Intracerebral Pathogenicity Index Research Articles

Assessment of PPMV-1 Genotype VI Virulence in Pigeons and Chickens and Protective Effectiveness of Paramyxovirus Vaccines in Pigeons.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 106 EID50/0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

RT-PCR based detection and pathotypical characterization of Newcastle disease virus isolated from layer chickens in Mymensingh, Bangladesh

The Newcastle disease virus (NDV) poses a significant threat to commercial chicken operations and is prevalent in Bangladesh. This rapidly spreading viral illness has severe economic implications for the poultry industry globally. In Bangladesh, the velogenic strain of NDV is particularly widespread. This study utilized primers from the Fusion (F) and Matrix (M) genes along with haemagglutination (HA) testing and reverse transcription-polymerase chain reaction (RT-PCR) to isolate and identify NDV. Post-mortem examinations provided 12 samples (from brain, trachea, and proventriculus) taken from four birds suspected of having NDV from Trishal and Mymensingh Sadar. The samples were injected into 10-day-old embryonated chicken eggs using the chorioallantoic membrane technique. Embryos and allantoic fluid (AF) were collected at 48 and 96 hours post-infection. NDV-infected embryos showed edema and hemorrhage. Significant hemagglutination was observed in the allantoic fluid tested with HA. Through the HA test and lesion observation, four NDV isolates were identified. Pathotypic characterization of the field isolates using the Intracerebral Pathogenicity Index (ICPI) and Mean Death Time (MDT) indicated velogenic strains (ICPI: 1.5–2.0, MDT: <60). RT-PCR confirmed the NDV status of all four isolates using primers for partial amplification of the F gene (839 bp) and the common sequence of the ‘F-M’ genes (970 bp). This study demonstrates that RT-PCR is an effective molecular technique for the rapid and confirmatory identification of velogenic NDV strains, particularly prevalent in Mymensingh at the field level.

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

ABSTRACT Newcastle disease virus (NDV) is among the most important viruses of poultry; however, variants also cause clinical diseases in birds of the family Columbidae. From 2014 onwards, notable mortality rates have been observed in racing and rock pigeons, with a correlation to subgenotypes XXI.1.1 and XXI.1.2 of genotype XXI in Pakistan. In this study, we performed isolation and genetic characterization of 29 pigeon-derived NDV isolates of sub-genotypes XXI.1.1, XXI.1.2 and VII.2. Phylogenetic analysis based on complete fusion gene sequences classified seven (n = 7) isolates into sub-genotype XXI.1.1 and they were genetically closely related to isolates previously characterized in Pakistan (0.05–0.43% nucleotide divergence), and in Iran, Russia, Egypt, Nigeria, Kazakhstan and Ukraine (3.75% to 6.67% nucleotide divergence) in the period from 2005–2018. In addition, nineteen (n = 19) isolates were clustered with viruses previously isolated in Pakistan (2014–2018) in sub-genotype XXI.1.2. In addition, three (n = 3) F-gene sequences assigned to subgenotype VII.2 were identified along with viruses repeatedly isolated (2011–2020) from several poultry and non-poultry avian species in Pakistan. The analysis showed that all isolates of sub-genotypes XXI.1.1 and XXI.1.2 were mesogenic (1.42–1.49) and the isolates belonging to sub-genotype VII.2 were velogenic (1.63–1.71) in nature according to the intracerebral pathogenicity index (ICPI). This study delivers a comprehensive description of the prevalence of sub-genotypes XXI.1.1 and XXI.1.2 in Pakistan since 2014. Additionally, it underscores the role of free-range pigeons as effective carriers of virulent sub-genotype VII.2 viruses, impacting both poultry and non-poultry avian species reared in open environments. 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.

Characterization of the dominant strain (G-VII) of Newcastle disease viruses isolated from commercial chickens in Bangladesh during recent outbreaks.

Newcastle disease virus genotype VII (NDV-GVII), an extremely infectious pathogen, has been causing severe economic consequences for the chicken industry. The current study aimed to isolate and characterize NDV-GVII from commercial chickens in Bangladesh during a recent outbreak. From clinically suspected chickens from 70 commercial poultry farms, a total of 420 samples (trachea, lungs, and brain tissue) were collected. The samples were cultivated in 9-10 day-old seronegative embryonated chicken eggs (ECEs) after evaluating them using the rapid Newcastle disease virus (NDV) antigen detection kit. The hemagglutination (HA) inhibition test, agar gel immune diffusion (AGID) test, molecular detection by reverse transcription-polymerase chain reaction (RT-PCR), and phylogenetic studies using gene sequences of fusion (F) protein. The HA pattern of isolated NDV was determined using different avian and mammalian red blood cells (RBCs). The pathogenicity of the isolated virus was evaluated using mean death time (MDT), intravenous pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). The study found 87 NDV samples positive using the rapid NDV Ag detection kit and then 60 positives for virus isolation in ECEs. All 60 isolates were positive for NDV by HI, AGID, and RT-PCR. Phylogenetic tree analysis indicated that recent NDV isolates belong to genotype VII and exhibit a similarity of 99.7%-98.5% with isolates from Bangladesh, Iran, and India. The new isolates, identified as velogenic strains of NDV, possess an F protein cleavage site with 112-R-T-K-R-F-117 amino acid motifs. The isolated NDV showed diversified HA activity while using RBCs from birds and mammals. The results of ICPI, IVPI, and MDT indicated that the recent NDV isolates were very virulent. This study concluded that NDV-GVII is prevalent in commercial poultry farms in Bangladesh.

Comparative protective efficacy of a newly generated live recombinant thermostable highly attenuated vaccine rK148/GVII-F using a single regimen against lethal NDV GVII.1.1

ABSTRACT The ongoing global spread of Newcastle disease underscores the crucial need for continued research on the efficacy of current vaccines against various circulating strains of Newcastle disease virus (NDV). The fusion gene of a representative Egyptian genotype VII.1.1 strain was used to substitute its corresponding gene in the K148/08 vaccinal strain after site directly mutating its cleavage site from 112RRQKRF117 to 112GKQGRL117. Fusion gene exchange between GVII and GI did not affect the thermostability of GI K148/08. Attenuation of the rescued virus was confirmed by mean death time 144 h with an intracerebral pathogenicity index of 0.00. Survival analysis after the challenge experiment confirmed that 107 EID50 was the protective dose of rK148/GVII-F. The haemagglutination inhibition level of antibodies required for full clinical protection was > 3.3 log2 for rK148/GVII-F and > 4.1 log2 for both K148/08 and LaSota. Oropharyngeal viral shedding was reduced on the 5th and 7th days post-challenge in the rK148/GVII-F vaccinated group. Replication and tropism investigations confirmed the respirotropic nature of LaSota, enterotropic nature of K148/08, and further attenuation of rK148/GVII-F. Altogether, rK148/GVII-F is a thermostable, safe, effective, and genetically stable vaccine candidate that could be adequate for use in countries that encounter GVII.1.1 and in those with tropical climate, such as most Middle Eastern countries. RESEARCH HIGHLIGHTS A thermostable, safe, and effective NDV GVII recombinant vaccine was generated. Fusion gene replacement with GVII did not affect GI K148/08 virus thermostability. Strain rK148/GVII-F provided adequate protection against a lethal NDV challenge. Oropharyngeal shedding was significantly reduced on post-challenge days 5 and 7.

Isolation and Biological Characterization of Newcastle Disease Virus (NDV) Field Isolate Pigeon (Columba livia) from Live Bird Market, East Java in 2019

Background: Avian Paramyxovirus (APMV) type-1 is the leading cause of Newcastle Disease (ND) and taxonomically belongs to the family Paramyxoviridae, genus Avulavirus. Due to its high transmission rate, Newcastle Disease (ND) is included in the A list by the OIE. Purpose: To determine the biological characterize the Newcastle Disease Virus (NDV) field isolate of pigeons (Columba livia) using Mean Death Time (MDT), Intracerebral Pathogenicity Index (ICPI), and Intravenous Pathogenicity Index (IVPI). Methods: Twenty pigeon organ samples were obtained from bird markets in East Java, and one was used as a positive control (LaSota). Organs were isolated from embryonated chicken eggs, identified by the HA test, and then confirmed by the HI test. Furthermore, positive samples were tested for MDT with a 10-1-10-18 dilution (0.1 ml and observed for eight days. The ICPI test used a fresh NDV suspension (0.05 ml and was observed for eight days. The IVPI test used a dose of 0.1 ml and was observed for ten days. Result: The MDT values of isolates MB1/NDV/19, MB2/NDV/19, MB3/NDV/19, and MG1/NDV/19 were 91.2 hours, 112.8 hours, 110.4 hours, and 124,8 hours. The ICPI values of isolate MB1/NDV/19 was 0.2375, MB2/NDV/19 was 0.375, MB3/NDV/19 was 0.5375, and MG1/NDV/19 was 0.3. The IVPI value of isolate MB1/NDV/19 was 0, MB2/NDV/19 was 0, MB3/NDV/19 was 0, and MG1/NDV/19 was 0. Conclusion: All four field samples were positive for NDV as a lentogenic strain based on the MDT, ICPI, and IVPI tests.

Virulent Newcastle disease virus genotypes V.3, VII.2, and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania

Oropharyngeal (OP) and cloacal (CL) swabs from 2049 adult backyard chickens collected at 12 live bird markets, two each in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga regions of Tanzania were screened for Newcastle disease virus (NDV) using reverse transcription real-time PCR (rRT-PCR). The virus was confirmed in 25.23% of the birds (n = 517; rRT-PCR CT ≤ 30), with the highest positivity rates observed in birds from Dar es Salaam region with higher prevalence during the dry season (September–November 2018) compared to the rainy season (January and April–May 2019). Next-generation sequencing of OP/CL samples of 20 out of 32 birds that had high amounts of viral RNAs (CT ≤ 25) resulted in the assembly of 18 complete and two partial genome sequences (15,192 bp and 15,045–15,190 bp in length, respectively) of NDV sub-genotypes V.3, VII.2 and XIII.1.1 (n = 1, 13 and 4 strains, respectively). Two birds had mixed NDV infections (V.3/VII.2 and VII.2/XIII.1.1), and nine were coinfected with viruses of families Astroviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, Pneumoviridae, and Reoviridae. Of the coinfecting viruses, complete genome sequences of two avastroviruses (a recombinant chicken astrovirus antigenic group-Aii and avian nephritis virus genogroup-5) and two infectious bronchitis viruses (a turkey coronavirus-like recombinant and a GI-19 virus) were determined. The fusion (F) protein F1/F2 cleavage sites of the Tanzanian NDVs have the consensus motifs 112 RRRKR↓F 117 (VII.2 strains) and 112 RRQKR↓F 117 (V.3 and XIII.1.1 strains) consistent with virulent virus; virulence was confirmed by intracerebral pathogenicity index scores of 1.66–1.88 in 1-day-old chicks using nine of the 20 isolates. Phylogenetically, the complete F-gene and full genome sequences regionally cluster the Tanzanian NDVs with, but distinctly from, other strains previously reported in eastern and southern African countries. These data contribute to the understanding of NDV epidemiology in Tanzania and the region.

Genomic and biological characterization of Pigeon Paramyxovirus Type-1 isolated from Indian pigeons: First report on the six nucleotide insertion in the non-coding region of nucleoprotein gene

Genotypic analysis of Pigeon Paramyxovirus Type-1 (PPMV-1) virus, in corroboration with pathotyping, underpins the knowledge on the occurrence of PPMV-1 mediated Newcastle disease in poultry. In the present study, two PPMV-1 isolates, namely D167 and D168, obtained from feral pigeons of India in the year 2014 were analysed for their genotypic and pathotypic characteristics. Phylogenetic analysis of Fusion (F) gene grouped D167 and D168 under sub-genotypes VI.2.2.2. and VI.2.1.1.2.2. respectively. The separate positioning of these isolates within genotype VI is corroborated with the variation in restriction enzyme sites analysed through virtual restriction mapping. Analysis of FPCS region showed that both D167 and D168 possessed 112RRQKRF117 amino-acid pattern. Intra-cerebral pathogenicity index (ICPI) of D167 and D168 was 1.57 and 1.3, and mean death time (MDT) was 48 and 70 hrs respectively, designating D168 as a mesogenic pathotype, and D167 as a velogenic pathotype. Histopathological analysis of proventriculus, intestine, lungs and spleen of chickens infected with D167 and D168 isolates through intramuscular (I/M), subcutaneous (S/C) and oculo-nasal/oral (O/N) routes of inoculation showed similar severity of tissue damage. This study shows that PPMV-1 isolates belonging to two different sub-genotypes without a common ancestor, can occur together in a particular geographical region. It also shows that PPMV-1 gains virulence in chickens, which needs to be addressed in order to avoid emergence of PPMV-1 as new velogenic variant causing Newcastle disease in chickens.

The genetic and biological characterization of the first avian paramyxovirus serotype 14 isolated from chicken in China.

In October 2020, an avian paramyxovirus serotype 14 (APMV-14)-designated chicken/Fujian/2160/2020 (FJ2160) was isolated from tracheal and cloacal swab sample of chicken collected from live bird market in Fujian province in China during the active surveillance program. The complete genome of FJ2160 comprised 15,444 nucleotides (nt) complying with the paramyxovirus "rule of six" and encoded six non-overlapping structural proteins in the order of 3'-NP-P-M-F-HN-L-'5. The complete genome sequence analysis showed that FJ2160 had the highest identity (90.0%) with the APMV-14 isolated from Japan, while the nucleotide sequence identities of FJ2160 and other APMVs ranged from 42.4 to 51.1%. The F protein cleavage site was TREGR↓L, which resembled a lentogenic strain of APMV-1. Phylogenetic analysis revealed that the FJ2160 closest relative was APMV-14. The intracerebral pathogenicity index (ICPI) tests indicated that the virus was lentogenic. This is the first report of APMV-14 in China. These results provide evidence that APMV-14 could infect chickens and reveal the genetic characteristics and biological properties of the virus, which can help to better understand this new emerging APMV-14.

Genome Sequencing and Characterization of an Avian Orthoavulavirus 1 VG/GA-like Isolate with a Unique Fusion Cleavage Site Motif.

A complete genome sequence of a VG/GA -like strain of avian orthoavulavirus 1 (AOAV-1) was identified by nontargeted next-generation sequencing of an oropharyngeal swab sample collected from a carcass of a 12-mo-old backyard chicken. The isolate has a fusion (F) protein cleavage site motif consistent with a low virulent AOAV-1, but it has a unique motif with phenylalanine at position 117 (112G-R-Q-G-R↓F117), which is typical for virulent AOAV-1 strains. The one nucleotide difference at the cleavage site compared to other low-virulence viruses made the isolate detectable by F-gene-specific real-time reverse transcription-PCR (rRT-PCR) developed as a diagnostic test to specifically detect virulent strains. The mean death time determined in eggs and intracerebral pathogenicity index determined in chickens classified the isolate as lentogenic. This is the first report of a lentogenic VG/GA-like virus with a phenylalanine residue at position 117 of the F protein cleavage site in the United States. In addition to concern for potential pathogenic shift of the virus through additional changes at the cleavage site, our finding warrants increased awareness of diagnosticians of potential false positive F-gene rRT-PCR tests.

Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

Construction of Novel Thermostable Chimeric Vaccine Candidates for Genotype VII Newcastle Disease Virus.

Genotype VII Newcastle Disease Virus (NDV) has caused a pandemic in many countries and usually causes fatal consequences in infected chickens. Although current commercial attenuated NDV vaccines can provide an ideal protection against genotype VII NDV, they cannot completely prevent the infection and viral shedding, and the genotype of some vaccine strains cannot match with the prevalent strain. In this study, in order to construct a thermostable and genotype VII-matched live attenuated vaccine, we used a thermostable genotype VIII virulent HR09 strain as the backbone and replaced its F gene with that of the genotype VII DT-2014 strain. Meanwhile, the cleavage site of F gene of DT-2014 was mutated to that of class I F protein and avirulent class II F protein, respectively. The results showed that the two chimeric viruses, designated rcHR09-CI and rcHR09-CII, shared a similar growth kinetics and thermostability with their parental HR09 strain. Mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests showed that the two chimeric viruses were highly attenuated. Though both chimeric NDVs and La Sota vaccine strain could provide complete protection to immunized chickens against the challenge of virulent genotype VII ZJ1 strain, the two chimeric NDVs could induce a higher level of antibody response against ZJ1 strain and could significantly reduce the viral shedding compared with La Sota vaccine strain. In conclusion, our study constructed two chimeric thermostable genotype VII-matched NDV vaccine candidates, which provided complete protection against the challenge of virulent genotype VII NDV.

The First Report of a Virulent Newcastle Disease Virus of Genotype VII.2 Causing Outbreaks in Chickens in Bangladesh.

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020-2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV.

Thermostability and Immunogenicity of Genotype II Avian Orthoavulavirus (AOaV-1) Isolates from Duck (Anas platyrhynchos) and Parrot (Eclectusroratus).

Newcastle disease (ND) is a highly contagious viral disease of poultry causing significant economic losses worldwide. Vaccination is considered the most reliable approach to curb the economic menace that is ND, but the thermolabile nature of Newcastle disease virus (NDV) vaccination poses a significant threat to its protective efficacy. This study aimed to profile the thermostability of NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) and evaluate their immunogenic potential in chicks. Fusion protein cleavage site (FPCS) and phylogenetic analysis demonstrated the lentogenic nature of both the isolates/strains and classified them as class II genotype II NDV. The characterized NDV isolates were adapted in specific-pathogen-free (SPF) chicks by serially passaging. Biological pathogenicity assessment of chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) revealed both the isolates to be avirulent with a mean death time (MDT) of more than 90 h and an intracerebral pathogenicity index (ICPI) ranging from 0.2 to 0.4. Both of the NDV isolates displayed varied thermostability profiles. PSD44C was the most thermostable strain as compared to PSP91C and the commercially available LaSota vaccine strain. The immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Based on these results, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects. Therefore, PSD44C is suitable for further research and vaccine development.

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

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. We aimed to characterise genetically, biologically and epidemiologically an exotic virulent ND virus (NDV) detected in Iran. 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. 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. 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.

A molecular, epidemiological and pathogenicity analysis of pigeon paramyxovirus type 1 viruses isolated from live bird markets in China in 2014–2021

To expand our understanding of the epidemiology of pigeon paramyxovirus type 1 (PPMV-1) in China, risk-based active surveillance was undertaken with pigeon swabs collected from live bird markets in 2014–2021. Seventy-six PPMV-1 strains were isolated from 12 provinces (60%) of the 20 provinces surveyed, and the positive rates of PPMV-1 varied from 0.50% to 3.19% annually. The complete genomic sequences of 18 representative viruses were analyzed, revealing a genome of 15,192 nucleotides, with the gene order 3′-NP-P-M-F-HN-L-5′. All isolates contained the 112RRQKRF117 cleavage site in the fusion (F) protein, a characteristic generally associated with virulent Newcastle disease viruses (NDVs), and the intracerebral pathogenicity index values (1.05–1.41) of four isolates indicated their virulence. A challenge experiment also demonstrated that all four isolates are pathogenic to pigeons, with morbidity rates of 60–100% and mortality rates of 0–30%. A further analysis of the functional domains of the F and HN proteins revealed several mutations in the fusion peptide, signal peptide, neutralizing epitopes, heptad repeat region, and transmembrane domains, and the substitution of cysteine residue 25 (C25Y) and substitutions in the HRb region (V287I) of the F protein and the transmembrane domain (V45A) of the HN protein may play important roles in PPMV-1 virulence. In a phylogenetic analysis based on the complete sequences of the F gene, all eighteen isolates all clustered into sub-genotype VI.2.1.1.2.2 (VIb) in class II, and shared high nucleotide sequence identity, indicating that the PPMV-1 strains in sub-genotype VI.2.1.1.2.2 are the predominant PPMV-1 viruses in pigeons in China and that the variations in these viruses have been relatively stable over the past 8 years. This study identifies the genetic and pathogenicity characteristics of the PPMV-1 strains prevalent in China and extends our understanding of the prevalence of this virus in China.

Molecular Detection and Pathotyping of Newcastle Disease Virus in Pigeons from Karnataka, India

Background: Newcastle disease (ND) is most important devastating diseases of poultry and it causes huge economic losses to poultry farmers throughout the world. One of the limiting factors in controlling the disease’s spread is the wide host range. While pigeons are highly susceptible and produce neurological signs of the disease. ND was suspected in five dead pigeons and detection of Avian Paramyxovirus-1 (APMV-1) in pigeons (Columba livia domestica) was confirmed by Reverse Transcriptase PCR (RT-PCR). Methods: Spleen, lung and trachea were collected and lysates were propagated in allantoic fluids for haemagglutination (HA) by chicken erythrocytes and pigeon convalescent serum inhibited haemagglutination (antibody titers greater than 1/16). Result: A 356 bp product was amplified from infected allantoic fluid by targeting a partial Fusion protein gene, including its cleavage site. Pigeon sample isolates were grown in specific pathogen Free (SPF) embryonated chicken eggs (ECE) showed virulent pathotype based on embryonic mean death time (MDT) of 42 hours and the intra-cerebral pathogenicity index (ICPI) in day-old chicks was 1.8. This is the first instance of APMV-1 identification in Karnataka with the possibility of a disease spillover in pigeons. In this scenario, more epidemiological works are suggested for surveillance of NDV in pigeons in this area of work along with implementation of suitable prevention and control measures.

Assessment of the virulence for chickens of Newcastle Disease virus with an engineered multi-basic cleavage site in the fusion protein and disrupted V protein gene

Newcastle Disease virus (NDV) has shown promise as an oncolytic virus for treatment of a wide range of tumours. NDV with a multi-basic cleavage site (MBCS) in the fusion (F) protein (NDV F3aa) has increased oncolytic efficacy in several tumour models, but also increased virulence in chickens compared to non-virulent NDV F0, raising potential environmental safety issues. Previously, we generated a variant of NDV F3aa with a disrupted V protein gene and a substitution of phenylalanine to serine at position 117 of the F protein (NDV F3aa-S-STOPV). Compared to NDV F3aa this virus had decreased virulence in embryonated chicken eggs. In this study, the virulence of the virus was evaluated upon inoculation of six-week-old chickens through a natural infection route and by determination of the intracerebral pathogenicity index (ICPI). Based on these data NDV F3aa-S-STOPV classified as a non-virulent virus. Although NDV F3aa was classified as a virulent virus based on the ICPI, the virus was also less pathogenic than NDV F0 upon inoculation of six-week-old chickens. These data indicate that NDV with a MBCS is not necessarily pathogenic in chickens. In addition, these data show that F3aa-S-STOPV is safe to use in viro-immunotherapies without posing a threat for chickens upon accidental exposure.

Emergence of XIII.2.2 genotype of Avian Avulavirus-1 with unique FPCS site in India

In the present study, two isolates namely D162 and D165 obtained from Newcastle disease outbreaks inbackyard poultry in Tamil Nadu were subjected to phylogenetic analysis using complete Fusion (F) gene sequenceto understand the circulation and evolution of Avian Avulavius-1 (AAv1). Further, these isolates were pathotypedbased on mean death time and intra-cerebral pathogenicity index. Pathotyping and genotyping of D162 and D165showed that these isolates belonged to velogenic pathotype and XIII.2.2 sub-genotype. In addition to this, the Fusion Protein Cleavage Site (FPCS) of F gene was analysed for amino acid sequence pattern. The amino acid sequence of FPCS of both D162 and D165 was 112RRRKRF117, which is unique among XIII.2.2 genotypes of AAv1, whereas it is common among Pigeon Paramyxovirus. It was hypothesized that cross-species infection by AAv1 has led to emergence of isolates with increased virulence.

Molecular techniques are better choice as compared to conventional methods for pathotypic characterization of Newcastle disease virus in poultry birds

Newcastle disease (ND) has importance in the poultry industry across the world. Despite vaccination, outbreaks of ND have been reported from various parts of Pakistan. The purpose of current study was to characterize the Newcastle disease virus (NDV) through different methods i.e., molecular techniques and conventional methods and evaluate their efficiency. To isolate pathogenic NDV, samples were taken from the commercial poultry farms after an outbreak, while for the non-pathogenic NDV isolation commercially available Lasota vaccine was used. In the first step NDV was tested by F gene sequence through RT-PCR. The predicted sequence of the amino acid in fusion protein indicates the following motif 112R-R-Q-R-R-F117 in pathogenic and 112G-R-Q-G-R-L117 in non-pathogenic NDV strains. While in the second phase, the pathotypic characterization of NDV was done through conventional methods like Intracerebral pathogenicity index (ICPI) and Mean death time (MDT). The results from conventional methods were also in agreement with standard values. The findings of current study suggest that for the assessment of genetic nature and the rapid diagnosis of NDV; molecular techniques are better choice than conventional methods.