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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.)

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.)

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.)

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.)

In order to characterize the evolutionary adaptations of avian paramyxovirus 1 (APMV-1) genomes, we have compared codon usage and codon adaptation indexes among groups of Newcastle disease viruses that differ in biological, ecological, and genetic characteristics. We have used available GenBank complete genome sequences, and compared codon usage of class I (CI-29 sequences containing 132,675 codons) and class II (CII-259 sequences containing 1,184,925 codons) APMV-1 genomes. We also compared available complete fusion protein gene sequences (CI-175 sequences containing 96,775 codons; CII-1166 sequences containing 644,798 codons). Adaptation to Gallus gallus was compared among the different classes of viruses, among different genomic regions based on transcriptional levels, or among the fusion gene. Interestingly, distinctive codon usage determined by differences in relative synonymous codon usage and by codon adaptation indexes was observed for the two APMV-1 classes and for different transcriptional regions within classes. Furthermore, differential use of the third codon position and preferential use of codon pairs were seen for the two different classes and for selected genotypes of class II despite the fact that there were no large differences in nucleotide composition. The data suggest that codon usage has changed significantly since the two APMV-1 classes diverged, however, these changes are not significantly pronounced among viruses of the same genotype, suggesting that codon adaptation in APMV-1 occurs through a slow evolutionary process., (Published by Elsevier B.V.)

Newcastle disease is caused by virulent forms of avian paramyxovirus of serotype 1 (APMV-1) and has global economic importance. The disease reached panzootic proportions within two decades after first being identified in 1926 in the United Kingdom and Indonesia and still remains endemic in many countries across the world. Here we review information on the host, temporal, and geographic distribution of APMV-1 genetic diversity based on the evolutionary systematics of the complete coding region of the fusion gene. Strains of APMV-1 are phylogenetically separated into two classes (class I and class II) and further classified into genotypes based on genetic differences. Class I viruses are genetically less diverse, generally present in wild waterfowl, and are of low virulence. Class II viruses are genetically and phenotypically more diverse, frequently isolated from poultry with occasional spillovers into wild birds, and exhibit a wider range of virulence. Waterfowl, cormorants, and pigeons are natural reservoirs of all APMV-1 pathotypes, except viscerotropic velogenic viruses for which natural reservoirs have not been identified. Genotypes I and II within class II include isolates of high and low virulence, the latter often being used as vaccines. Viruses of genotypes III and IX that emerged decades ago are now isolated rarely, but may be found in domestic and wild birds in China. Containing only virulent viruses and responsible for the majority of recent outbreaks in poultry and wild birds, viruses from genotypes V, VI, and VII, are highly mobile and have been isolated on different continents. Conversely, virulent viruses of genotypes XI (Madagascar), XIII (mainly Southwest Asia), XVI (North America) and XIV, XVII and XVIII (Africa) appear to have a more limited geographic distribution and have been isolated predominantly from poultry., (Published by Elsevier B.V.)

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in 1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics., (Published by Elsevier B.V.)

Different genotypes of avian paramyxovirus serotype-1 virus (APMV-1) circulate in many parts of the world. Traditionally, Newcastle disease virus (NDV) is recognized as having two major divisions represented by classes I and II, with class II being further divided into sixteen genotypes. Although all NDV are members of APMV-1 and are of one serotype, antigenic and genetic diversity is observed between the different genotypes. Reports of vaccine failure from many countries and reports by our lab on the reduced ability of classical vaccines to significantly decrease viral replication and shedding have created renewed interest in developing vaccines formulated with genotypes homologous to the virulent NDV (vNDV) circulating in the field. We assessed how the amount and specificity of humoral antibodies induced by inactivated vaccines affected viral replication, clinical protection and evaluated how non-homologous (heterologous) antibody levels induced by live NDV vaccines relate to transmission of vNDV. In an experimental setting, all inactivated NDV vaccines protected birds from morbidity and mortality, but higher and more specific levels of antibodies were required to significantly decrease viral replication. It was possible to significantly decrease viral replication and shedding with high levels of antibodies and those levels could be more easily reached with vaccines formulated with NDV of the same genotype as the challenge viruses. However, when the levels of heterologous antibodies were sufficiently high, it was possible to prevent transmission. As the level of humoral antibodies increase in vaccinated birds, the number of infected birds and the amount of vNDV shed decreased. Thus, in an experimental setting the effective levels of humoral antibodies could be increased by (1) increasing the homology of the vaccine to the challenge virus, or (2) allowing optimal time for the development of the immune response., (Published by Elsevier Ltd.)

Newcastle disease (ND) remains a constant threat to poultry producers worldwide, in spite of the availability and global employment of ND vaccinations since the 1950s. Strains of Newcastle disease virus (NDV) belong to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus, are contained in one serotype and are also known as avian paramyxovirus serotype-1 (APMV-1). They are pleomorphic in shape and are single-stranded, non-segmented, negative sense RNA viruses. The virus has been reported to infect most orders of birds and thus has a wide host range. Isolates are characterized by virulence in chickens and the presence of basic amino acids at the fusion protein cleavage site. Low virulent NDV typically produce subclinical disease with some morbidity, whereas virulent isolates can result in rapid, high mortality of birds. Virulent NDV are listed pathogens that require immediate notification to the Office of International Epizootics and outbreaks typically result in trade embargos. Protection against NDV is through the use of vaccines generated with low virulent NDV strains. Immunity is derived from neutralizing antibodies formed against the viral hemagglutinin and fusion glycoproteins, which are responsible for attachment and spread of the virus. However, new techniques and technologies have also allowed for more in depth analysis of the innate and cell-mediated immunity of poultry to NDV. Gene profiling experiments have led to the discovery of novel host genes modulated immediately after infection. Differences in virus virulence alter host gene response patterns have been demonstrated. Furthermore, the timing and contributions of cell-mediated immune responses appear to decrease disease and transmission potential. In view of recent reports of vaccine failure from many countries on the ability of classical NDV vaccines to stop spread of disease, renewed interest in a more complete understanding of the global immune response of poultry to NDV will be critical to developing new control strategies and intervention programs for the future., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The avian paramyxovirus type 1 (APMV-1), or Newcastle disease virus (NDV), comprise a diverse group of viruses with a single-stranded, negative-sense RNA genome. Historically, two systems have been simultaneously used to classify NDV isolates into lineages or genotypes, generating confusion in the nomenclature and discrepancies in the assignment of genetic groups. In the present study we assessed the genetic diversity of the avian paramyxovirus type-1 (APMV-1) and propose a unified nomenclature and a classification system based on objective criteria to separate NDV into genotypes. Complete F gene sequences of class I (n = 110) and class II (n = 602) viruses were used for the phylogenetic reconstruction and to identify distinct taxonomic groups. The mean interpopulational evolutionary distance was estimated (10%) and set as the cutoff value to assign new genotypes. Results of our study revealed that class I viruses comprise a single genotype, while class II contains 15 genetic groups including 10 previously established (I-IX, and XI) and five new genotypes (X, XII, XIII, XIV and XV). Sub-genotypes were identified among class I and class II genotypes. Adoption of a unified nomenclature and of objective criteria to classify NDV isolates will facilitate studies on NDV epidemiology, evolution, disease control and diagnostics., (Published by Elsevier B.V.)

The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks varies between different viruses and is affected by the age of the ducks, with younger ducks presenting a more severe disease. In order to better understand the pathobiology of H5N1 HPAI in ducks including the role of host responses, 2 and 5-week-old Pekin ducks were infected with three different H5N1 HPAI viruses. Virus-induced pathology ranged from no clinical signs to severe disease and mortality, with the 2-week-old ducks being more severely affected by the more virulent viruses. However, these more virulent viruses induced higher body temperatures in the 5-week-old ducks than in the 2-week-old ducks indicating possible differences in innate immune responses. To analyze the ducks host responses to H5N1 HPAI virus infection, expression of innate immune-related genes was measured in the spleens and lungs of infected ducks at the peak of virus infection. IFN-α, RIG-I, and IL-6 RNA levels were increased in spleens regardless of the virus given and the age of the ducks, however differences were observed in the levels of up-regulation of IFN-α and RIG-I between the 2 and the 5-week-old ducks with the more virulent virus. Differences in IL-2 gene expression were also observed. In the lungs, the levels of expression of innate immune-related genes were lower than in the spleen, with mostly up-regulation of RIG-I and IL-6 and down-regulation of IFN-α and IL-2; no significant difference in expression was found between the 2 and the 5-week-old ducks. The differences observed in the innate immune responses to infection with H5N1 HPAI viruses could explain in part the differences in pathogenicity found between the 2 and 5-week-old ducks, however earlier time points after infection and additional innate immune-related genes should be examined., (Published by Elsevier B.V.)

Since the discovery of Newcastle disease virus (NDV) in 1926, nine genotypes of class I viruses and ten of class II have been identified, representing a diverse and continually evolving group of viruses. The emergence of new virulent genotypes from global epizootics and the year-to-year changes observed in the genomic sequence of NDV of low and high virulence implies that distinct genotypes of NDV are simultaneously evolving at different geographic locations across the globe. This vast genomic diversity may be favored by the large variety of avian species susceptible to NDV infection and by the availability of highly mobile wild bird reservoirs. The genomic diversity of NDV increases the possibility of diagnostic failures, resulting in unidentified infections. Constant epidemiological surveillance and pro-active characterization of circulating strains are needed to ensure that the immunological and PCR reagents are effective in identifying NDV circulating worldwide. For example, in the United States, the widely used real-time reverse transcription polymerase chain reaction (RRT-PCR) matrix gene assay for the identification of NDV often fails to detect low virulence APMV-1 from waterfowl, while the RRT-PCR fusion gene assay, used to identify virulent isolates, often fails to detect certain virulent NDV genotypes. A new matrix-polymerase multiplex test that detects most of the viruses currently circulating worldwide and a modified fusion test for the identification of virulent pigeon viruses circulating in the U.S. and Europe have recently been developed. For newly isolated viruses with unknown sequences, recently developed random priming sequencing methods need to be incorporated into the diagnostic arsenal. In addition, the current system of classifying NDV into genotypes or lineages is inadequate. Here, we review the molecular epidemiology and recent diagnostic problems related to viral evolution of NDV and explain why a new system, based on objective criteria, is needed to categorize genotypes., (Published by Elsevier B.V.)

Strains of Newcastle disease virus (NDV) can be separated into genotypes based on genome differences even though they are antigenically considered to be of a single serotype. It is widely recognized that an efficacious Newcastle disease (ND) vaccine made with any NDV does induce protection against morbidity and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from infection and viral shed from such a challenge. Vaccines prepared from ND viruses corresponding to five different genotypes were compared to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced and the amount of challenge virus shed. Six groups of 4-week-old specific pathogen-free Leghorn chickens were given oil-adjuvanted vaccines prepared from one of five different inactivated ND viruses including strains B1, Ulster, CA02, Pigeon84, Alaska 196, or an allantoic fluid control. Three weeks post-vaccination, serum was analyzed for antibody content using a hemagglutination inhibition assay against each of the vaccine antigens and a commercial NDV ELISA. After challenge with virulent CA02, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for virus shedding. All vaccines except for the control induced greater than 90% protection to clinical disease and mortality. The vaccine homologous with the challenge virus reduced oral shedding significantly more than the heterologous vaccines. NDV vaccines formulated to be phylogenetically closer to potential outbreak viruses may provide better ND control by reducing virus transmission from infected birds.

Objectives: The haphazard distribution of fibrous tissue can interfere with quantitative methods for evaluating hepatic fibrosis. Inter-sample variation may represent a crucial issue when hydroxyproline measurement is used to quantify fibrosis. A comparative study of the hydroxyproline levels in normal and fibrotic rats is herein reported., Material and Methods: Twelve normal and 20 Capillaria hepatica-infected Wistar rats were used. Two fragments of the liver (A and B) of each rat were taken from separate areas and hydroxyproline measurements were made. Calculated differences in hydroxyproline measurements between samples from the same liver were analyzed by BOOTSTRAP., Results: Differences in normal rats varied from 0.026 to 1.85 micromol of HP/g, in ten rats, the difference was less than 0.50 micromol. In infected rats, it varied from 0.04 to 2.86 micromol HP/g. Differences higher than 0.69 micromol/g were significant for normal rats (p<0.05) and above 1.22 micromol/g (p<0.05) for fibrotic rats., Conclusions: Hydroxyproline ratio in a normal liver kept a fair degree of reproducibility. In the presence of hepatic fibrosis, the levels of hydroxyproline may vary significantly between samples from a single liver and may have limited value in quantifying the extent of fibrosis.