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Avian orthoreoviruses (ARV) are an emerging threat for the poultry industry, both in the United States (US) and globally. ARV infections in turkeys have been associated with arthritis, lameness and neurological disorders, and cost the US economy approximately USD 33 million per year. There is not a commercial vaccine available and the shortage of turkey ARV (TRV) genomic data hinders the efforts to explore the molecular epidemiology of this virus, although several studies suggest a close relationship between European TRVs and TRVs present in the US. This study shows a snapshot of the genomic diversity of Avian orthoreoviruses (ARV) circulating in Germany in the mid-2000s. Through a deep genomic analysis of 18 ARV isolates recovered from sick turkeys, we observed that co-infection was a common condition. 80% of the samples showed signs of a simultaneous infection with a TRV and a chicken ARV (CRV). We believe this is the first reported evidence of CRV and TRV naturally occurring co-infections in commercial turkeys. These co-infection events were identified due to the significant genomic diversity observed among ARV infecting various production bird species. Our phylogenetic analysis revealed a consistent host-associated ARV clustering, with three main clades: (i) a TRV clade, (ii) a CRV clade, and (iii) a Duck ARV (DRV)/Goose ARV (GRV) clade. Furthermore, our findings indicate that German TRVs have interacted with their European and American counterparts, suggesting active mobilization of the virus, likely through the commercial trading of live animals. However, we also consider the potential role of migratory birds in the international movement of ARV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo . Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid "aa" in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Background: Hemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures., Methods: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions., Results: Researchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3' rapid amplification of cDNA ends (3' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated., Conclusions: Similar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs., (© 2024. The Author(s).)

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type ( wt ) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Lymphoproliferative disease virus (LPDV) was first documented in wild turkeys in North America in 2009. LPDV infection is often subclinical but can manifest as lymphoid proliferation or round cell neoplasia. Despite high prevalence across many sampled areas corresponding to declining populations of wild turkeys, knowledge regarding LPDV pathogenesis, risk factors for disease development, and associated impacts on population dynamics are unknown. To understand transmission, viral shedding, and tissue tropism, we inoculated 21 domestic turkeys via the oral cavity, crop, nasal cavity, subcutis, or coelomic cavity. For 12 weeks, oropharyngeal swabs, cloacal swabs, and whole blood were collected weekly. At 1 week postinoculation, 3 turkeys (3/21; 14%) had detectable LPDV proviral DNA in blood by polymerase chain reaction, and 10 developed DNAemia (50%; 10/20) by 12 weeks. LPDV proviral DNA was intermittently detected in oropharyngeal and cloacal swabs. Splenomegaly was the most consistent gross finding in DNAemic birds (8/11; 73%). Lymphoid hyperplasia in the spleen was the most significant microscopic finding (9/11; 82%). Three turkeys (3/11; 27%) developed round cell neoplasia characterized by sheets of pleomorphic, round to polygonal cells in the adrenal gland, bone marrow, skin, small intestine, and/or spleen. LPDV was detected in the spleen and bone marrow from all turkeys with DNAemia and all neoplasms. Our study establishes that infection and disease with North American LPDV from wild turkeys can be experimentally reproduced in domestic turkeys, laying the groundwork for future investigations into LPDV pathogenesis, development of diagnostic techniques, and understanding the impacts of LPDV on wild turkey populations., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Adenoviruses are a diverse group of viruses that can cause a variety of diseases in poultry, including respiratory and gastrointestinal infections. In turkeys (Meleagris gallopavo), adenoviruses commonly cause hemorrhagic enteritis and, rarely, inclusion body hepatitis. In this study, we investigated fowl adenoviruses (FAdVs) circulating in turkeys in Egypt. Following clinical examination of 500 birds, a portion of the hexon gene was amplified from four out of 50 samples from diseased birds (8%), and one amplicon that produced a strong band was selected for sequencing. Molecular and phylogenetic analysis revealed that the virus in that sample belonged to serotype FAdV-8b. Histopathological and immunohistochemical examinations of prepared tissue sections were performed to confirm the pathological findings. Diseased birds exhibited ruffled feathers, low body weight, a crouching posture, and diarrhea. Gross examination revealed petechial hemorrhage on the spleen, swollen pale liver, and congested intestine. Microscopic examination revealed the presence of eosinophilic and basophilic intranuclear inclusion bodies, nuclear pyknosis, and apoptotic bodies in the liver, congestion, hemorrhage, and fibrosis in the lungs, and desquamation of enterocytes. The presence of viral antigens in the liver, lungs, and intestine was confirmed by immunohistochemistry. To our knowledge, this is the first report of the characterization of an outbreak of inclusion body hepatitis in turkeys (hybrid converter breeds) due to FAdV-8b in Egypt. This finding raises an epidemiological alarm, necessitating further studies, including full-genome sequencing, to trace the virus's origin and genetic diversity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: In this study, we investigated the prevalence of respiratory viruses in four Hybrid Converter Turkey (Meleagris gallopavo) farms in Egypt. The infected birds displayed severe respiratory signs, accompanied by high mortality rates, suggesting viral infections. Five representative samples from each farm were pooled and tested for H5 & H9 subtypes of avian influenza viruses (AIVs), Avian Orthoavulavirus-1 (AOAV-1), and turkey rhinotracheitis (TRT) using real-time RT-PCR and conventional RT-PCR. Representative tissue samples from positive cases were subjected to histopathology and immunohistochemistry (IHC)., Results: The PCR techniques confirmed the presence of AOAV-1 and H5 AIV genes, while none of the tested samples were positive for H9 or TRT. Microscopic examination of tissue samples revealed congestion and hemorrhage in the lungs, liver, and intestines with leukocytic infiltration. IHC revealed viral antigens in the lungs, liver, and intestines. Phylogenetic analysis revealed that H5 HA belonged to 2.3.4.4b H5 sublineage and AOAV-1 belonged to VII 1.1 genotype., Conclusions: The study highlights the need for proper monitoring of hybrid converter breeds for viral diseases, and the importance of vaccination programs to prevent unnecessary losses. To our knowledge, this is the first study that reports the isolation of AOAV-1 and H5Nx viruses from Hybrid Converter Turkeys in Egypt., (© 2024. The Author(s).)

Avian reoviruses continue to cause disease in turkeys with varied pathogenicity and tissue tropism. Turkey enteric reovirus has been identified as a causative agent of enteritis or inapparent infections in turkeys. The new emerging variants of turkey reovirus, tentatively named turkey arthritis reovirus (TARV) and turkey hepatitis reovirus (THRV), are linked to tenosynovitis/arthritis and hepatitis, respectively. Turkey arthritis and hepatitis reoviruses are causing significant economic losses to the turkey industry. These infections can lead to poor weight gain, uneven growth, poor feed conversion, increased morbidity and mortality and reduced marketability of commercial turkeys. To combat these issues, detecting and classifying the types of reoviruses in turkey populations is essential. This research aims to employ clustering methods, specifically K-means and Hierarchical clustering, to differentiate three types of turkey reoviruses and identify novel emerging variants. Additionally, it focuses on classifying variants of turkey reoviruses by leveraging various machine learning algorithms such as Support Vector Machines, Naive Bayes, Random Forest, Decision Tree, and deep learning algorithms, including convolutional neural networks (CNNs). The experiments use real turkey reovirus sequence data, allowing for robust analysis and evaluation of the proposed methods. The results indicate that machine learning methods achieve an average accuracy of 92%, F1-Macro of 93% and F1-Weighted of 92% scores in classifying reovirus types. In contrast, the CNN model demonstrates an average accuracy of 85%, F1-Macro of 71% and F1-Weighted of 84% scores in the same classification task. The superior performance of the machine learning classifiers provides valuable insights into reovirus evolution and mutation, aiding in detecting emerging variants of pathogenic TARVs and THRVs., (© The Author(s) 2024. Published by Oxford University Press.)

Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Wild birds are the reservoir for all avian influenza viruses (AIV). In poultry, the transition from low pathogenic (LP) AIV of H5 and H7 subtypes to highly pathogenic (HP) AIV is accompanied mainly by changing the hemagglutinin (HA) monobasic cleavage site (CS) to a polybasic motif (pCS). Galliformes, including turkeys and chickens, succumb with high morbidity and mortality to HPAIV infections, although turkeys appear more vulnerable than chickens. Surprisingly, the genetic determinants for virulence and pathogenesis of HPAIV in turkeys are largely unknown. Here, we determined the genetic markers for virulence and transmission of HPAIV H7N1 in turkeys, and we explored the host responses in this species compared to those of chickens. We found that recombinant LPAIV H7N1 carrying pCS was avirulent in chickens but exhibited high virulence in turkeys, indicating that virulence determinants vary in these two galliform species. A transcriptome analysis indicated that turkeys mount a different host response than do chickens, particularly from genes involved in RNA metabolism and the immune response. Furthermore, we found that the HA glycosylation at residue 123, acquired by LP viruses shortly after transmission from wild birds and preceding the transition from LP to HP, had a role in virus fitness and virulence in chickens, though it was not a prerequisite for high virulence in turkeys. Together, these findings indicate variable virulence determinants and host responses in two closely related galliformes, turkeys and chickens, after infection with HPAIV H7N1. These results could explain the higher vulnerability to HPAIV of turkeys compared to chickens. IMPORTANCE Infection with HPAIV in chickens and turkeys, two closely related galliform species, results in severe disease and death. Although the presence of a polybasic cleavage site (pCS) in the hemagglutinin of AIV is a major virulence determinant for the transition of LPAIV to HPAIV, there are knowledge gaps on the genetic determinants (including pCS) and the host responses in turkeys compared to chickens. Here, we found that the pCS alone was sufficient for the transformation of a LP H7N1 into a HPAIV in turkeys but not in chickens. We also noticed that turkeys exhibited a different host response to an HPAIV infection, namely, a widespread downregulation of host gene expression associated with protein synthesis and the immune response. These results are important for a better understanding of the evolution of HPAIV from LPAIV and of the different outcomes and the pathomechanisms of HPAIV infections in chickens and turkeys.

Background: Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens., Results: This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64-100% and 99.79-100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies., Conclusions: These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping., (© 2022. The Author(s).)

Understanding the amount of virus shed at the flock level by birds infected with low pathogenicity avian influenza virus (LPAIV) over time can help inform the type and timing of activities performed in response to a confirmed LPAIV-positive premises. To this end, we developed a mathematical model which allows us to estimate viral shedding by 10,000 turkey toms raised in commercial turkey production in the United States, and infected by H7 LPAIV strains. We simulated the amount of virus shed orally and from the cloaca over time, as well as the amount of virus in manure. In addition, we simulated the threshold cycle value (Ct) of pooled oropharyngeal swabs from birds in the infected flock tested by real-time reverse transcription polymerase chain reaction. The simulation model predicted that little to no shedding would occur once the highest threshold of seroconversion was reached. Substantial amounts of virus in manure (median 1.5×108 and 5.8×109; 50% egg infectious dose) were predicted at the peak. Lastly, the model results suggested that higher Ct values, indicating less viral shedding, are more likely to be observed later in the infection process as the flock approaches recovery.

Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. This study was designed to examine the effect of virulent ND infection in turkey's tissues and the tissue tropism of the virus. During the previous study period, poults were inoculated at 32 days of age with 10 5 EID50 virulent Newcastle disease virus. Three poults on days 0, 1, 2, 3, 4, 6, 7, and 14 postinoculations (PI) were selected from each group. They were euthanized by intravenous sodium pentobarbital injection. After macroscopic observation, to histopathological and immunohistochemical studies, the spleen, bursa, cecal tonsils, intestine, proventriculus, lung, kidney, and brain were sampled. Clinically, the infected turkeys exhibited loss of appetite, severe depression, down on hock joint, white to greenish (sometimes bloody) diarrhea, nervous signs, and mild respiratory problems. Out of 45 birds inoculated, 9 (20%) died. Histopathological effects in lymphoid tissues included necrosis and penetration of mononuclear cells on day 4 PI, and subsequent follicular lymphoid depletion on days 6 and 8 PI was observed. Based on the immunohistochemical test, on day 3 in cecal tonsils and spleen, and on day 8 PI, all of them were positive for virus antigen. In conclusion, the NDV circulating in Iranian chicken flocks has the potential to cause severe illness in commercial turkeys., (© 2021. Sociedade Brasileira de Microbiologia.)

An outbreak caused by H7N3 low pathogenicity avian influenza virus (LPAIV) occurred in commercial turkey farms in the states of North Carolina (NC) and South Carolina (SC), United States in March of 2020. Subsequently, H7N3 high pathogenicity avian influenza virus (HPAIV) was detected on a turkey farm in SC. The infectivity, transmissibility, and pathogenicity of the H7N3 HPAIV and two LPAIV isolates, including one with a deletion in the neuraminidase (NA) protein stalk, were studied in turkeys and chickens. High infectivity [<2 log 10 50% bird infectious dose (BID 50 )] and transmission to birds exposed by direct contact were observed with the HPAIV in turkeys. In contrast, the HPAIV dose to infect chickens was higher than for turkeys (3.7 log 10 BID 50 ), and no transmission was observed. Similarly, higher infectivity (<2-2.5 log 10 BID 50 ) and transmissibility were observed with the H7N3 LPAIVs in turkeys compared to chickens, which required higher virus doses to become infected (5.4-5.7 log 10 BID 50 ). The LPAIV with the NA stalk deletion was more infectious in turkeys but did not have enhanced infectivity in chickens. These results show clear differences in the pathobiology of AIVs in turkeys and chickens and corroborate the high susceptibility of turkeys to both LPAIV and HPAIV infections.

Turkey coronavirus (TCoV) can cause a highly contagious enteric disease in turkeys with severe economic losses in the global turkey industry. To date, no commercial vaccines are available for control of the disease. In the present study, we isolated a field strain (NC1743) of TCoV and evaluated its pathogenicity in specific-pathogen-free (SPF) turkey poults to establish a TCoV disease model. The results showed that the TCoV NC1743 isolate was pathogenic to turkey poults with a minimal infectious dose at 10 6 EID 50 /bird. About 50 % of one-day-old SPF turkeys infected with the virus's minimal infectious dose exhibited typical enteric disease signs and lesions from 6 days post-infection (dpi) to the end of the experiment (21 dpi). In contrast, fewer than 20 % of older turkeys (1- or 2-week-old) infected with the same amount of TCoV displayed enteric disease signs, which disappeared after 15-18 dpi. Although all infected turkeys, regardless of age, shed TCoV, the older turkeys shed less virus than the younger birds, and 50 % of the 2-week-old birds even cleared the virus at 21 dpi. Furthermore, the viral infection caused day-old turkeys more body-weight-gain reduction than older birds. The overall data demonstrated that the TCoV NC1743 isolate is a highly pathogenic strain and younger turkeys are more susceptible to TCoV infection than older birds. Thus, one-day-old turkeys infected with the minimal infectious dose of TCoV NC1743 could be used as a TCoV disease model to study the disease pathogenesis, and the TCoV NC1743 strain could be used as a challenge virus to evaluate a vaccine protective efficacy., (Published by Elsevier B.V.)

Marek's disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus ( Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus ( Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses ; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera ( Mardivirus vs. Iltovirus ) and not the host ( Gallid vs. Meleagrid ).

Highly-pathogenic avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) incurred into Europe in late 2017 and was predominantly detected in wild birds, with very few terrestrial poultry cases. Pekin ducks directly-infected with a UK virus (H5N6-2017) were donors of infection to investigate contact transmission to three recipient species: Ducks, chickens and turkeys. H5N6-2017 transmission to ducks was 100% efficient, but transmission to in-contact galliforme species was infrequent and unpredictable, thereby reflecting the European 2017-2018 H5N6 epidemiology. Although only two of 28 (7%) infected ducks died, the six turkeys and one chicken which became infected all died and displayed systemic H5N6-2017 dissemination, while pathogenesis in ducks was generally milder. Analysis of H5N6-2017 progeny in the contacts revealed no emergent polymorphisms in an infected duck, but the galliforme species included changes in the polymerase (PB2 A199T, PA D347A), matrix (M1 T218A) and neuraminidase genes (T88I). H5N6-2017 environmental contamination was associated with duck shedding., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Infectious diseases are a major obstacle to profitable poultry production in Nigeria due to the mortality and severe economic losses they cause. In particular, they are a potent threat to attainment of the food security goals of government and national self-sufficiency in food production. Thus, there is a need for continuous monitoring of the nation's poultry population for these diseases. As part of an ongoing investigation of enteric viruses associated with poor performance or hatchery diseases in commercial poultry in southwestern Nigeria, intestinal contents from 97 condemned or runted day-old commercial turkey poults were examined for turkey astroviruses, infectious bronchitis virus, chicken astrovirus (CAstV), avian nephritis virus, avian rotavirus, avian reovirus, fowl adenovirus, and chicken parvovirus by virus isolation, electron microscopy (EM), polymerase chain reaction (PCR), and reverse transcription PCR. The samples were collected from five commercial hatcheries and five farms located in southwestern Nigeria. While all samples tested negative for other viruses, CAstV was detected in the majority (83.5%) of the birds, although some pleomorphic virus-like particles with surface projections that appeared fringed or fimbriated were observed in five of the cell culture samples by EM. Phylogenetic analysis revealed these CAstV strains belonged to the Bi clade. These findings not only implicate CAstV as the major cause of hatchery condemnations in commercial turkeys in southwestern Nigeria but also highlight the need for experimental studies to further establish its role in this disease condition.

Outbreaks of avian influenza virus (AIV) infection included the spread of highly pathogenic AIV in commercial poultry and backyard flocks in the spring of 2015. This resulted in estimated losses of more than $8.5 million from federal government expenditures, $1.6 billion from direct losses to produces arising from destroyed turkey and chicken egg production, and economy-wide indirect costs of $3.3 billion from impacts on retailers and the food service industries. Additionally, these outbreaks resulted in the death or depopulation of nearly 50 million domestic birds. Domesticated male ferrets (Mustela putorius furo) were trained to display a specific conditioned behavior (i.e. active scratch alert) in response to feces from AIV-infected mallards in comparison to feces from healthy ducks. In order to establish that ferrets were identifying samples based on odors associated with infection, additional experiments controlled for potentially confounding effects, such as: individual duck identity, housing and feed, inoculation concentration, and day of sample collection (post-infection). A final experiment revealed that trained ferrets could detect AIV infection status even in the presence of samples from mallards inoculated with Newcastle disease virus or infectious laryngotracheitis virus. These results indicate that mammalian biodetectors are capable of discriminating the specific odors emitted from the feces of non-infected versus AIV infected mallards, suggesting that the health status of waterfowl can be evaluated non-invasively for AIV infection via monitoring of volatile fecal metabolites. Furthermore, in situ monitoring using trained biodetectors may be an effective tool for assessing population health., Competing Interests: The authors have declared that no competing interests exist.

H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.

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 6 EID 50 each of H5N1, H5N2 or H5N8 HPAI virus at 28 days of age. The birds were monitored for clinical protection and virus shedding during a 10-day postchallenge period. Clinical protection levels were 90%, 90% and 80% following challenge with the H5N1, H5N2 and H5N8 field isolates, respectively. Challenge virus shedding was significantly reduced in vaccinated groups, with up to 40%, 30% and 20% of non-shedders, and 3.8, 3.3 and 2.8 log 10 reduction in the amount of excreted virus following challenge with H5N1, H5N2 and H5N8 viruses, respectively. Analyses of the amino acid sequences of the HA proteins of challenge viruses and serological relatedness with the vaccine insert revealed significant antigenic divergences between the vaccine and the challenge viruses. These results provide further evidence of the potential of HVT-based recombinant H5 vaccine to provide cross-protection against antigenically drifted HPAI H5Nx viruses with strong control on virus shedding.

ABSTRACT Low pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Hemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes an acute clinical disease characterized by hemorrhagic gastroenteritis in 4-week-old turkeys and older. Recurrent incidence of secondary infections (e.g., systemic bacterial infections, cellulitis, and elevated mortality), may be associated with the presence of field-type HEV in Canadian turkey farms. We speculate that field-type HEV and vaccine/vaccine-like strains can be differentiated through analysis of the viral genomes, hexon genes, and the specific virulence factors (e.g., ORF1, E3, and fib knob domain). Nine out of sixteen spleens obtained from cases suspected of immunosuppression by HEV were analyzed. The limited data obtained showed that: (1) field-type HEV circulates in many non-vaccinated western Canadian flocks; (2) field-type HEV circulates in vaccinated flocks with increased recurrent bacterial infections; and (3) the existence of novel point mutations in hexon, ORF1, E3, and specially fib knob domains. This is the first publication showing the circulation of wild-type HEV in HEV-vaccinated flocks in Western Canada, and the usefulness of a novel procedure that allows whole genome sequencing of HEV directly from spleens, without passaging in cell culture or passaging in vivo. Further studies focusing more samples are required to confirm our observations and investigate possible vaccination failure.

The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-Δ vNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-Δ vNr-13 showed similar growth kinetics; however, at early time points, HVT-Δ vNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-Δ vNr-13 -infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells. IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek's disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication., (Copyright © 2020 Reddy et al.)

Low-pathogenicity avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate to highly pathogenic (HPAI) virus variants, causing high mortality in poultry. The highly pathogenic phenotype is caused by mutation of the hemagglutinin (HA) cleavage site, but additional mutations may play a role. Evidence from the field for the switch to high pathogenicity remains scarce. This study provides direct evidence for LPAI-to-HPAI virus mutation during H7N3 infection of a turkey farm in the Netherlands. No severe clinical symptoms were reported at the farm, but deep sequencing of isolates from the infected turkeys revealed a minority of HPAI virus sequences (0.06%) in the virus population. The HPAI virus contained a 12-nucleotide insertion in the HA cleavage site that was likely introduced by a single event as no intermediates with shorter inserts were identified. This suggests nonhomologous recombination as the mechanism of insertion. Analysis of different organs of the infected turkeys showed the largest amount of HPAI virus in the lung (4.4%). The HPAI virus was rapidly selected in experimentally infected chickens after both intravenous and intranasal/intratracheal inoculation with a mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified additional mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus population. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence and selection of HPAI viruses. IMPORTANCE Low-pathogenicity avian influenza (LPAI) viruses circulate in wild birds and can be transmitted to poultry. LPAI viruses can mutate to become highly pathogenic avian influenza (HPAI) viruses causing severe disease and death in poultry. Little is known about this switch to high pathogenicity. We isolated an LPAI H7N3 virus from an infected turkey farm and showed that this contains small amounts of HPAI virus. The HPAI virus rapidly outcompeted the LPAI virus in chickens that were experimentally infected with this mixture of viruses. We analyzed the genome sequences of the LPAI and HPAI viruses and identified several changes that may be important for a virus to become highly pathogenic. This knowledge may be used for timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field., (Copyright © 2020 American Society for Microbiology.)

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Itsevolution and adaptation may be affected by multiple host and ecological factors, which are stillpoorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model toinvestigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with theclinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a doseof 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission andseroconversion were evaluated, and swabs collected at selected time-points were characterized indeep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for theexamined bird species, but differences in shedding patterns, seroconversion and clinical outcomewere noted. The highest heterogeneity of the virus population as measured by the number of singlenucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail,followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus duringreplication in ducks, which can be explained by its poor adaptation and stronger selection pressurein waterfowl. The high within-host virus diversity in quail with high level of respiratory sheddingand asymptomatic course of infection may contribute to our understanding of the role of quail asan intermediate host for adaptation of AIV to other species of poultry. In contrast, low viruscomplexity was observed in cloacal swabs, mainly from turkeys, showing that the within-hostdiversity may vary between different replication sites. Consequences of these observations on thevirus evolution and adaptation require further investigation.

Using viral metagenomics, the complete genome sequence of an infectious bronchitis virus (IBV) strain (named ahysx-1) from a fecal sample from a healthy chicken in Anhui province, China, was determined. The genome sequence of ahysx-1 was found to be very similar to that of IBV strain ck/CH/LLN/131040 (KX252787), except for the spike gene region, which is similar to that of a turkey coronavirus strain (EU022526), suggesting that ahysx-1 is a recombinant. Recombination analysis and phylogenetic analysis based on the genomic sequences of ahysx-1 and other related strains confirmed that ahysx-1 appears to be a recombinant resulting from a recombination event that occurred between a chicken coronavirus and a turkey coronavirus. Further studies need to be performed to determine whether this recombinant IBV strain is pathogenic and whether it is transmitted between chickens and turkeys.

Influenza A viruses of H5 and H7 subtype in poultry can circulate subclinically and subsequently mutate from low to high pathogenicity with potentially devastating economic and welfare consequences. European Union Member States undertake surveillance of commercial and backyard poultry for early detection and control of subclinical H5 and H7 influenza A infection. This surveillance has moved towards a risk-based sampling approach in recent years; however, quantitative measures of relative risk associated with risk factors utilized in this approach are necessary for optimization. This study describes serosurveillance for H5 and H7 influenza A in domestic and commercial poultry undertaken in the European Union from 2004 to 2010, where a random sampling and thus representative approach to serosurveillance was undertaken. Using these representative data, this study measured relative risk of seropositivity across poultry categories and spatially across the EU. Data were analysed using multivariable logistic regression. Domestic waterfowl, game birds, fattening turkeys, ratites, backyard poultry and the 'other' poultry category holdings had relatively increased probability of H5 and/or H7 influenza A seropositivity, compared to laying-hen holdings. Amongst laying-hen holdings, free-range rearing was associated with increased probability of H7 seropositivity. Spatial analyses detected 'hotspots' for H5 influenza A seropositivity in western France and England, and H7 influenza A seropositivity in Italy and Belgium, which may be explained by the demographics and distribution of poultry categories. Findings suggest certain poultry category holdings are at increased risk of subclinical H5 and/or H7 influenza A circulation, and free-range rearing increases the likelihood of exposure to H7 influenza A. These findings may be used in further refining risk-based surveillance strategies and prioritizing management strategies in influenza A outbreaks., (© 2019 Blackwell Verlag GmbH.)

H5N8 highly-pathogenic avian influenza viruses (HPAIVs, clade 2.3.4.4) have spread globally via migratory waterfowl. Pekin ducks infected with a UK virus (H5N8-2014) served as the donors of infection in three separate cohousing experiments to attempt onward transmission chains to sequentially introduced groups of contact ducks, chickens and turkeys. Efficient transmission occurred among ducks and turkeys up to the third contact stage, with all (100%) birds becoming infected. Introduction of an additional fourth contact group of ducks to the turkey transmission chain demonstrated retention of H5N8-2014's waterfowl-competent adaptation. However, onward transmission ceased in chickens at the second contact stage where only 13% became infected. Analysis of viral progeny at this contact stage revealed no emergent polymorphisms in the intra-species (duck) transmission chain, but both terrestrial species included changes in the polymerase and accessory genes. Typical HPAIV pathogenesis and mortality occurred in infected chickens and turkeys, contrasting with 5% mortality among ducks., Competing Interests: Declaration of competing interest None of the authors have any competing interests with respect to this study., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Background: Haemorrhagic enteritis (HE) of turkeys was first described in 1937 in the USA, while in Poland it was first diagnosed in 1987. Polish haemorrhagic enteritis virus (HEV) isolates are usually low pathogenic and trigger a subclinical disease. Unfortunately, even the low- pathogenic HEV strains cause severe immunosuppression leading to secondary bacterial infections and huge economic losses. The objective of this study was to evaluate if the influence of Met on HEV infected turkeys immune response can be differentiated by both its level and source. Met is one of the amino acids that not only play a nutritional role but also participate in and regulate key metabolic pathways and immune response. In our study, the birds were assigned to 4 dietary treatments which differed in Met levels (0.55 and 0.78% in weeks 1-4 of age and 0.45 and 0.65% in weeks 5-8 of age, respectively) and sources (DL-methionine (DLM) or DL-methionine hydroxy analogue (MHA))., Results: The HEV added the percentage of CD4 + cells and decreased the percentage of IgM + cells in the blood, spleen and caecal tonsils (CTs) of turkeys. In addition, it increased the percentage of CD4 + CD25 + cells in blood, and interleukin-6 (IL-6) level in plasma. The higher dose of Met led to a significant decrease in the percentages of CD4 + , CD8 + and CD4 + IL-6 + cell subpopulations in the blood of HEV-infected and uninfected turkeys and to an increase in the percentage of IgM + B cells in CTs. Turkeys administered feeds with an increased Met content displayed a decrease in plasma IL-6 levels and an increase in plasma IgA levels., Conclusions: The results of this study indicate that HEV infection impairs the immune function in turkeys. Met content in the feed has a moderate effect on the immune response in HEV-infected turkeys. The source of this amino acid appears not be as important as its dose, because value of the analysed parameters did not differ significantly between turkeys receiving feeds with DLM or MHA. In the uninfected turkeys, the higher by 40% (than recommended by NRC) level of Met in the feeds had a positive effect on humoral immunity parameters.

The process of avian influenza virus (AIV) evolution in a new host was investigated in the experiment in which ten serial passages of a turkey-derived H9N2 AIV were carried out in specific pathogen free chickens (3 birds/group) inoculated by oculonasal route. Oropharyngeal swabs collected 3 days post infection were used for inoculation of birds in the next passage and subjected to analysis using deep sequencing. In total, eight mutations in the consensus sequence were found in the viral pool derived from the 10th passage: four mutations (2 in PB1 and 2 in HA) were present in the inoculum as minority variants while the other four (2 in NP, 1 in PA and 1 in HA) emerged during the passages in chickens. The detected fluctuations in the genetic heterogeneity of viral pools from consecutive passages were most likely attributed to the selective bottleneck. The genes known for bearing molecular determinants of the AIV host specificity (HA, PB2, PB1, PA) contributed most to the overall virus diversity. In some cases, a fast selection of the novel variant was noticed. For example, the amino-acid substitution N337K in the haemagglutinin (HA) cleavage site region detected in the 6th passage as low frequency variant had undergone rapid selection and became predominant in the 7th passage. Interestingly, detection of identical mutation in the field H9N2 isolates 1-year apart suggests that this substitution might provide the virus with a selective advantage. However, the role of specific mutations and their influence on the virus adaptation or fitness are mostly unknown and require further investigations., (Copyright © 2019. Published by Elsevier B.V.)

The genotype of chickens is assumed to be associated with variable immune responses. In this study a modern, moderate performing dual-purpose chicken line (DT) was compared with a high-performing layer-type (LT) as well as a broiler-type (BT) chicken line. One group of each genotype was vaccinated in ovo with a recombinant herpesvirus of turkeys expressing the virus protein VP2 of the infectious bursal disease virus (HVT-IBD) while one group of each genotype was left HVT-IBD unvaccinated (control group). Genotype associated differences in innate and adapted immune responses between the groups were determined over five weeks post hatch. HVT-IBD vaccination significantly enhanced humoral immune responses against subsequently applied live vaccines compared to non-HVT-IBD vaccinated groups at some of the investigated time points (P < 0.05). In addition HVT-IBD vaccination had depending on the genotype a significant impact on splenic macrophage as well as bursal CD4 + T-cell numbers (P < 0.05). On the other hand, the detectable genotype influence on Interferon (IFN) γ and nitric oxide (NO) release of ex vivo stimulated spleen cells was independent of HVT-IBD vaccination. The results of our study suggest considering a genotype specific vaccination regime in the field., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.

Reticuloendotheliosis virus (REV) is an immunosuppressive and sometimes oncogenic avian retrovirus that establishes lifelong infection in a wide range of avian species. REV-infected wild birds roaming near at-risk captive flocks, such as is the case for the highly endangered Attwater's Prairie Chicken (APC; Tympanuchus cupido attwateri ), could act as a reservoir for viral transmission. In wild birds, prevalence rates of REV are low and appearance of associated disease is uncommon. During 2016-17, nearly half of all captive adult APC mortality at Fossil Rim Wildlife Center captive breeding facility in Glen Rose, Texas, US was attributed to REV infection. The unusually high REV prevalence rate prompted us to survey for this virus in wild galliforms throughout the region. From 2016-17, 393 blood samples collected from two subspecies of Wild Turkeys ( Meleagris gallopavo ) were tested for REV proviral DNA through amplification of the viral 3' long terminal repeat and segments of the viral pol gene. In REV-affected counties, 5% (5/98) of native Rio Grande Wild Turkeys ( Meleagris gallopavo intermedia ) were identified as REV-positive. In addition, we detected REV in one of 62 Eastern Wild Turkeys ( Meleagris gallopavo silvestris ) that had been imported during conservation efforts. To better determine protective measures, continued surveillance, including collection and genetic analysis of REV-infected samples, is necessary to identify sources of REV outbreaks in captive APC flocks.

Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Highly pathogenic avian influenza (HPAI) is a viral disease with devastating consequences to the poultry industry as it results in high morbidity, mortality and international trade restrictions. In the present study, we characterized age-related differences in terms of pathology in commercial white broad breasted turkeys inoculated with A/turkey/Minnesota/12582/2015 (H5N2) HPAIV clade 2.3.4.4A, a virus from the largest HPAI poultry outbreak that affected the Unites States in 2014-2015. Turkeys infected at 6-weeks of age showed inapparent to little clinical signs with rapid disease progression, reaching 100% mortality at 3 days post infection (dpi). In contrast, turkeys infected at 16-weeks of age developed ataxia and lethargy and reached 100% mortality by 5 dpi. Infection in the 6-weeks old turkeys resulted in peracute lesions consistent of extensive hemorrhages, edema and necrosis, but inflammation was not prominent. In the 16-weeks old turkeys, necrosis and hemorrhages in tissues were accompanied by a more prominent subacute inflammatory infiltrate. Both age groups showed presence of avian influenza virus (AIV) nucleoprotein (NP) in multiple cell types including neurons, glial cells, ependymal cells, respiratory epithelial cells, air capillary epithelium and pulmonary macrophages, cardiac myocytes, smooth muscle fibers, pancreatic acini and ductal cells. Cells of the vascular walls stained strongly positive for viral antigens, but no positivity was found in the endothelial cells of any organs. These findings indicate that age is a determinant factor in the progression of the disease and delay of mortality during infection with the H5N2 clade 2.3.4.4A HPAI virus in naïve white broad breasted turkeys., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Low pathogenic avian influenza (LPAI) viruses can silently circulate in poultry and wild aquatic birds and potentially mutate into highly pathogenic avian influenza (HPAI) viruses. In the U.S., recent emergence and spread of H7N8 and H7N9 HPAI viruses not only caused devastating losses to domestic poultry but also underscored the capability of LPAI viruses to mutate into HPAI viruses. Therefore, in this study, we evaluated pathogenicity and transmissibility of H7N8 and H7N9 LPAI viruses (the progenitors of HPAI viruses) in chickens and turkeys. We also included H7N2 isolated from an outbreak of LPAI in commercial chickens. H7 viruses replicated more efficiently in the respiratory tract than in the gastrointestinal tract, suggesting that their replication is restricted to the upper respiratory tract. Specifically, H7N2 replicated most efficiently in two-week-old chickens and turkeys. In contrast, H7N8 replicated least efficiently in those birds. Further, replication of H7N2 and H7N9 was restricted in the upper respiratory tract of four-week-old specific-pathogen-free (SPF) and broiler chickens. Despite their restricted replication, the two viruses efficiently transmitted from infected to naïve birds by direct contact, leading to seroconversion of contacted chickens. Our findings suggest the importance of continuous monitoring and surveillance of LPAI viruses in the fields.

The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The successful reintroduction of Wild Turkeys ( Meleagris gallopavo) to Ontario, Canada, has led to established populations in southern portions of the province and currently allows for biannual hunting seasons. These populations geographically overlap Domestic Turkey farms, an important sector of the provincial agri-food industry. Potential pathogen transmission between Wild Turkeys and Domestic Turkeys ( Meleagris gallopavo) is a concern, because they are susceptible to infection with many of the same pathogens and have direct and indirect contact in outdoor or open farm settings and contaminated environmental substrates. However, data concerning potential poultry pathogens in Wild Turkeys in Canada are scarce. Thus, we assessed the prevalence and geographic distribution of geographically relevant viruses in Ontario Wild Turkeys. Oropharyngeal and cloacal swabs were tested for avian influenza viruses (AIV) by real-time reverse transcriptase (RT)-PCR ( n=207), pooled tissues for lymphoproliferative disease virus (LPDV; n=183) and reticuloendotheliosis virus ( n=119) by PCR, and gross skin lesions by real-time RT-PCR for avian poxvirus ( n=8). We sequenced a fragment of the gag polyprotein (p31) gene of LPDV on a subset ( n=10) of LPDV-positive samples for phylogenetic analysis and tested additional upland game bird species ( n=39) and domestic fowl for LPDV ( n=17). To the best of our knowledge, we document the first detection of LPDV in Wild Turkeys in Canada, with a prevalence of 65% (119/183). Phylogenetic analysis revealed that LPDV sequences from Ontario were genetically similar to other North American strains and did not group into separate clades. Reticuloendotheliosis virus was detected in 4% (5/119) of LPDV-positive Wild Turkeys. Grossly evident skin lesions from five Wild Turkeys tested positive for poxvirus, and all turkeys tested negative for AIV. This study provides evidence of LPDV circulation in Canada and provides a baseline for comparison with future Wild Turkey pathogen surveillance and monitoring in Ontario and elsewhere.

In January 2016, highly pathogenic avian influenza (HPAI) A(H7N8) virus and low pathogenicity avian influenza (LPAI) A(H7N8) virus were detected in commercial turkey flocks in Dubois County, Indiana. The Indiana State Department of Health (ISDH) and the Dubois County Health Department (DCHD) coordinated the public health response to this outbreak, which was the first detection of HPAI A(H7N8) in any species (1). This response was the first to fully implement unpublished public health monitoring procedures for HPAI responders that were developed by the U.S. Department of Agriculture (USDA) and CDC in 2015 (Sonja Olsen, CDC, personal communication, October 2017). No cases of zoonotic avian influenza infection in humans were detected during the outbreak., Competing Interests: All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Respiratory diseases continue to have a major impact on poultry health, welfare and productivity. However, little information is available on their current status in UK poultry flocks. We investigated the presence of four economically important respiratory pathogens in healthy or problematic flocks; infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms). Samples from 131 UK poultry flocks were received during the 12 month study period. Oropharyngeal (OP) swabs were taken from eight birds per flock and accompanied with flock health information. The study included 118 chicken, 6 pheasant and 5 turkey flocks, and 1 quail and 1 partridge flock. Chicken flocks were of layers (n = 98), broilers (n = 15), breeders (n = 3) and undisclosed (n = 2). Flock ages ranged from 3 to 72 weeks old, and the average flock size was 17,633 birds. PCR detected 65 (49.6%), 59 (45%) and 8 (6.1%) flocks as positive for IBV, Mg/Ms and aMPV respectively. Analysis of the mgc2 gene of the Mg isolates revealed high similarities to Mg TS-11 and Mg 6/85. Further gene analysis found that the TS-11-like isolates were unrelated to the TS-11 vaccine. Multi-locus sequence typing (MLST) analysis identified the majority of positive Ms as ST21, along with ST2 (MS-H-like), ST6 and ST43. IBV S1 gene sequencing identified strains as 793B (66.7%), Arkansas (23.8%) and Massachusetts (9.5%). All aMPV positive samples belonged to subtype B. Findings indicate that over half of the flocks sampled were positive for at least one of the four vaccine or field strains of mycoplasmas or viruses., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

A 12-month pilot project for notifiable avian disease (NAD) exclusion testing in chicken and turkey flocks in Great Britain (GB) offered, in partnership with industry, opportunities to carry out differential diagnosis in flocks where NAD was not suspected, and to identify undetected or undiagnosed infections. In May 2014, clinical samples received from a broiler breeder chicken premises that had been experiencing health and production problems for approximately one week tested positive by avian influenza (AI) real-time reverse transcription polymerase chain reaction (RRT-PCR). Following immediate escalation to an official, statutory investigation to rule out the presence of notifiable AI virus (AIV; H5 or H7 subtypes), a non-notifiable H4N6 low pathogenicity (LP) AIV was detected through virus isolation in embryonated specific pathogen free (SPF) fowls' eggs, neuraminidase inhibition test, cleavage site sequencing and AIV subtype H4-specific serology. Premises movement restrictions were lifted, and no further disease control measures were implemented as per the United Kingdom (UK) legislation. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the virus revealed closest relationships to viruses from Mallard ducks in Sweden during 2007 and 2009. In June 2014, clinical suspicion of NAD was reported in a flock of free-range laying chickens elsewhere in GB, due to increasing daily mortality and reduced egg production over a five-day period. An H4N6 LPAIV with an intravenous pathogenicity index of 0.50 was isolated. This virus was genetically highly similar, but not identical, to the virus detected during May 2014. Full viral genome analyses showed characteristics of a strain that had not recently transferred from wild birds, implying spread within the poultry sector had occurred. A stalk deletion in the neuraminidase gene sequence indicated an adaptation of the virus to poultry. Furthermore, there was unexpected evidence of systemic spread of the virus on post-mortem. No other cases were reported. Infection with LPAIVs often result in variable clinical presentation in poultry, making detection of disease more difficult., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Samples from 45 chickens, two turkeys, one peacock and one quail with symptoms of fowlpox were collected in Mozambique between November 2016 and January 2018. Phylogenetic analysis revealed that the samples contained avipoxviruses belonging to both clade A1 and clade A2. In addition, all of the Clade A1 viruses were positive by PCR for the integration of reticuloendotheliosis virus, while the clade A2 avipoxvirus samples were negative. This study confirms the circulation of clade A1 avipoxviruses in Mozambique in addition to identifying clade A2 for the first time in the country.

The China-origin H7N9 low pathogenicity avian influenza virus (LPAIV) emerged as a zoonotic threat in 2013 where it continues to circulate in live poultry markets. Absence of overt clinical signs in poultry is a typical LPAIV infection outcome, and has contributed to its insidious maintenance in China. This study is the first description of H7N9 LPAIV (A/Anhui/1/13) infection in turkeys, with efficient transmission to two additional rounds of introduced contact turkeys which all became infected during cohousing. Surprisingly, mortality was observed in six of eight (75%) second-round contact turkeys which is unusual for LPAIV infection, with unexpected systemic dissemination to many organs beyond the respiratory and enteric tracts, but interestingly no accompanying mutation to highly pathogenic AIV. The intravenous pathogenicity index score for a turkey-derived isolate (0.39) affirmed the LPAIV phenotype. However, the amino acid change L235Q in the haemagglutinin gene occurred in directly-infected turkeys and transmitted to the contacts, including those that died and the two which resolved infection to survive to the end of the study. This polymorphism was indicative of a reversion from mammalian to avian adaptation for the H7N9 virus. This study underlined a new risk to poultry in the event of H7N9 spread beyond China.

Avian influenza virus (AIV) H9N2 subtype is endemic in Iran and causes substantial economic loss to the growing poultry industry within the country. In this study, a cross-sectional analysis was carried out to determine the sero-prevalence of H9N2 in several commercial farms between the years 2014 and 2015. The comparison of the mean of serum titers and the ratio of sero-positive birds between all units were analyzed using one-way ANOVA test. In 2014, a total of 77 farms (58 turkey farms, 14 quail farms, and 5 partridge farms) and 894 birds (682 turkeys, 154 quails, and 58 partridges) were sampled while in 2015, a total of 69 farms (54 turkey farms, 8 quail farms, and 7 partridge farms) and 856 birds (675 turkeys, 105 quails, and 76 partridges) were sampled. Of that, 52 of 77 sampled farms (67.5%) and 437 of 894 samples (48.9%) were positive for H9N2 in 2014 while. Forty-one of 69 farms (59.4%) and 307 of 856 sera (35.9%) were positive in 2015. Furthermore, the mean titer of partridge farms was significantly lower than that of turkey farms (p < 0.01) and the mean percentage of sero-positive turkey farms was significantly higher than partridge farms (p < 0.01) in 2014. In 2015, no significant difference was observed between the mean sera titer amongst farms and percentage of sero-positive birds (p > 0.05). Our results indicated that H9N2 is circulating in these farms. Since many more such farms are being established for operations, in addition to the threat of emergence and continuous reemergence of the disease in these farms, enhanced veterinary biosecurity measures on farms are required for mitigation.

Rotavirus G (RVG) strains have been detected in a variety of avian species, but RVG genomes have been published from only a single pigeon and two chicken strains. Two turkey RVG strains were identified and characterized, one in a hatchery with no reported health issues and the other in a hatchery with high embryo/poult mortality. The two turkey RVG strains shared only an 85.3 % nucleotide sequence identity in the VP7 gene while the other genes possessed high nucleotide identity among them (96.3-99.9 %). Low nucleotide percentage identities (31.6-87.3 %) occurred among the pigeon and chicken RVG strains. Interestingly, potential recombination events were detected between our RVG strains and a human RVB strain, in the VP6 and NSP3 segments. The epidemiology of RVG in avian flocks and the pathogenicity of the two different RVG strains should be further investigated to understand the ecology and impact of RVG in commercial poultry flocks.