GenBank. All the analysed strains showed 100% sequence identity in the meq gene, which encodes a Meq protein of 339 aa. The Meq protein includes four PPPP motifs in the transactivation domain and an interruption of a PPPP motif due to a proline-to-serine substitution at position 218. These features are typically encountered in highly virulent isolates. Phylogenetic analysis revealed that the analysed strains belonged to a cluster that includes high-virulence GaHV-2 strains detected in Italian backyard flocks and a hypervirulent Polish strain. Our results support the hypothesis that the virulence of field isolates can be suggested by meq aa sequence analysis.

Molecular characterization of a Marek's disease virus strain detected in tumour-bearing turkeys.

Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2), which primarily affects chickens. However, the virus is also able to induce tumours in turkeys, albeit less frequently than in chickens. This study reports the molecular characterization of a GaHV-2 strain detected in a flock of Italian meat-type turkeys exhibiting visceral lymphomas. Sequencing and phylogenetic analysis of the meq gene revealed that the turkey GaHV-2 has molecular features of high virulence and genetic similarity with GaHV-2 strains recently detected in Italian commercial and backyard chickens. GaHV-2 is ubiquitous among chickens despite vaccination, and chicken-to-turkey transmission is hypothesized due to the presence of broilers in neighbouring pens.RESEARCH HIGHLIGHTS A GaHV-2 strain from Italian turkeys was molecularly characterized.The turkey strain presented molecular characteristics of high virulence in its meq gene.The turkey strain was closely related to previously detected chicken strains.

Inoculation of specific pathogen-free chickens with an infectious bursal disease virus of the ITA genotype (G6) leads to a high and persistent viral load in lymphoid tissues and to a delayed antiviral response.

Infectious Bursal Disease Virus (IBDV) of the ITA genotype (G6) was shown to have peculiar molecular characteristics and, despite a subclinical course, aggressiveness towards lymphoid tissues after experimental infection of specific-pathogen-free (SPF) chickens. The aim of the present study was to evaluate and compare with a Classical IBDV strain, ITA IBDV distribution and persistence in various tissues (bursa of Fabricious, spleen, thymus, bone marrow, caecal tonsils, Harderian gland, kidney, liver and proventriculus), its cloacal shedding and the involvement of gut TLR-3 in duodenum tissues. The 35-day-old SPF chickens were experimentally infected and sampled up to 28 days post infection (dpi) for IBDV detection and TLR-3 quantification by qRT-PCR. The ITA IBDV strain was detected in lymphoid and most non-lymphoid tissues up to the end of the trial, with higher loads compared to the Classical IBDV. Most of those differences were found during the first 2 weeks post-infection. Notably, bone marrow and caecal tonsils presented higher viral loads until 28 dpi, allowing to speculate that these organs may serve as non-bursal lymphoid tissues supporting virus replication. Differences in relative TLR-3 gene expression between ITA IBDV-infected birds and Classical-IBDV infected ones were observed at 4, 14 and 21 dpi, being initially higher in Classical group and later in ITA group. Our results provide new insights into IBDV pathogenesis showing that IBDV of ITA genotype leads to a high and persistent viral load in lymphoid tissues and to a delayed antiviral response.

Molecular characterization of the meq gene of Marek's disease viruses detected in unvaccinated backyard chickens reveals the circulation of low- and high-virulence strains.

Marek's disease (MD) is an important lymphoproliferative disease of chickens, caused by Gallid alphaherpesvirus 2 (GaHV-2). Outbreaks are commonly reported in commercial flocks, but also in backyard chickens. Whereas the molecular characteristics of GaHV-2 strains from the commercial poultry sector have been reported, no recent data are available for the rural sector. To fill this gap, 19 GaHV-2 strains detected in 19 Italian backyard chicken flocks during suspected MD outbreaks were molecularly characterized through an analysis of the meq gene, the major GaHV-2 oncogene. The number of four consecutive prolines (PPPP) within the proline-rich repeats of the Meq transactivation domain, the proline content, and the presence of amino acid (aa) substitutions were determined. Phylogenetic analysis was performed using the Maximum Likelihood method. Sequence analysis revealed a heterogeneous population of GaHV-2 strains circulating in Italian backyard flocks. Seven strains, detected from birds affected by classical MD, showed a unique meq isoform of 418 aa with a very high number of PPPP motifs. Molecular and clinical features are suggestive of a low oncogenic potential of these strains. The remaining 12 strains, detected from flocks experiencing acute MD, transient paralysis, or sudden death, had shorter Meq protein isoforms (298 or 339 aa) with a lower number of PPPP motifs and point mutations interrupting PPPP. These features allow us to assert the high virulence of these strains. These findings reveal the circulation of low- and high-virulence GaHV-2 strains in the Italian rural sector.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

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-pathogen-free 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.

First evidence of avian metapneumovirus subtype A infection in turkeys in Egypt.

Although avian metapneumovirus (aMPV) infection has been reported in most regions of the world, to date, only subtype B has been detected in Egypt. At the end of November 2013, dry oropharyngeal swabs were collected during an outbreak of respiratory diseases in a free-range, multi-age turkey dealer farm in Northern Upper Egypt. The clinical signs that appeared when turkeys were 3 weeks-old were characterized by ocular and nasal discharge and swelling of sinuses. aMPV of subtype A was detected by real-time reverse transcription-polymerase chain reaction. In order to confirm the results and obtain more information on the molecular characteristics of the virus, F and G protein genes were partially sequenced and compared with previously published sequences deposited in GenBank by using BLAST. Subtype of the strain was confirmed by sequencing of partial F and G protein genes. The highest percentages of identity were observed when G sequence of the Egyptian strain was compared with the sequence of an aMPV-A isolated in Nigeria (96.4 %) and when the F sequence was compared with strains isolated respectively in Italy and in UK (97.1 %). Moreover, the alignment of the sequences with commercial subtype A vaccine or vaccine-derived strains showed differences in the Egyptian strain that indicate its probable field origin. The detection of aMPV in the investigated turkey flock highlights some relevant epidemiological issues regarding the role that multi-age farms and dealers may play in perpetuating aMPV infection within and among farms. To our knowledge, this is the first report of aMPV subtype A in Egypt.

Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

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(-0.41) median infectious dose/ml and 10(1.15) median 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 (R (2)>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.

Avian metapneumoviruses expressing Infectious Bronchitis virus genes are stable and induce protection.

The study investigates the ability of subtype A Avian metapneumovirus (AMPV) to accept foreign genes and be used as a vector for delivery of Infectious bronchitis virus (IBV) QX genes to chickens. Initially the GFP gene was added to AMPV at all gene junctions in conjunction with the development of cassetted full length DNA AMPV copies. After recombinant virus had been recovered by reverse genetics, GFP positions supporting gene expression while maintaining virus viability in vitro, were determined. Subsequently, either S1 or nucleocapsid (N) genes of IBV were positioned between AMPV M and F genes, while later a bivalent recombinant was prepared by inserting S1 and N at AMPV MF and GL junctions respectively. Immunofluorescent antibody staining showed that all recombinants expressed the inserted IBV genes in vitro and furthermore, all recombinant viruses were found to be highly stable during serial passage. Eyedrop inoculation of chickens with some AMPV-IBV recombinants at one-day-old induced protection against virulent IBV QX challenge 3 weeks later, as assessed by greater motility of tracheal cilia from chickens receiving the recombinants. Nonetheless evidence of AMPV/IBV seroconversion, or major recombinant tracheal replication, were largely absent.

Italian field survey reveals a high diffusion of avian metapneumovirus subtype B in layers and weaknesses in the vaccination strategy applied.

The current information on the prevalence of avian metapneumovirus (aMPV) infection in layers is fragmentary and its true impact on egg production often remains unknown or unclear. In order to draw an epidemiologic picture of aMPV presence in layer flocks in Italy, a survey was performed on 19 flocks of pullets and layers based on longitudinal studies or sporadic samplings. aMPV was detected by reverse transcription (RT)-PCR, and blood samples were collected for serology by aMPV ELISA. Occurrences of respiratory signs and a drop in egg production were recorded. Possible involvement of infectious bronchitis (IB) and egg drop syndrome (EDS) viruses that could have caused loss of egg production we ruled out for IB virus by RT-PCR, and EDS virus was ruled out by hemagglutination-inhibition (HI). Only subtype B of aMPV was found in both pullet and layer farms. Surveys of pullets showed that most groups became infected prior to the onset of lay without showing clear respiratory signs. At the point of lay, these groups were serologically positive to aMPV. In two layer flocks, egg drops were observed and could be strongly linked to the presence of aMPV infection. Results were correlated with aMPV vaccination programs applied to the birds in three flocks on the same farm. Only a vaccination program which included two live and one killed vaccines gave complete protection from aMPV infection to the birds, while a single live vaccine application was not efficacious. The current study gives an inside view of field aMPV diffusion in Italy and its control in layers.

Avian metapneumovirus (AMPV) attachment protein involvement in probable virus evolution concurrent with mass live vaccine introduction.

Avian metapneumoviruses detected in Northern Italy between 1987 and 2007 were sequenced in their fusion (F) and attachment (G) genes together with the same genes from isolates collected throughout western European prior to 1994. Fusion protein genes sequences were highly conserved while G protein sequences showed much greater heterogeneity. Phylogenetic studies based on both genes clearly showed that later Italian viruses were significantly different to all earlier virus detections, including early detections from Italy. Furthermore a serine residue in the G proteins and lysine residue in the fusion protein were exclusive to Italian viruses, indicating that later viruses probably arose within the country and the notion that these later viruses evolved from earlier Italian progenitors cannot be discounted. Biocomputing analysis applied to F and G proteins of later Italian viruses predicted that only G contained altered T cell epitopes. It appears likely that Italian field viruses evolved in response to selection pressure from vaccine induced immunity.