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The consequences for the health of the work are the result of the interaction of the body with agents/factors existing in the working environment, characterizing the occupational exposure. The dentists are exposed to various occupational hazards, whether chemical, physical, mechanical, ergonomic or accidents. The aim of this study was identify whether dentists and dental students know the risks that they are exposed to chemicals during their practical work. A questionnaire was given to 33 dentists and 33 students of final year of Dentistry, about his knowledge of the chemical agents which dental professionals are exposed and what the risks posed by exposure to each of these agents. With the exception of ether and formaldehyde, most students are exposed to all the chemicals studied, while dentists are less exposed to ether, formaldehyde and surgical cement. The majority of students do 16 chemicals associated with health problems, including alcohol, antibacterial liquid soap, latex, powder, glutaraldehyde, sodium alginate and glass ionomer. Most dentists not associated products with 11 health problems, like a list of the students. None dentist and only 6.1% of the students associated the risk of cancer with exposure to cement/plaster. Only 24.2% of dentists and 21.1% of students associated methyl methacrylate exposure with lung disorders. Dentists and dental students don’t have an adequate knowledge of the risks involved with exposure to the chemicals they use every day, and it may result in not using appropriate protective equipment and occupational diseases.

JUSTIFICATIVA E OBJETIVOS - Estudos clínicos e experimentais já demonstraram a eficácia e a segurança da ropivacaína aplicada a estruturas nervosas por via subaracnóidea. Por outro lado, a potência da ropivacaína em raquianestesia é aproximadamente igual à metade da potência de bupivacaína, considerando-se soluções hiperbáricas. O objetivo deste estudo foi comparar as características do bloqueio subaracnóideo com ropivacaína ou bupivacaína hiperbáricas, em doses equipotentes. MÉTODO - Vinte pacientes com idades entre 20 e 60 anos, estado físico I ou II (ASA), programados para cirurgias eletivas de membros inferiores, perineais e herniorrafias inguinais, foram distribuídos de forma aleatória em dois grupos. No grupo R (n = 10), receberam 4 ml de ropivacaína hiperbárica a 0,5% (dose = 20 mg) e no grupo B (n = 10), receberam 2 ml de bupivacaína hiperbárica a 0,5% (dose = 10 mg) por via subaracnóidea. A punção foi praticada com agulha calibre 25G na posição sentada. Monitorização não-invasiva de PAS, PAD, PAM, FC, ECG, SpO2. Foram avaliadas características de instalação e regressão dos bloqueios sensorial e motor, a evolução dos parâmetros cardiovasculares e respiratórios, a incidência de eventos adversos. RESULTADOS: Os grupos foram homogêneos quanto aos dados demográficos. Não houve diferenças significativas entre os grupos R e B quanto a: latência do bloqueio sensorial (174,4 ± 75,9 vs 191 ± 51,7 s); latência do bloqueio motor (373,6 ± 214,6 vs 240 ± 60 s); nível superior do bloqueio sensorial T8-T10 (90% dos pacientes no grupo R vs 70% no grupo B); bloqueio motor grau 3 (50% dos pacientes no grupo R vs 30% no grupo B); tempo para regressão completa do bloqueio sensorial (178,5 ± 65,2 vs 181 ± 26,9 minutos); tempo para regressão completa do bloqueio motor (192 ± 50,7 vs 162,5 ± 37,8 min); tempo para a primeira queixa espontânea de dor (183,9 ± 37,1 vs 206,5 ± 46,6 minutos). CONCLUSÕES: Neste estudo as características clínicas do bloqueio subaracnóideo com ropivacaína ou bupivacaína hiperbáricas em doses equipotentes foram semelhantes. Os dados parecem confirmar observações anteriores de que nestas condições a potência da ropivacaína é aproximadamente igual à metade daquela da bupivacaína.

Abstract Background Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing. Results We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter. Conclusion The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

Infectious bronchitis virus (IBV) causes a highly contagious, acute upper respiratory disease in chickens characterized by nasal discharge, coughing, and rales. Here, the complete genome sequence of a recombinant GI-13 IBV strain ck/IN/A2332039-001/24 was sequenced from a choanal sample of a commercial broiler chicken in India using nontargeted next-generation sequencing.

In this case study, we describe an outbreak of highly pathogenic avian influenza (HPAI) virus subtype H7N3 in an ecological reserve in Chiapas, Mexico, affecting captive and wild birds. The virus was detected mainly in plain chachalacas displaying respiratory and gastrointestinal clinical signs and death within 24 hours. Mortality in white-fronted parrots and a clay-colored thrush was also recorded. We describe control strategies implemented to prevent virus dissemination and active surveillance within the risk area. Phylogenetic analysis revealed that the HPAI H7N3 virus detected in affected birds shared a close genetic relationship with Mexican H7N3 isolates from 2012., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no competing interest in this work. Ethics statement: All animal procedures were performed in strict compliance with Mexican legislation on the use and care of animals., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Here, we report the coding-complete genomic sequences of two chicken caliciviruses from US poultry flocks in 2003 and 2004. They show the same genomic organization as that of other members of the Bavovirus genus and have the highest nucleotide identity (~88%) with strains from clinically normal chickens from Germany in 2004 and Netherlands in 2019., Competing Interests: The authors declare no conflict of interest.

Metagenomics offers the potential to replace and simplify classical methods used in the clinical diagnosis of human and veterinary infectious diseases. Metagenomics boasts a high pathogen discovery rate and high specificity, advantages absent in most classical approaches. However, its widespread adoption in clinical settings is still pending, with a slow transition from research to routine use. While longer turnaround times and higher costs were once concerns, these issues are currently being addressed by automation, better chemistries, improved sequencing platforms, better databases, and automated bioinformatics analysis. However, many technical options and steps, each producing highly variable outcomes, have reduced the technology's operational value, discouraging its implementation in diagnostic labs. We present a case for utilizing non-targeted RNA sequencing (NT-RNA-seq) as an ideal metagenomics method for the detection of infectious disease-causing agents in humans and animals. Additionally, to create operational value, we propose to identify best practices for the "core" of steps that are invariably shared among many human and veterinary protocols. Reference materials, sequencing procedures, and bioinformatics standards should accelerate the validation processes necessary for the widespread adoption of this technology. Best practices could be determined through "implementation research" by a consortium of interested institutions working on common samples.

Direct-targeted next-generation sequencing (tNGS), with its undoubtedly superior diagnostic capacity over real-time PCR (RT-PCR), and direct-non-targeted NGS (ntNGS), with its higher capacity to identify and characterize multiple agents, are both likely to become diagnostic methods of choice in the future. tNGS is a rapid and sensitive method for precise characterization of suspected agents. ntNGS, also known as agnostic diagnosis, does not require a hypothesis and has been used to identify unsuspected infections in clinical samples. Implemented in the form of multiplexed total DNA metagenomics or as total RNA sequencing, the approach produces comprehensive and actionable reports that allow semi-quantitative identification of most of the agents present in respiratory, cloacal, and tissue samples. The diagnostic benefits of the use of direct tNGS and ntNGS are high specificity, compatibility with different types of clinical samples (fresh, frozen, FTA cards, and paraffin-embedded), production of nearly complete infection profiles (viruses, bacteria, fungus, and parasites), production of "semi-quantitative" information, direct agent genotyping, and infectious agent mutational information. The achievements of NGS in terms of diagnosing poultry problems are described here, along with future applications. Multiplexing, development of standard operating procedures, robotics, sequencing kits, automated bioinformatics, cloud computing, and artificial intelligence (AI) are disciplines converging toward the use of this technology for active surveillance in poultry farms. Other advances in human and veterinary NGS sequencing are likely to be adaptable to avian species in the future.

We report the complete genome sequences of seven virulent Newcastle disease viruses (NDVs) that were isolated from chickens from live bird markets in the Arusha, Iringa, Mbeya, and Tanga regions of Tanzania in 2012. Phylogenetic analysis revealed that all isolates belong to sub-genotype XIII.1.1., Competing Interests: The authors declare no conflict of interest.

Avian metapneumovirus (aMPV) causes a highly contagious upper respiratory and reproductive disease in chickens, turkeys, and ducks. Here, complete genome sequences of aMPV-B vaccine strains BR/1890/E1/19 (PL21, Nemovac; Boehringer Ingelheim Animal Health, Brazil) and BR/1891/E2/19 (1062; Hipraviar, France) were sequenced and compared with the pathogenic field strain VCO3/60616., Competing Interests: The authors declare no conflict of interest.

We report the complete genome sequence of an avian orthoavulavirus 13 strain, isolated from a white-fronted goose in the Odesa region of Ukraine in 2013. The detection of avian orthoavulavirus 13 in Ukraine confirms that the geographic distribution of this virus extends beyond Asia., Competing Interests: The authors declare no conflict of interest.

Newcastle disease virus (NDV) infects a wide range of bird species worldwide and is of importance to the poultry industry. Although certain virus genotypes are clearly associated with wild bird species, the role of those species in the movement of viruses and the migratory routes they follow is still unclear. In this study, we performed a phylogenetic analysis of nineteen NDV sequences that were identified among 21,924 samples collected from wild and synanthropic birds from different regions of Ukraine from 2006 to 2015 and compared them with isolates from other continents. In synanthropic birds, NDV strains of genotype II, VI, VII, and XXI of class II were detected. The fusion gene sequences of these strains were similar to strains detected in birds from different geographical regions of Europe and Asia. However, it is noteworthy to mention the isolation of vaccine viruses from synanthropic birds, suggesting the possibility of their role in viral transmission from vaccinated poultry to wild birds, which may lead to the further spreading of vaccine viruses into other regions during wild bird migration. Moreover, here we present the first publicly available complete NDV F gene from a crow (genus Corvus ). Additionally, our phylogenetic results indicated a possible connection of Ukrainian NDV isolates with genotype XXI strains circulating in Kazakhstan. Among strains from wild birds, NDVs of genotype 1 of class I and genotype I of class II were detected. The phylogenetic analysis highlighted the possible exchange of these NDV strains between wild waterfowl from the Azov-Black Sea region of Ukraine and waterfowl from different continents, including Europe, Asia, and Africa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Goraichuk, Gerilovych, Bolotin, Solodiankin, Dimitrov, Rula, Muzyka, Mezinov, Stegniy, Kolesnyk, Pantin-Jackwood, Miller, Afonso and Muzyka.)

The avian gamma-coronavirus infectious bronchitis virus (AvCoV, IBV; Coronaviridae family) causes upper respiratory disease associated with severe economic losses in the poultry industry worldwide. Here, we report for the first time in Kenya and the Eastern African region two novel AvCoVs, designated IBV/ck/KE/1920/A374/2017 (A374/17) and AvCoV/ck/KE/1922/A376/2017 (A376/17), inadvertently discovered using random nontargeted next-generation sequencing (NGS) of cloacal swabs collected from indigenous chickens. Despite having genome organization (5'UTR-[Rep1a/1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR), canonical conservation of essential genes and size (~27.6 kb) typical of IBVs, the Kenyan isolates do not phylogenetically cluster with any genotypes of the 37 IBV lineages and 26 unique variants (UVs). Excluding the spike gene, genome sequences of A374/17 and A376/17 are only 93.1% similar to each other and 86.7-91.4% identical to genomes of other AvCoVs. All five non-spike genes of the two isolates phylogenetically cluster together and distinctly from other IBVs and turkey coronaviruses (TCoVs), including the indigenous African GI-26 viruses, suggesting a common origin of the genome backbone of the Kenyan isolates. However, isolate A376/17 contains a TCoV-like spike (S) protein coding sequence and is most similar to Asian TCoVs (84.5-85.1%) compared to other TCoVs (75.6-78.5%), whereas isolate A374/17 contains an S1 gene sequence most similar to the globally distributed lineage GI-16 (78.4-79.5%) and the Middle Eastern lineage GI-23 (79.8-80.2%) viruses. Unanswered questions include the actual origin of the Kenyan AvCoVs, the potential pathobiological significance of their genetic variations, whether they have indeed established themselves as independent variants and subsequently spread within Kenya and to the neighboring east/central African countries that have porous live poultry trade borders, and whether the live-attenuated Mass-type (lineage GI-1)-based vaccines currently used in Kenya and most of the African countries provide protection against these genetically divergent field variants.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples ( n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples ( n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19-34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12-37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Butt, Kariithi, Volkening, Taylor, Leyson, Pantin-Jackwood, Suarez, Stanton and Afonso.)

Avian metapneumoviruses (aMPV subtypes A-D) are respiratory and reproductive pathogens of poultry. Since aMPV-A was initially reported in Mexico in 2014, there have been no additional reports of its detection in the country. Using nontargeted next-generation sequencing (NGS) of FTA card-spotted respiratory samples from commercial chickens in Mexico, seven full genome sequences of aMPV-A (lengths of 13,288-13,381 nucleotides) were de novo assembled. Additionally, complete coding sequences of genes N ( n = 2), P and M ( n = 7 each), F and L ( n = 1 each), M2 ( n = 6), SH ( n = 5) and G ( n = 2) were reference-based assembled from another seven samples. The Mexican isolates phylogenetically group with, but in a distinct clade separate from, other aMPV-A strains. The genome and G-gene nt sequences of the Mexican aMPVs are closest to strain UK/8544/06 (97.22-97.47% and 95.07-95.83%, respectively). Various amino acid variations distinguish the Mexican isolates from each other, and other aMPV-A strains, most of which are in the G ( n = 38), F ( n = 12), and L ( n = 19) proteins. Using our sequence data and publicly available aMPV-A data, we revised a previously published rRT-PCR test, which resulted in different cycling and amplification conditions for aMPV-A to make it more compatible with other commonly used rRT-PCR diagnostic cycling conditions. This is the first comprehensive sequence analysis of aMPVs in Mexico and demonstrates the value of nontargeted NGS to identify pathogens where targeted virus surveillance is likely not routinely performed.

New variants of infectious bronchitis viruses (IBVs; Coronaviridae ) continuously emerge despite routine vaccinations. Here, we report genome sequence variations of IBVs identified by random non-targeted next generation sequencing (NGS) of vaccine and field samples collected on FTA cards from commercial flocks in Mexico in 2019-2021. Paired-ended sequencing libraries prepared from rRNA-depleted RNAs were sequenced using Illumina MiSeq. IBV RNA was detected in 60.07% ( n = 167) of the analyzed samples, from which 33 complete genome sequences were de novo assembled. The genomes are organized as 5'UTR-[Rep1a-Rep1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR, except in eight sequences lacking non-structural protein genes (accessory genes) 4b, 4c, and 6b. Seventeen sequences have auxiliary S2' cleavage site located 153 residues downstream the canonically conserved primary furin-specific S1/S2 cleavage site. The sequences distinctly cluster into lineages GI-1 (Mass-type; n = 8), GI-3 (Holte/Iowa-97; n = 2), GI-9 (Arkansas-like; n = 8), GI-13 (793B; n = 14), and GI-17 (California variant; CAV; n = 1), with regional distribution in Mexico; this is the first report of the presence of 793B- and CAV-like strains in the country. Various point mutations, substitutions, insertions and deletions are present in the S1 hypervariable regions (HVRs I-III) across all 5 lineages, including in residues 38, 43, 56, 63, 66, and 69 that are critical in viral attachment to respiratory tract tissues. Nine intra-/inter-lineage recombination events are present in the S proteins of three Mass-type sequences, two each of Holte/Iowa-97 and Ark-like sequence, and one each of 793B-like and CAV-like sequences. This study demonstrates the feasibility of FTA cards as an attractive, adoptable low-cost sampling option for untargeted discovery of avian viral agents in field-collected clinical samples. Collectively, our data points to co-circulation of multiple distinct IBVs in Mexican commercial flocks, underscoring the need for active surveillance and a review of IBV vaccines currently used in Mexico and the larger Latin America region., Competing Interests: JV and CA were employed by BASE2BIO. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kariithi, Volkening, Leyson, Afonso, Christy, Decanini, Lemiere and Suarez.)

Background and Purpose: Krabbe disease (KD), or globoid cell leukodystrophy (Online Mendelian Inheritance in Man #245200), is an autosomal recessive lysosomal storage disease caused by mutations in GALC leading to galactocerebrosidase deficiency. Age at onset can vary from early infancy (3-6 months of age) to adulthood, which has rarely been reported. Little is known about the natural history and early manifestations of adult onset KD (AOKD)., Methods: Here, we report a patient with an incidental diagnosis of AOKD and discuss management options in this scenario., Results: A 32-year-old woman came to medical attention because of headache and had brain magnetic resonance imaging findings compatible with AOKD, two pathogenic variants in GALC, and reduced activity of galactocerebrosidase. The jury is still out about the best management of such cases, and clinicians should be aware of this diagnosis, as AOKD is a potentially treatable condition., Conclusions: AOKD is a rare and potentially treatable condition. More studies on natural history of AOKD are urgently needed to guide the best management of this disease., (© 2022 European Academy of Neurology.)

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

Background: There is evidence that chicken IL4 (chIL4) functions similarly to its mammalian analogue by enhancing type 2 T helper (Th2) humoral immunity and promoting protection against parasitic infections; however, no studies have been performed to assess the effect of chIL4 on the pathogenesis of Newcastle disease (ND). To assess the role of chIL4 in velogenic NDV pathogenesis we created a vNDV infectious clone expressing chIL4. We hypothesized that co-expression of chIL4 during virus replication would result in decreased inflammation caused by the Th1 response and thereby increasing survival to challenge with vNDV., Methods: To evaluate the effect of chIL4 during early infection with velogenic Newcastle disease virus (NDV) in chickens, recombinant NDV clones expressing either chIL4 (rZJ1-IL4) or a control expressing green fluorescent protein (rZJ1-GFP) were created by inserting an expression cassette in an intergenic region of the NDV genome. The pathogenesis of rZJ1-IL4 was assessed in 4-week-old specific pathogen free chickens. The extent of virus replication was evaluated by titration in mucosal secretions and immunohistochemistry in multiple tissues. Expression of chIL4 was confirmed in tissues using immunohistochemistry., Results: Infection of birds with the rZJ1-IL4 resulted in successful viral replication in vivo and in vitro and generation of the chIL4 in tissues. All birds were clinically normal 2 DPI, with one bird in each group showing conjunctival swelling and enlarged spleens grossly. At 5 DPI, moderate or severe depression was observed in birds infected with rZJ1-GFP or rZJ1-IL4, respectively. Neurological signs and thymic atrophy were observed in one bird infected with rZJ1-IL4. Grossly, conjunctival swelling, mottled spleen and proventricular hemorrhages were observed at 5 DPI in one bird from each group. At 5 DPI, severe necrosis in the spleen, bursa and cecal tonsils were observed in birds infected with rZJ1-GFP, along with minimal evidence of chIL4 expression. In contrast, splenic atrophy, and moderate necrosis in the bursa and cecal tonsils were observed in birds infected with rZJ1-IL4. In addition, chIL4 signal was found in all tissues of rZJ1-IL4 birds at 5DPI., Conclusions: The production of chIL4 by a recombinant NDV strain resulted in the activation of the positive feedback loop associated with IL4 production. Insertion of chIL4 into NDV may decrease necrosis to lymphoid organs while increasing the severity of lymphoid atrophy and prolonged disease. However, with a low number of birds it is difficult to determine whether these results are significant to disease outcome., (Copyright © 2021. Published by Elsevier Ltd.)

In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 10 4.5 or 10 3.5 50% embryo infectious dose (EID 50 /egg) and chickens were challenged at 21 days post-hatch (DPH). In the second experiment, 10 3.5 EID 50 /egg of each vaccine was administered at 19 DOE, and chickens were challenged at 14 DPH. Chickens vaccinated with 10 3.5 EID 50 /egg of rZJ1*L-IL4R had hatch rates comparable to the group that received diluent alone, whereas other groups had significantly lower hatch rates. All vaccinated chickens survived challenge without displaying clinical disease, had protective hemagglutination inhibition titers, and shed comparable levels of challenge virus. The recombinant rZJ1*L-IL4R vaccine yielded lower post-vaccination mortality rates compared with the other in ovo NDV live vaccine candidates as well as provided strong protection post-challenge.

Here, we report near-complete genome sequences of sicinivirus from U.S. poultry flocks in 2003 to 2005 and Mexico in 2019. They show highest nucleotide identity (84.5 to 85.5%) with other members of the Sicinivirus genus. These sequences update knowledge on diversity and contribute to a better understanding of the molecular epidemiology of sicinivirus.

Here, we report the complete genome sequence of an Avian coronavirus strain GA08-like isolate from a fecal sample from a broiler chicken collected in Georgia in 2004. The viral genome in this 15-year-old sample provides evidence for the circulation of the GA08-like strain at least 4 years before its first report in 2008.

The hypothesis that host adaptation in virulent Newcastle disease viruses (NDV) has been accompanied by virulence modulation is reviewed here. Historical records, experimental data, and phylogenetic analyses from available GenBank sequences suggest that currently circulating NDVs emerged in the 1920-1940's from low virulence viruses by mutation at the fusion protein cleavage site. These viruses later gave rise to multiple virulent genotypes by modulating virulence in opposite directions. Phylogenetic and pathotyping studies demonstrate that older virulent NDVs further evolved into chicken-adapted genotypes by increasing virulence (velogenic-viscerotropic pathotypes with intracerebral pathogenicity indexes [ICPIs] of 1.6 to 2), or into cormorant-adapted NDVs by moderating virulence (velogenic-neurotropic pathotypes with ICPIs of 1.4 to 1.6), or into pigeon-adapted viruses by further attenuating virulence (mesogenic pathotypes with ICPIs of 0.9 to 1.4). Pathogenesis and transmission experiments on adult chickens demonstrate that chicken-adapted velogenic-viscerotropic viruses are more capable of causing disease than older velogenic-neurotropic viruses. Currently circulating velogenic-viscerotropic viruses are also more capable of replicating and of being transmitted in naïve chickens than viruses from cormorants and pigeons. These evolutionary virulence changes are consistent with theories that predict that virulence may evolve in many directions in order to achieve maximum fitness, as determined by genetic and ecologic constraints.

Kenyan poultry consists of ~80% free-range indigenous chickens kept in small flocks (~30 birds) on backyard poultry farms (BPFs) and they are traded via live bird markets (LBMs). Newcastle disease virus (NDV) was detected in samples collected from chickens, wild farm birds, and other domestic poultry species during a 2017-2018 survey conducted at 66 BPFs and 21 LBMs in nine Kenyan counties. NDV nucleic acids were detected by rRT-PCR L-test in 39.5% (641/1621) of 1621 analyzed samples, of which 9.67% (62/641) were NDV-positive by both the L-test and a fusion-test designed to identify the virulent virus, with a majority being at LBMs (64.5%; 40/62) compared to BPFs (25.5%; 22/62). Virus isolation and next-generation sequencing (NGS) on a subset of samples resulted in 32 complete NDV genome sequences with 95.8-100% nucleotide identities amongst themselves and 95.7-98.2% identity with other east African isolates from 2010-2016. These isolates were classified as a new sub-genotype, V.3, and shared 86.5-88.9% and 88.5-91.8% nucleotide identities with subgenotypes V.1 and V.2 viruses, respectively. The putative fusion protein cleavage site ( 113 R-Q-K-R↓F 117 ) in all 32 isolates, and a 1.86 ICPI score of an isolate from a BPF chicken that had clinical signs consistent with Newcastle disease, confirmed the high virulence of the NDVs. Compared to genotypes V and VI viruses, the attachment (HN) protein of 18 of the 32 vNDVs had amino acid substitutions in the antigenic sites. A time-scaled phylogeographic analysis suggests a west-to-east dispersal of the NDVs via the live chicken trade, but the virus origins remain unconfirmed due to scarcity of continuous and systematic surveillance data. This study reveals the widespread prevalence of vNDVs in Kenyan backyard poultry, the central role of LBMs in the dispersal and possibly generation of new virus variants, and the need for robust molecular epidemiological surveillance in poultry and non-poultry avian species.

Here, we report the complete genome sequence of fowl aviadenovirus D (FAdV-D) isolated from a preserved 24-year-old pancreas sample of a broiler chicken embryo. The results of the sequence showed that the viral genome is 44,079 bp long.

Runting stunting syndrome (RSS) in commercial chickens has been reported worldwide, and although several studies have attempted to clarify the cause and describe the lesions, there are gaps in knowledge of the epidemiology, pathogenesis, and etiology. The study objective was to use commercial chicks naturally affected by RSS to describe the histologic changes of RSS in all segments of the small intestine in chicks of different ages and to identify viral gene sequences in affected chicks and their association with histologic lesions. Chicks lacking clinical signs but from the same houses and from unaffected houses were used as controls. The average weight of affected chicks was significantly lower than expected for their flocks. Macroscopically, the small intestines had paler serosa, with watery, mucoid, or foamy contents and poorly digested food. Histologic lesions were characterized by necrotic crypts, crypt dilation, and flattening of the crypt epithelium. Histomorphometry of the intestines revealed villous atrophy especially in the jejunum and ileum. Histologic changes in other organs were not observed. Random next-generation sequencing of total RNA extracted from formalin-fixed paraffin-embedded tissues detected avian nephritis virus, avian rotavirus, and picornavirus in jejunal segments from 7-day-old chicks. No viruses were detected in the jejunum of 1-day-old chicks. Detection of picornaviral reads was significantly associated ( P < .05) with histologic lesions of RSS. Sequence analysis of the picornavirus revealed genetic similarity with the genus Gallivirus . Using in situ hybridization for galliviral nucleic acid sequences, the signal was associated with crypt lesion severity, although signal was detected both in chicks with and without RSS.

Parvoviruses are commonly found in U.S. poultry and are associated with clinical disease. Here, we report the complete coding sequences of three chicken parvoviruses from broiler chickens from commercial farms in the state of Georgia.

Poultry production plays an important role in the economy and livelihoods of rural households in Kenya. As part of a surveillance program, avian influenza virus (AIV)-specific real-time RT-PCR (RRT-PCR) was used to screen 282 oropharyngeal swabs collected from chickens at six live bird markets (LBMs) and 33 backyard poultry farms in Kenya and 8 positive samples were detected. Virus was isolated in eggs from five samples, sequenced, and identified as H9N2 low pathogenic AIV (LPAIV) G1 lineage, with highest nucleotide sequence identity (98.6-99.9%) to a 2017 Ugandan H9N2 isolate. The H9N2 contained molecular markers for mammalian receptor specificity, implying their zoonotic potential. Virus pathogenesis and transmissibility was assessed by inoculating low and medium virus doses of a representative Kenyan H9N2 LPAIV isolate into experimental chickens and exposing them to naïve uninfected chickens at 2 -days post inoculation (dpi). Virus shedding was determined at 2/4/7 dpi and 2/5 days post placement (dpp), and seroconversion determined at 14 dpi/12 dpp. None of the directly-inoculated or contact birds exhibited any mortality or clinical disease signs. All directly-inoculated birds in the low dose group shed virus during the experiment, while only one contact bird shed virus at 2 dpp. Only two directly-inoculated birds that shed high virus titers seroconverted in that group. All birds in the medium dose group shed virus at 4/7 dpi and at 5 dpp, and they all seroconverted at 12/14 dpp. This is the first reported detection of H9N2 LPAIV from Kenya and it was shown to be infectious and transmissible in chickens by direct contact and represents a new disease threat to poultry and potentially to people., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All authors read and approved the final version of the manuscript., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Newcastle disease (ND) is a major constraint to Kenya's poultry production, which is comprised of approximately 80% indigenous chickens (ICs; caged and free-range system) and 20% exotic chickens (intensive system). This study analyzed cases reported as suspected ND in Kenya between 2005 and 2015. Of the suspected 332 ND reported cases from the three production systems in 27 locations within six Kenyan Agro-Ecological Zones (AEZs), 140 diagnosed as infected with avian orthoavulavirus 1 (AOaV-1; formerly Newcastle disease virus) were present in every year in all AEZs. The numbers of AOaV-1-positive cases differed significantly (p < 0.05) between the production systems across the years depending on the season, climate, and location. In the free-range system, both ambient temperatures and season associated significantly (p = 0.001 and 0.02, respectively) with the number of cases, while in the intensive and caged systems, the positive cases correlated significantly with season and relative humidity, respectively (p = 0.05). Regardless of the production systems, the numbers of clinically sick birds positively correlated with the ambient temperatures (r = 0.6; p < 0.05). Failure to detect AOaV-1 in 58% of the ND cases reported, and mortalities exceeding the observed numbers of clinically sick birds suggest deficiencies in the current ND reporting and diagnostic system. Intensive farmers were the slowest in reporting the cases and diagnostic deficiencies were most evident by failure to test the exposure of ICs to natural infection with AOaV-1 and for the AOaV-1-negative cases lack of testing for other pathogens and/or AOaV-1 variants. This study indicates a need for improved surveillance and diagnostics in Kenyan domestic poultry.

Here, we report the complete genome sequence of Avian coronavirus strain ArkDPI of the GI-9 lineage, isolated from broiler chickens in North Georgia in 1994. This is the complete genome sequence of this vaccine strain, reisolated from broilers in the United States.

Avian coronavirus , also known as infectious bronchitis virus, is a highly contagious respiratory pathogen of chickens that is responsible for major economic losses to the poultry industry around the globe. Here, we report the complete genome sequence of strain GA08 of the GI-27 lineage, isolated from a fecal sample from a broiler chicken collected in Georgia in 2015.

We report the complete genome sequences of 11 virulent Newcastle disease viruses. The isolates were obtained from vaccinated broiler and layer chickens in three different provinces of Indonesia in 2013 and 2014. Phylogenetic analysis revealed that all isolates belong to subgenotype VII.2 in the class II cluster.

Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE., (Published by Elsevier Ltd.)

U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Highly pathogenic avian influenza (HPAI) virus subtype H7N3 has been circulating in poultry in Mexico since 2012 and vaccination has been used to control the disease. In this study, eight Mexican H7N3 HPAI viruses from 2015-2017 were isolated and fully sequenced. No evidence of reassortment was detected with other avian influenza (AI) viruses, but phylogenetic analyses show divergence of all eight gene segments into three genetic clusters by 2015, with 94.94 to 98.78 percent nucleotide homology of the HA genes when compared to the index virus from 2012. The HA protein of viruses from each cluster showed a different number of basic amino acids (n = 5-7) in the cleavage site, and six different patterns at the predicted N-glycosylation sites. Comparison of the sequences of the Mexican lineage H7N3 HPAI viruses and American ancestral wild bird AI viruses to characterize the virus evolutionary dynamics showed that the nucleotide substitution rates in PB2, PB1, PA, HA, NP, and NS genes greatly increased once the virus was introduced into poultry. The global nonsynonymous and synonymous ratios imply strong purifying selection driving the evolution of the virus. Forty-nine positively selected sites out of 171 nonsynonymous mutations were identified in the Mexican H7N3 HPAI viruses, including 7 amino acid changes observed in higher proportion in North American poultry origin AI viruses isolates than in wild bird-origin viruses. Continuous monitoring and molecular characterization of the H7N3 HPAI virus is important for better understanding of the virus evolutionary dynamics and further improving control measures including vaccination., Competing Interests: The authors have declared that no competing interests exist.

Background: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally., Results: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels., Conclusion: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Our prior work has shown that live poultry vaccines have been intermittently isolated from wild birds sampled during field surveillance studies for Newcastle disease virus (NDV). Thus, we experimentally investigated the susceptibility of four native agriculturally associated wild bird species to the NDV LaSota vaccine and evaluated the shedding dynamics, potential transmission from chickens, and humoral antibody responses. To test susceptibility, we inoculated wild-caught, immunologically NDV-naïve house finches ( Haemorhous mexicanus ; n = 16), brown-headed cowbirds ( Molothrus ater ; n = 9), northern cardinals ( Cardinalis cardinalis ; n = 6), and American goldfinches ( Spinus tristis ; n = 12) with 0.1 ml (10 6.7 mean embryo infectious doses [EID 50/ml ]) of NDV LaSota vaccine via the oculo-nasal route. To test transmission between chickens and wild birds, adult specific-pathogen-free white leghorn chickens were inoculated similarly and cohoused in separate isolators with two to five wild birds of the species listed above. This design resulted in three treatments: wild bird direct inoculation (five groups) and wild bird exposure to one (two groups) or two inoculated chickens (six groups), respectively. Blood and oropharyngeal and cloacal swabs were collected before and after infection with the live vaccine. All wild birds that were directly inoculated with the LaSota vaccine shed virus as demonstrated by virus isolation (VI). Cardinals were the most susceptible species based on shedding viruses from 1 to 11 days postinoculation (dpi) with titers up to 10 4.9 EID 50 /ml. Although LaSota viruses were shed by all inoculated chickens and were present in the drinking water, most noninoculated wild birds cohoused with these chickens remained uninfected for 14 days as evidenced by VI. However, one American goldfinch tested positive for vaccine transmission by VI at 7 dpi and one house finch tested positive for vaccine transmission by real-time reverse-transcription PCR at 13 dpi. Only one directly inoculated cowbird (out of three) and two cardinals (out of two) developed NDV-specific hemagglutination inhibition antibody titers of 16, 16, and 128, respectively. No clinical signs were detected in the chickens or the wild birds postinoculation.

Avian infectious bronchitis virus is the causative agent of a highly contagious disease that results in severe economic losses to the poultry industry worldwide. Here, we report the first coding-complete genome sequence of strain DMV/1639 of the GI-17 lineage, isolated from broiler chickens in Georgia in 2019.

Five, class II, virulent Newcastle disease virus (vNDV) isolates of different genotypes from different host species were evaluated for their ability to infect, cause disease, and transmit to naïve chickens. Groups of five birds received a low, medium, or high dose, by the oculonasal route, of one of the following vNDV: three chicken-origin, one cormorant-origin, and one pigeon-origin. Three naïve birds were added to each group at two days post-inoculation (DPI) to evaluate transmission. Virus shedding was quantified from swabs (2/4/7 DPI), and seroconversion was evaluated at 14 DPI. All inoculated and contact birds in the chicken-origin vNDV groups succumbed to infection, displaying clinical signs typical of Newcastle disease and shed virus titers above 6 log 10 EID 50 /ml. Birds receiving a high and medium dose of the cormorant virus showed primarily neurological clinical signs with 80% and 60% mortality, respectively. The chickens showing clinical disease shed virus at titers below 4 log 10 EID 50 /ml, and the remaining bird in the high dose group seroconverted with a high HI titer. For the pigeon-origin virus, no clinical signs were observed in any of the birds, but all 5 chickens in the high challenge dose and one bird in the medium challenge group shed virus at mean titers of 3.1 and 2.2 log 10 EID 50 /ml, respectively. Overall, the chicken-origin viruses infected chickens and efficiently transmitted to naïve birds, while the cormorant- and pigeon-origin viruses infected chickens only at the higher doses and did not transmit to other birds., (Published by Elsevier B.V.)

Newcastle disease virus (NDV) has a wide avian host range and a high degree of genetic variability, and virulent strains cause Newcastle disease (ND), a worldwide concern for poultry health. Although NDV has been studied in Nigeria, genetic information about the viruses involved in the endemicity of the disease and the transmission that likely occurs at the poultry-wildlife interface is still largely incomplete. Next-generation and Sanger sequencing was performed to provide complete (n = 73) and partial genomic sequence data (n = 38) for NDV isolates collected from domestic and wild birds in Nigeria during 2002-2015, including the first complete genome sequences of genotype IV and subgenotype VIh from the African continent. Phylogenetic analysis revealed that viruses of seven different genotypes circulated in that period, demonstrating high genetic diversity of NDV for a single country. In addition, a high degree of similarity between NDV isolates from domestic and wild birds was observed, suggesting that spillovers had occurred, including to three species that had not previously been shown to be susceptible to NDV infection. Furthermore, the first spillover of a mesogenic Komarov vaccine virus is documented, suggesting a previous spillover and evolution of this virus. The similarities between viruses from poultry and multiple bird species and the lack of evidence for host adaptation in codon usage suggest that transmission of NDV between poultry and non-poultry birds occurred recently. This is especially significant when considering that some viruses were isolated from species of conservation concern. The high diversity of NDV observed in both domestic and wild birds in Nigeria emphasizes the need for active surveillance and epidemiology of NDV in all bird species.

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99-100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3-98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000-2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

A Newcastle disease virus was isolated from a chicken from a live bird market in the Mbeya region of Tanzania. Complete genome characterization of the isolate identified it as a member of subgenotype Vd. This is the first complete genome sequence of this subgenotype.

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing. RESEARCH HIGHLIGHTS A multi-allelic assay identified nine ILTV genotypes circulating in the US Single-allele genotyping is congruent with whole genome phylogenetic partitioning US ILTV strains can be grouped into five genotypes using the single-allele assay The single-allele assay can be done using MinION sequencing of barcoded amplicons.

Newcastle disease (ND) infects wild birds and poultry species worldwide, severely impacting the economics of the poultry industry. ND is especially problematic in Latin America (Mexico, Colombia, Venezuela, and Peru) where it is either endemic or re-emerging. The disease is caused by infections with one of the different strains of virulent avian Newcastle disease virus (NDV), recently renamed Avian avulavirus 1. Here, we describe the molecular epidemiology of Latin American NDVs, current control and prevention methods, including vaccines and vaccination protocols, as well as future strategies for control of ND. Because the productive, cultural, economic, social, and ecological conditions that facilitate poultry endemicity in South America are similar to those in the developing world, most of the problems and control strategies described here are applicable to other continents.

Background: Avian avulavirus (commonly known as avian paramyxovirus-1 or APMV-1) can cause disease of varying severity in both domestic and wild birds. Understanding how viruses move among hosts and geography would be useful for informing prevention and control efforts. A Bayesian statistical framework was employed to estimate the evolutionary history of 1602 complete fusion gene APMV-1 sequences collected from 1970 to 2016 in order to infer viral transmission between avian host orders and diffusion among geographic regions. Ancestral states were estimated with a non-reversible continuous-time Markov chain model, allowing transition rates between discrete states to be calculated. The evolutionary analyses were stratified by APMV-1 classes I (n = 198) and II (n = 1404), and only those sequences collected between 2006 and 2016 were allowed to contribute host and location information to the viral migration networks., Results: While the current data was unable to assess impact of host domestication status on APMV-1 diffusion, these analyses supported the sharing of APMV-1 among divergent host taxa. The highest supported transition rate for both classes existed from domestic chickens to Anseriformes (class I:6.18 transitions/year, 95% highest posterior density (HPD) 0.31-20.02, Bayes factor (BF) = 367.2; class II:2.88 transitions/year, 95%HPD 1.9-4.06, BF = 34,582.9). Further, among class II viruses, domestic chickens also acted as a source for Columbiformes (BF = 34,582.9), other Galliformes (BF = 34,582.9), and Psittaciformes (BF = 34,582.9). Columbiformes was also a highly supported source to Anseriformes (BF = 322.0) and domestic chickens (BF = 402.6). Additionally, our results provide support for the diffusion of viruses among continents and regions, but no interhemispheric viral exchange between 2006 and 2016. Among class II viruses, the highest transition rates were estimated from South Asia to the Middle East (1.21 transitions/year; 95%HPD 0.36-2.45; BF = 67,107.8), from Europe to East Asia (1.17 transitions/year; 95%HPD 0.12-2.61; BF = 436.2) and from Europe to Africa (1.06 transitions/year, 95%HPD 0.07-2.51; BF = 169.3)., Conclusions: While migration appears to occur infrequently, geographic movement may be important in determining viral diversification and population structure. In contrast, inter-order transmission of APMV-1 may occur readily, but most events are transient with few lineages persisting in novel hosts.

In May of 2018, virulent Newcastle disease virus was detected in sick, backyard, exhibition chickens in southern California. Since, the virus has affected 401 backyard and four commercial flocks, and one live bird market in California, and one backyard flock in Utah. The pathogenesis and transmission potential of this virus, along with two genetically related and widely studied viruses, chicken/California/2002 and chicken/Belize/2008, were evaluated in both 3-week- and 62-week-old chickens given a low, medium, or high challenge dose. All three viruses were highly virulent causing clinical signs, killing all the chickens in the medium and high dose groups, and efficiently transmitting to contacts. The three viruses also replicated in the reproductive tract of the adult hens. Virus shedding for all viruses was detected 24 hours after challenge, peaking with high titers at day 4 post challenge. Although not genetically identical, the studied isolates were shown to be phenotypically very similar, which allows the utilization of the available literature in the control of the current outbreak., (Copyright © 2019. Published by Elsevier Inc.)