Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola.

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009-2012).

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3'-NP-P-M-F-HN-L-5'. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai's meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

A review of current knowledge on avian Newcastle infection in commercial poultry in the Kingdom of Saudi Arabia.

Newcastle disease (ND) is a tremendously contagious avian infection with extensive monetary ramifications for the chicken zone. To reduce the effect of ND on the Saudi rooster enterprise, our analysis emphasizes the necessity of genotype-particular vaccinations, elevated surveillance, public recognition campaigns, and stepped-forward biosecurity. Data show that one-of-a-kind bird species, outdoor flocks, and nearby differences in susceptibility are all vulnerable. The pathogenesis consists of tropism in the respiratory and gastrointestinal structures and some genotypes boom virulence. Laboratory diagnostics use reverse transcription-polymerase chain reaction, sequencing, and serotyping among different strategies. Vital records are supplied through immune responses and serological trying out. Vaccination campaigns, biosecurity protocols, and emergency preparedness are all covered in prevention and manipulation techniques. Notably, co-circulating genotypes and disparities in immunization regulations worry Saudi Arabia. The effect of ND in Saudi Arabia is tested in this paper, with precise attention paid to immunological reaction, pathogenesis, susceptibility elements, laboratory analysis, and preventative and manipulation measures. Saudi Arabia can shield its bird region and beef up its defences against Newcastle's ailment, enforcing those hints into its policies.

Newcastle disease virus: the past and current situation in Indonesia.

The Newcastle disease virus (NDV) outbreak was first reported in Java Island, Indonesia, in 1926, which was then reported further in Newcastle-upon-Tyne, England. Nevertheless, the NDV is still endemic in Indonesia, with outbreaks occurring in free-range and commercial chicken farms. The dynamic evolution of the NDV has led to the further development of vaccines and diagnostic tools for more effective control of this virus. This paper discusses the history of the NDV occurrence, vaccines, the development of diagnostic tools, and the epidemiological condition of the NDV in Indonesia. Indonesia, which has the largest poultry population in the world after China, has challenges in preventing and controlling this virus that causes economic losses to the farmers and has an impact on the welfare of the poultry farming community in Indonesia.

Geographic distribution and genetic diversity of Newcastle disease virus in pigeons from Pakistan.

RESEARCH HIGHLIGHTS: Virulent NDV genotypes were repeatedly isolated from pigeons.Evidence of epidemiological links among viruses isolated from various locations.Distinct phylogenetic branches suggest separate, simultaneous evolution of NDVs.Study information could be helpful in the development of an effective vaccine.

Molecular characterization of Newcastle disease virus obtained from Mawenzi live bird market in Morogoro, Tanzania in 2020-2021.

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

Fatal Human Neurologic Infection Caused by Pigeon Avian Paramyxovirus-1, Australia.

Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts.

Metagenomic characterization reveals virus coinfections associated with Newcastle disease virus among poultry in Kenya.

Newcastle disease (ND) is an endemic viral disease affecting poultry and causing massive economic losses. This cross-sectional purposive study detected coinfections that are associated with the Newcastle disease virus among poultry from selected regions in Kenya. Cloacal (n = 599) and oral-pharyngeal (n = 435) swab samples were collected and pooled into 17 and 15 samples, respectively. A total of 17,034,948 and 7,751,974 paired-end reads with an average of 200 nucleotides were generated from the cloacal and oral-pharyngeal swab samples, respectively. Analysis of the de novo assembled contigs identified 177 and 18 cloacal and oral-pharyngeal contigs, respectively with hits to viral sequences, as determined by BLASTx and BLASTn analyses. Several known and unknown representatives of Coronaviridae, Picobirnaviridae, Reoviridae, Retroviridae, and unclassified Deltavirus were identified in the cloacal swab samples. However, no Newcastle disease virus (family Paramyxoviridae) was detected in the cloacal swabs, although they were detected in the oropharyngeal swabs of chickens sampled in Nairobi, Busia, and Trans Nzoia. Additionally, sequences representative of Paramyxoviridae, Coronaviridae, and Retroviridae were identified in the oral-pharyngeal swab samples. Infectious bronchitis virus and rotavirus were chickens' most prevalent coinfections associated with the Newcastle disease virus. The detection of these coinfections suggests that these viruses are significant threats to the control of Newcastle disease as the Newcastle disease virus vaccines are known to fail because of these coinfections. Therefore, this study provides important information that will help improve disease diagnosis and vaccine development for coinfections associated with the Newcastle disease virus.

Isolation, full sequence analysis, and in situ hybridization of pigeon paramyxovirus-1 genotype VI.2.1.1.2.2 from oriental turtle doves (Streptopelia orientalis).

Pigeon paramyxovirus-1 (PPMV-1), a genetic variant of avian paramyxovirus-1 (APMV-1), has been identified in Columbiformes and is the primary cause of diseases in captive and free-ranging pigeons. However, it has also been reported that PPMV-1 can infect chickens naturally and experimentally, thus posing a potential threat to the poultry industry. This study investigated a lethal outbreak of paramyxovirus infection that occurred among 16 oriental turtle doves (Streptopelia orientalis) in a walk-in aviary at a zoo from March to April 2021. Necropsies were performed, and histopathological findings revealed mild to moderate lymphoplasmacytic infiltration in several organs, such as the pancreas, liver, kidneys, and lungs. Reverse transcription polymerase chain reaction (RT-PCR) using formalin-fixed paraffin-embedded tissue blocks, virus isolation from fresh tissue, and in situ hybridization against the fusion (F) protein confirmed the diagnosis for PPMV-1 infection. The isolated strain NTU/C239/21 was fully sequenced by next-generation sequencing, and the results of phylogenetic analyses revealed that the F protein of NTU/C239/21 shared 98.8% nucleotide sequence identity with Pigeon/Taiwan/AHRI121/2017, which was isolated from a feral pigeon in Taiwan. The present study is the first to identify PPMV-1 infection in Streptopelia orientalis and suggests that Streptopelia orientalis may also play an important role in spreading the infection, similar to pigeons in APMV-1 spreading.

Phylogenetic analysis of Newcastle disease virus detected in Eritrea between 2017 and 2021.

Thirty-five samples collected from chickens in 13 commercial farms in Eritrea between 2017 and 2021 following reports of disease were screened for Newcastle disease virus. Seventeen samples (50%) were shown to be positive by RT-PCR. An initial analysis of partial fusion (F) gene sequences of 10 representative samples indicated that the viruses belonged to subgenotype VII.1.1. Subsequently, full F gene sequence analysis of four of these representative samples confirmed the genotype of the viruses but also revealed that they were not identical to each other suggesting different origins of the VII.1.1 subgenotype viruses circulating in Eritrea. These data have implications for the control of Newcastle disease within the poultry population in Eritrea.

Understanding the disease and economic impact of avirulent avian paramyxovirus type 1 (APMV-1) infection in Great Britain.

Newcastle disease (ND) is a notifiable disease affecting chickens and other avian species caused by virulent strains of Avian paramyxovirus type 1 (APMV-1). While outbreaks of ND can have devastating consequences, avirulent strains of APMV-1 generally cause subclinical infections or mild disease. However, viruses can cause different levels of disease in different species and virulence can evolve following cross-species transmission events. This report describes the detection of three cases of avirulent APMV-1 infection in Great Britain (GB). Case 1 emerged from the 'testing to exclude' scheme in chickens in Shropshire while cases 2 and 3 were made directly from notifiable avian disease investigations in chicken broilers in Herefordshire and on premises in Wiltshire containing ducks and mixed species, respectively). Class II/genotype I.1.1 APMV-1 from case 1 shared 99.94% identity to the Queensland V4 strain of APMV-1. Class II/genotype II APMV-1 was detected from case 2 while the class II/genotype I.2 virus from case 3 aligned closely with strains isolated from Anseriformes. Exclusion of ND through rapid detection of avirulent APMV-1 is important where clinical signs caused by avirulent or virulent APMV-1s could be ambiguous. Understanding the diversity of APMV-1s circulating in GB is critical to understanding disease threat from these adaptable viruses.

Alternative method using Real Time PCR for evaluation of inactivated Newcastle disease viral vaccine

The present work aims to establish a new alternative protocol to evaluate in vitro potency of inactivated Newcastle disease virus vaccine using Real Time PCR. Aqueous phases of seven inactivated Newcastle disease virus vaccines batches of different manufacturers were extracted by isopropyl myristate. The Newcastle disease virus antigen of each vaccine sample was determined by a standard Real Time PCR assay. Vaccines were inoculated into separate groups of 3-week-old specific pathogen free chickens using the recommended dose of vaccine. The immunogenicity was assessed for each vaccine by the Newcastle disease virus hemagglutination inhibition antibody titers. Individual serum samples were collected 4 weeks post vaccination, then vaccine efficacy and protection rates were recorded after challenge test of birds vaccinated with the virulent Newcastle disease virus. There is the possibility of using the Real Time PCR as an in vitro assay for vaccine evaluation. The Cycle Threshold values were ranged between 21.17 and 25.23. On the other hand, the hemagglutination inhibition titers ranged between 7.1 log2 to 6.2. The comparison between the Cycle Threshold values of the antigen extracts and the corresponding results of challenge test and in vivo hemagglutination inhibition assays using sera of vaccinated birds proved a strong correspondence between the in vitro and in vivo results(AU)

El presente trabajo pretende establecer un nuevo protocolo alternativo para la evaluación in vitro de la potencia de la vacuna de virus inactivado contra la enfermedad de Newcastle mediante PCR en tiempo real. Las fases acuosas de siete lotes de vacunas inactivadas contra el virus de la enfermedad de Newcastle de distintos fabricantes se extrajeron mediante miristato de isopropilo. El antígeno del virus de la enfermedad de Newcastle de cada muestra de vacuna se determinó mediante un ensayo estándar de PCR en tiempo real. Las vacunas se inocularon en grupos separados de pollos libres de patógenos específicos de 3 semanas de edad utilizando la dosis recomendada de vacuna. La inmunogenicidad se evaluó para cada vacuna mediante los títulos de anticuerpos de inhibición de la hemaglutinación del virus de la enfermedad de Newcastle. Se recogieron muestras individuales de suero 4 semanas después de la vacunación y, a continuación, se registraron la eficacia de la vacuna y los índices de protección tras la prueba de reto de las aves vacunadas con el virus virulento de la enfermedad de Newcastle. Existe la posibilidad de utilizar la PCR en tiempo real como ensayo in vitro para la evaluación de vacunas. Los valores del umbral de ciclo oscilaron entre 21,17 y 25,23. Por otra parte, los títulos de anticuerpos inhibidores de la hemaglutinación oscilaron entre 7,1 log2 y 6,2. La comparación entre los valores del umbral de ciclo de los extractos de antígeno con los resultados correspondientes de la prueba de reto y los ensayos de inhibición de la hemaglutinación in vivo, utilizando sueros de aves vacunadas, demostró una fuerte correspondencia entre los resultados in vitro e in vivo(AU)

Investigation of Newcastle Disease Virus Infection in Pet Birds in the Southwest of Iran.

Newcastle disease virus (NDV) is a serious threat to the international poultry industry. Therefore, to determine the role of pet birds (Psittaciformes and Passeriformes) in its spread and epidemiology, the presence of this virus in these birds was investigated. In this study, fecal and cloaca swabs from 63 Psittaciformes and 37 Passeriformes, along with tissue samples of dead birds, including proventriculus, trachea, lungs, and intestine, were collected from breeding and sales markets as well as the birds referred to Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Isolation of the virus was performed by injecting the suspension of the samples into the allantoic fluid of fertilized eggs, and NDV was detected in the achieved allantoic fluids by reverse transcription polymerase chain reaction. The NDV was detected in 13 allantoic samples. The partial F gene sequences of 10 positive samples were investigated, and their genetic relationship with each other as well as with other isolates in the gene bank was marked. Consequently, subgenotype VII.1.1 (VIId) was in the locus of all 10 viruses. By the amino acid cleavage site sequences of F protein, 10 isolates were determined as velogenic NDV. Moreover, all sequences were similar to each other and other Iranian isolates. Furthermore, the 112RRQKR/F117 pattern was the main amino acid (aa) sequence in the F-protein Cleavage site for VIId genotype isolates.

Newcastle Disease Genotype VII Prevalence in Poultry and Wild Birds in Egypt.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019-2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70-99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

First detection and characterisation of sub-genotype XIII.2.1 Newcastle disease virus isolated from backyard chickens in Iran.

BACKGROUND: Newcastle disease (ND) is an economically significant poultry disease worldwide. During field surveillance for ND in 2010 in Iran, a backyard chicken flock showed clinical signs of ND with 100% mortality. OBJECTIVES: We aimed to characterise genetically, biologically and epidemiologically an exotic virulent ND virus (NDV) detected in Iran. METHODS: After observing high mortality, dead birds were sampled and then disposed of by burial, and the chicken house was disinfected. Tissue samples were molecularly tested for NDV. The genetic homogeneity of the isolate RT30/2010 was tested by plaque assay, and then a large virus plaque was used for the second step of plaque purification. Fusion and matrix complete genes were sequenced and used for genotyping and epidemiological tracing. We tested biological pathotypes using mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. RESULTS: The isolate formed heterogeneous plaques in chicken embryo fibroblast cells. The second step of plaque purification produced homogeneous and large plaques. Phylogenetic analysis using both genes classified the virus into sub-genotype XIII.2.1. Nucleic acid and amino acid identities of RT30/2010 fusion gene with the closest available isolate SPVC/Karachi/NDV/43 are 97.95% and 98.73%, respectively. Isolate has 112 RRRKRF117 motif at the fusion cleavage site, and pathogenicity tests showed MDT of 56.4 h and ICPI of 1.85. CONCLUSIONS: This study presents the first detection and characterisation of a velogenic NDV of sub-genotype XIII.2.1 from Iran. Our follow-up surveillance for ND shows that timely virus detection and carcass management have led to the cessation of virus transmission in Iran.

Epidemiology of Newcastle disease in chickens of Ethiopia: a systematic review and meta-analysis.

The objective of this systematic review was to estimate the overall pooled prevalence of Newcastle disease in chickens in Ethiopia and identify the sources of heterogeneity among and within studies. The seroprevalence of Newcastle disease was estimated using a single-group meta-analysis. Attempts were also made to identify study-level variables that could explain the heterogeneity in the apparent seroprevalence of the Newcastle disease. The findings were based on 16 published articles and 33 district-level reports and were limited to studies performed during 2005-2017. Due to the presence of heterogeneity, pooled analysis from different districts was conducted using random-effects meta-analysis. The single-group summary of Newcastle disease seroprevalence in chickens was estimated to be 21.47% (19.54-23.4%) with a 95% confidence interval. Our results indicated high inter-study variability (Cochran's Q statistic = 196.2, true variance (τ2) = 0.36, inverse variance index (I2) = 90.0%, p < 0.001). Of all variables analysed, diagnostic techniques and regions were the most significant predictors (p ˂ 0.05) of heterogeneity. According to the diagnostic technique-based meta-analysis of random pooled prevalence, the haemagglutination inhibition test had the highest prevalence, followed by the enzyme-linked immunosorbent assay. In conclusion, the high-pooled prevalence estimates of the disease, combined with the scarcity of published data for the entire country of Ethiopia, indicate a significant data gap on the distribution of Newcastle disease in the country. While the high pooled prevalence tells the need for intervention to control the disease, there is also a need to assess the disease prevalence in all other parts of the country.

Simultaneous detection of velogenic Newcastle disease virus of genotype XIII 2.2 from spot-billed pelican and backyard chicken: implications to the viral maintenance and spread.

Despite the widespread occurence of Newcastle disease virus (NDV) in different avian species, there has been scanty reports on genetic characterization of NDV strains from wild birds in India. During 2017-18, a total of forty eight cloacal swab samples were collected from apparently healthy migratory birds (painted storks, n = 32 and spot-billed pelicans, n = 16) at the Telineelapuram bird sanctuary of Andhra Pradesh, India. NDV was isolated from a spot-billed pelican (NDV/Pelican/Telineelapuram/2018) which is genetically identical to that isolated from a naturally infected backyard chicken flock (NDV/Chicken/SKLM-1/2018). The isolates are found to be velogenic based on mean death time, intracerebral pathogenicity index and the putative fusion protein cleavage site (112R-R-R-K-R-F117). Phylogenetic analysis based on full-length fusion gene classified the isolates into genotype XIII, sub-genotype 2.2, however these isolates demonstrated multiple amino acid substitutions in the critical domains of F and HN proteins. The pelican strain (MIG-9) was tested for its pathogenic and transmission potential in three-weeks-old broiler chickens and the isolate proved to be highly virulent to chickens. To the best of our knowledge, this is the first evidence for the role of spot-billed pelicans in the maintenance of virulent NDV and its transmission to chickens in India. This study further highlights the role of wild birds in NDV transmission and the need for enhanced biosecurity in commercial poultry operations. Keywords: Newcastle disease virus; Pelecanus philippensis; chicken; transmission; pathogenicity; India.

Surveillance and Assessment of Risk Factors for Newcastle Disease Virus from Live Bird Retail Stalls in Lahore District of Pakistan.

Live bird markets (LBMs) in Asian countries are considered hubs for the spread of several poultry viruses. In Pakistan, there is a lack of uniformity in practices used in LBMs, which leads to the spread of poultry diseases. A cross-sectional survey was conducted in June-October 2017 to determine the circulation of Newcastle disease virus (NDV) in chickens being sold in live bird retail stalls (LBRSs) and to identify potential risk factors associated with estimated prevalence. A total of 189 stalls (n = 1134 birds) distributed in eight administrative towns of Lahore were visited. A pool of six oropharyngeal swabs was collected from each stall and tested by real-time reverse transcriptase PCR for the presence of NDV. Forty-two out of 189 swabs were found positive with an overall prevalence of 22.22% (95% confidence interval [Cl]: 16.88%-28.67%). Data for 11 potential risk factors acquired through questionnaires were analyzed by survey-weighted logistic regression and prevalence odds ratios (ORs) for associated risk factors were calculated. A final multivariable model identified three risk factors for NDV prevalence in LBRSs, including trading other poultry breeds alongside broilers (OR = 2.41; 95% confidence interval [CI] = 1.5-6.1), purchasing birds from mixed sources (OR = 3.12; 95% CI = 1.4-11.9), and number of birds sold per day (OR = 6.32; 95% CI = 1.9-23.5). Additionally, 24 selected samples were sequenced and phylogenetic analysis of the complete fusion gene (1662 bp) revealed that all isolates belonged to Subgenotype VII.2. This study provides important information on the epidemiology of NDV in Pakistan and highlights the importance of implementing surveillance and biosecurity practices in LBRSs.

Vigilancia y evaluación de factores de riesgo para el virus de la enfermedad de Newcastle en puestos de venta al menudeo de aves vivas en el distrito de Lahore en Pakistán. Los mercados de aves vivas (LBM, por sus siglas en inglés) en los países asiáticos se consideran centros de propagación de varios virus aviares. En Pakistán, existe una falta de uniformidad en las prácticas utilizadas en los mercados de aves vivas, lo que conduce a la propagación de enfermedades avícolas. Se realizó una encuesta transversal de junio a octubre del 2017 para determinar la circulación del virus de la enfermedad de Newcastle (NDV) en pollos que se venden en puestos minoristas de aves vivas y para identificar posibles factores de riesgo asociados con la prevalencia estimada. Se visitó un total de 189 puestos (n = 1134 aves) distribuidos en ocho ciudades administrativas de Lahore. Se recolectó un grupo de seis hisopos orofaríngeos de cada puesto y se analizó mediante transcripción reversa y PCR en tiempo real para detectar la presencia del virus de Newcastle. Cuarenta y dos de los 189 hisopos resultaron positivos con una prevalencia general del 22.22 % (intervalo de confianza [IC] del 95 % = 16.88­28.67). Los datos para 11 factores de riesgo potenciales adquiridos a través de cuestionarios se analizaron mediante regresión logística ponderada por encuesta y se calcularon las razones de probabilidad (OR) de prevalencia para los factores de riesgo asociados. Un modelo multivariable final identificó tres factores de riesgo para la prevalencia del virus de Newcastle en puestos minoristas de aves vivas, incluido el comercio de otras razas de aves de corral junto con pollos de engorde (OR = 2.41; IC del 95 % = 1.5­6.1), la compra de aves de fuentes mixtas (OR = 3.12; IC del 95 % = 1.4 ­11.9), y número de aves vendidas por día (OR = 6.32; IC 95% = 1.9­23.5). Además, se secuenciaron 24 muestras seleccionadas y el análisis filogenético del gene de fusión completo (1662 pb) reveló que todos los aislamientos pertenecían al subgenotipo VII.2. Este estudio brinda información importante sobre la epidemiología del virus de Newcastle en Pakistán y destaca la importancia de implementar prácticas de vigilancia y bioseguridad en los en puestos minoristas de aves vivas.

Active Surveillance and Genetic Characterization of Prevalent Velogenic Newcastle Disease and Highly Pathogenic Avian Influenza H5N8 Viruses Among Migratory Wild Birds in Southern Egypt During 2015-2018.

A total of 1007 samples (910 fecal droplets and 97 cloacal swabs) were collected from 14 species of migratory wild birds in most wetlands during 3 successive migration seasons from September to March (2015-2018) in Southern Egypt. The samples were propagated in embryonated chicken eggs and positive allantoic fluids by hemagglutination test were tested for Newcastle disease virus (NDV) and avian influenza virus (AIV) prevalence using RT-PCR and specific primers targeting the NDV fusion (F) and AIV matrix genes. Further subtyping of the AIV hemagglutinin (HA) and neuraminidase (NA) was conducted, and representative isolates were selected and sequenced for full F gene of NDVs and HA and NA genes of the AIV. Overall isolation rate of hemagglutinating viruses was 5.56% (56/1007), from them 5.36% (3/56) AIV, 85.71% (48/56) NDV and 8.93% (5/56) co-infection of NDV and AIV was detected. The sequences analysis of full F genes of 10 NDV isolates revealed that they have multi-basic amino acid motifs 111E/GRRQKR/F117 as velogenic strains with nucleotides and amino acids similarities of 96-100%. In addition, they phylogenetically clustered into groups and subgroups within genotype VII.1.1 and sub-genotype VIIj with a close relation to NDVs isolated from chickens in Egypt. The AIV H5N8 subtype was in clade 2.3.4.4b with a highly pathogenic nature and close relation to Egyptian domesticated H5N8 viruses rather than those from wild birds. The current data showed the contribution of migratory birds to the continuous circulation of virulent NDV and AIV H5N8 among domesticated chickens in Southern Egypt.

Epidemiological surveillance of Newcastle disease virus in Egypt - a 6-year cohort study.

Newcastle disease (ND) is one of the most important poultry diseases worldwide and can lead to annual losses of up to 80% of backyard chickens in Africa. A retrospective cohort of 6 years was planned to screen the NDV in intensive chicken and turkey flocks. The existence of velogenic NDV strain was screened in different poultry flocks showing suspected signs of NDV using real-time RT-PCR targeting the F gene of the velogenic strain. A total of 843 poultry flocks were screened during the cohort. Samples were classified based on the month and year as well as the poultry type. All flocks should be negative for avian influenza virus as an inclusion criterion of the study. The F gene of a randomly selected positive sample from each year as well as an archival sample from 2005 was sequenced. An overall of 52.4% (443/842) of the tested farms showed positive results for the velogenic NDV. The cumulative percentage of positive flocks to the total positive flocks per month ranged from 5.9 to 11.8%. The results revealed that NDV is circulating across all months annually without evidence of seasonal tendency of the disease. Most of the strains belong to genotype VII.1.1, with only two strains related to XXI.1.1 and XXI.2. All VII.1.1 strains possess arginine at 27 position while XXI.1.1 and XXI.2 strains showed cysteine at 27 and amino acid substitutions in the signal peptide, cleavage site, and neutralizing epitopes. In conclusion, the current molecular epidemiological surveillance confirms the enzootic nature of NDV. It circulates all year round with no evidence of seasonal incidence. Genotype VII is the most predominant NDV genotype in Egypt.