Velogenic Viscerotropic Newcastle Disease Virus Research Articles

Advancement in Vaccination Against Newcastle Disease: Recombinant HVT NDV Provides High Clinical Protection and Reduces Challenge Virus Shedding with the Absence of Vaccine Reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.

Characterization of avian paramyxovirus type 1 from migratory wild birds in chickens

Newcastle disease virus (NDV) is one of the most important infectious agents in the poultry industry, and vaccines against it have been widely used for prevention and control. Live vaccines, which can replicate in the respiratory and digestive systems, have been especially needed in areas with outbreaks of viscerotropic velogenic Newcastle disease. Towards the goal of searching for a new live vaccine candidate, avian paramyxovirus type 1 (APMV-1) was isolated from the faeces of wild birds. Three APMV-1 strains thus isolated were characterized in terms of phylogeny, pathogenicity, immunogenicity and tissue tropism, and on the basis of these analyses were classified as lentogenic genotype I NDV. CBU2179, one of the three APMV-1 strains, was selected and was evaluated in terms of its efficacy and safety in specific pathogen-free chickens and commercial broilers. The manufactured trial vaccine from this strain, also called CBU2179, induced similar immune responses to those of VG/GA and B1 commercial vaccines, and provided 100% protection against challenge from viscerotropic velogenic NDV, KJW/49 strain (the official challenge strain in Korea). Also, the CBU2179 virus was re-isolated and persisted as long as or longer than other vaccine strains in both the respiratory and alimentary tracts. Therefore, the CBU2179 strain may represent a good candidate for a live Newcastle disease vaccine to protect chickens against viscerotropic velogenic NDV.

Complete Genome and Clinicopathological Characterization of a Virulent Newcastle Disease Virus Isolate from South America

Newcastle disease (ND) is one of the most important diseases of poultry, negatively affecting poultry production worldwide. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), a negative-sense single-stranded RNA virus of the genus Avulavirus, family Paramyxoviridae. Although all NDV isolates characterized to date belong to a single serotype of APMV-1, significant genetic diversity has been described between different NDV isolates. Here we present the complete genome sequence and the clinicopathological characterization of a virulent Newcastle disease virus isolate (NDV-Peru/08) obtained from poultry during an outbreak of ND in Peru in 2008. Phylogenetic reconstruction and analysis of the evolutionary distances between NDV-Peru/08 and other isolates representing established NDV genotypes revealed the existence of large genomic and amino differences that clearly distinguish this isolate from viruses of typical NDV genotypes. Although NDV-Peru/08 is a genetically distinct virus, pathogenesis studies conducted with chickens revealed that NDV-Peru/08 infection results in clinical signs characteristic of velogenic viscerotropic NDV strains. Additionally, vaccination studies have shown that an inactivated NDV-LaSota/46 vaccine conferred full protection from NDV-Peru/08-induced clinical disease and mortality. This represents the first complete characterization of a virulent NDV isolate from South America.

Vaccination with newcastle disease vaccines strain i<sub>2</sub> and lasota in commercial and local chickens in Plateau State Nigeria

Vaccination trials and comparative immunogenicity study using Newcastle disease vaccine strain I2 (NDVI2) and NDV La Sota administered to commercial and local chickens through intraocular (i/o), intramuscular (i/m), drinking water (dw), untreated sorghum, parboiled sorghum, sorghum coated with gum Arabic or commercial chick mash feed as vaccine carriers was conducted. Newcastle disease vaccine strain I2 and NDV La Sota vaccines provided protection to commercial and local chickens vaccinated through i/o, i/m or dw. No significant difference (P.0.05) was observed in the antibody titre of commercial or local chickens vaccinated with either NDVI2 or NDV La Sota vaccines administered via commercial feed, parboiled sorghum, parboiled sorghum coated with gum Arabic and untreated sorghum. NDVI2 or NDV La Sota vaccines administered through commercial feed, parboiled sorghum, parboiled sorghum coated with gum Arabic and untreated sorghum gave no or limited protection (0-22%) to the birds when challenged with a local strain of velogenic viscerotropic Newcastle disease virus Kudu 113 strain. It was concluded that the vaccine carriers used in this study were not suitable for delivery of NDVI2 or NDVS La Sota vaccines to local or commercial chickens.

Protection and Differentiation of Infected from Vaccinated Animals by an Inactivated Recombinant Newcastle Disease Virus/Avian Influenza H5 Vaccine

Specific-pathogen-free chickens immunized at 14 days of age with either an inactivated recombinant Newcastle disease virus-LaSota/avian influenza H5 (K-rNDV-LS/AI-H5) vaccine or a killed Newcastle disease/avian influenza whole-virus vaccine (K-ND/AI) were protected from disease when challenged with either A/chicken/Queretaro/14588-19/95 (H5N2), a high pathogenicity avian influenza virus (HPAIV) strain isolated in Mexico in 1995, or with a Mexican velogenic viscerotropic Newcastle disease virus (VVNDV) strain 21 days postvaccination. All nonvaccinated chickens challenged with HPAIV or VVNDV succumbed to disease, while those vaccinated with K-rNDV-LS/AI-H5 or K-ND/AI were protected from severe clinical signs and death. Both vaccines induced hemagglutination-inhibition (HI) antibody responses against NDV and AIV. Antibodies against AIV nucleoprotein were not detected by enzyme-linked immunosorbent assay (ELISA) in birds vaccinated with the inactivated rNDV-LS/AI-H5 vaccine. These chickens became positive for AIV antibodies by ELISA only after challenge with HPAIV. The data clearly indicate that the inactivated rNDV-LS/AI-H5 vaccine confers protection comparable to that of the conventional killed whole-virus vaccine against both NDV and AIV, while still allowing differentiation of infected from vaccinated animals by HI and ELISA tests.

Genomic comparison of the complete coding and intergenic regions of the VG/GA Newcastle disease virus and its respirotropic clone 5

The complete genome of the Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) and that of a plaque purified clone (clone 5) exhibiting a respiratory phenotype were sequenced and analyzed. The VG/GA strain, isolated from the intestine of healthy turkeys, replicates in the respiratory and intestinal tract of chickens. It is used worldwide as a vaccine strain and its tissue tropism is extremely important for protection against velogenic viscerotropic NDV which targets both intestinal and respiratory epithelia, inducing severe gross and microscopic damage. The clone 5, a plaque purified clone from the VG/GA strain, cannot be recovered from the intestine of infected birds, suggesting a respirotropic nature. A modified primer sequence-independent amplification method was used to sequence the complete coding regions of both viruses and to assess phylogenetic relationships and genomic basis for phenotype differences. The phylogenetic analysis grouped the VG/GA strain and the clone 5 within class II, genotype II viruses and showed that they are greater than 99.9% identical with only 5 nucleotides differences. Both are closely related to classic vaccine strains, such as LaSota and B1. Only 3 amino acid differences at the fusion protein differentiated the VG/GA strain from the clone 5. These differences may explain the differential phenotype observed in the VG/GA strain and are discussed.

Sequence Analysis of Fusion and Matrix Protein Genes of the Velogenic Viscerotropic Newcastle Disease Virus Egyptian Strain sr/76

Sequence Analysis of Fusion and Matrix Protein Genes of the Velogenic Viscerotropic Newcastle Disease Virus Egyptian Strain sr/76

Pathogenesis of conjunctivitis caused by Newcastle disease viruses in specific-pathogen-free chickens

The pathogenesis of conjunctivitis caused by Newcastle disease viruses (NDVs) was investigated in 3-week-old specific-pathogen-free chickens. The chickens were inoculated intramuscularly, by eye drop or intranasally, with 107 plaque forming units of viscerotropic velogenic Newcastle disease virus (VVNDV), mesogenic NDV or lentogenic NDV. Macroscopically, lower palpebral conjunctivae appeared red with oedematous swelling in chickens inoculated with VVNDV. Histologically, mild lesions were focal hyperplasia of the conjunctival epithelial cells with cellular infiltration in the lamina propria of the conjunctivae in the chickens inoculated with VVNDV. Moderate lesions were vascular necrosis with congestion and haemorrhages, hyperplasia of conjunctival epithelial cells, and oedema and cellular infiltration in the lamina propria of the conjunctivae. Fibrin thrombi were observed in the capillaries of the lamina propria. In marked lesions, the whole conjunctiva was affected with more severe and extensive lesions. The mesogenic and lentogenic NDV strains induced no gross conjunctival lesions, but induced mild conjunctivitis without vascular necrosis.

Virulence of Pigeon-Origin Newcastle Disease Virus Isolates for Domestic Chickens

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.

Cloning and expression of the HN gene from the velogenic viscerotropic Newcastle disease virus strain AF2240 in Sf9 insect cells.

The haemagglutinin-neuraminidase (HN) gene ofNewcastle disease virus (NDV) strain AF2240, amplifiedfrom the viral genomic RNA ( approximately 1.8 kb) was directionallycloned and inserted into a baculovirus expressionvector system. The recombinant glycoprotein expressedin Spodoptera frugiperda (Sf9) cellsshowed haemagglutinin (HA), neuraminidase (NA) andhemadsorption activities. HA activity was detected inboth extra- and intra-cellular recombinant HN(recHNAF2240) samples. In addition, both HA andhemadsorption activities were inhibited by polyclonalanti-NDV sera. Furthermore, significant expression ofthe recombinant protein was observed on the surface ofinfected cells. SDS-PAGE analysis revealed thepresence of visually distinguishable bands between the70 and 80 kDa in size that were absent in thewild-type samples. Western blot analysis showed thatthe distinct approximately 63 kDa band and a approximately 75 kDa bandcorresponded to the unglycosylated and glycosylated HNglycoprotein respectively as reported in anotherstudy. These observations indicated that the HNrecombinant protein was not only expressed on thesurface of the infected cells as well as with theviral coat protein, but also appears to be functional.

Detection of a Macrophage-Specific Antigen and the Production of Interferon Gamma in Chickens Infected with Newcastle Disease Virus

Formalin-fixed, paraffin-embedded spleen and intestinal tissues were harvested at 2 days postinfection from 4-wk-old white rock chickens infected with five different strains of Newcastle disease virus (NDV). These tissues were examined for the presence of macrophage antigen expression, virus replication, and interferon gamma (IFN gamma) production. The five strains represented all three NDV pathotypes. Viral replication and IFN gamma, as determined by riboprobe in situ hybridization, were detected only in those chickens infected with velogenic viscerotropic NDV (VVNDV) strains. Macrophage antigen expression, an indicator of macrophage activation, was determined by immunohistochemistry with a macrophage-specific antibody, CVI-ChNL-68.1. Presence of macrophage antigen was most prominent in VVNDV-infected chickens. The distribution of this antigen within tissues was far more diffuse than the staining for viral mRNA. The presence of IFN gamma mRNA was detected in the spleen and intestinal lymphoid tissue of VVNDV-infected chickens. There was also increased macrophage antigen expression in the mesogen-infected birds, but it was less dramatic than in tissues from VVNDV-infected chickens. One of two lentogen-infected birds had evidence of increased macrophage antigen expression only in the spleen.

A Comparison of the Onset of Protection Induced by Newcastle Disease Virus Strain B1 and a Fowl Poxvirus Recombinant Newcastle Disease Vaccine to a Viscerotropic Velogenic Newcastle Disease Virus Challenge

Four-week-old specific-pathogen-free white rock chickens were immunized with either a commercial recombinant fowl poxvirus-vectored Newcastle disease vaccine (FPN) expressing the hemagglutinin-neuraminidase and fusion protein genes of Newcastle disease virus (NDV) strain B1 or live NDV B1. Vaccinates and controls were challenged by eyedrop and intranasal (E/I) route with a viscerotropic velogenic NDV at 14 days postvaccination to determine the time of clearance of challenge virus. In a subsequent experiment, chickens were challenged at 3, 6, or 10 days postvaccination to determine the onset of immunity. Chickens that received a recommended field dose (1x) or a 0.01x dose of FP-N subcutaneously (s.c.) and were seropositive by hemagglutination-inhibition test at 14 days postvaccination cleared the challenge virus by 14 days postchallenge. Clinical Newcastle disease and high challenge virus titers in tissues were seen only in seronegative FP-N 0.01x dose vaccinates and controls. In a comparison of vaccination with FP-N (1x, 10(4,9) median tissue culture infective dose) s.c., B1 (10(6) median egg infective dose [EID50]) s.c., or B1 (10(6) EID50) E/I, chickens vaccinated at 6 or 10 days before challenge with all vaccines were protected against clinical disease, but only those vaccinated with B1 E/I 10 days before challenge were protected against infection with the challenge virus. Vaccination at 3 days before challenge with B1 E/I provided early protection, but severe nervous signs developed later and reduced overall protection to 60%, whereas disease in chickens vaccinated with B1 s.c. and FP-N s.c. 3 days before challenge was similar to the challenge controls.

Comparison of Pathology-based Techniques for Detection of Viscerotropic Velogenic Newcastle Disease Virus in Chickens

Two pathology-based techniques, immunohistochemistry and riboprobe in-situ hybridization, were applied to formalin-fixed, paraffin wax-embedded tissues from chickens infected with three different isolates of velogenic viscerotropic Newcastle disease virus (VVNDV). With the immunohistochemical method, viral protein was consistently detectable in the spleen and caecum at the terminal phase of the infection. With in-situ hybridization, viral nucleic acid was consistently detected in the eyelid, spleen and caecum in both the acute and terminal phases. Hybridization with anti-sense probe to detect viral mRNA was often more intense than hybridization with sense probe to detect viral genomic RNA.

Exotic Newcastle Disease in a Game Chicken Flock

A sudden increase in mortality, preceded by a short history of respiratory signs and diarrhea, occurred in a backyard flock of 48 game chickens in the Central Valley of California. Necropsy findings included severe generalized linear hemorrhages and/or ulcers in the digestive tract, larynx, and trachea. Histology revealed severe multifocal hemorrhages and necrosis in the mucosa of the respiratory and digestive tracts, vasculitis, and necrosis of lymphoid tissue. The birds were serologically negative to Newcastle disease virus; this was consistent with an acute infection. The avian paramyxovirus type 1 isolated was characterized as velogenic viscerotropic Newcastle disease virus. A thorough epidemiologic investigation was carried out, and no other premises were found to have birds with clinical signs or evidence of exposure. The entire outbreak was limited to the original backyard flock and resolved within 14 days of the onset of clinical signs.

In ovo Vaccination of Chicken Embryos with Experimental Newcastle Disease and Avian Influenza Oil-Emulsion Vaccines

Inactivated oil-emulsion (OE) Newcastle disease (ND) and avian influenza (AI) vaccines were injected into 18-day-old white rock (WR) and white leghorn (WL) chicken embryos to evaluate their immunologic efficacy and their effects on hatchability. Embryonating eggs were inoculated at 1.5 inches depth with various vaccine volumes and antigen concentrations. Serum hemagglutination-inhibition (HI) titers were first detected in chickens at 2 wk posthatch. Protection against morbidity and mortality was demonstrated in all of 10 chickens vaccinated as embryos and challenged with viscerotropic velogenic ND virus at 53 days of age and also in all of eight in ovo- vaccinated chickens challenged with highly pathogenic AI virus at 34 days of age. All of five unvaccinated control chickens for each respective ND- and AI-vaccinated group died. In pooled groups from successive hatches, the hatchability of WR or WL embryos injected with 100 microliters of vaccine was not significantly different (P > 0.05) from unvaccinated hatchmate controls when needle gauges of 22, 20, and 18 were used. Seroconversion rates of chickens vaccinated as embryos ranged from 27% to 100% with ND vaccination and 85% to 100% for AI vaccination. For ND, geometric mean HI titers of chickens per vaccine group ranged from 11 to 733, and in pooled groups, the range was 49 to 531. Titers for AI vaccine groups ranged from 156 to 1178. This study demonstrated that acceptable hatchability, seroconversion rates, and protective immunity can be attained with in ovo inoculation of ND or AI OE vaccines if the vaccines are prepared with sufficient antigen and administered properly.

Occurrence of Velogenic Viscerotropic Newcastle Disease in Pet and Exotic Birds in 1991

In 1991, velogenic viscerotropic Newcastle disease (VVND) was diagnosed in domestic psittacine birds in six states: Illinois, Indiana, Michigan, Texas, California, and Nevada. In the first four states, the disease assumed outbreak proportions. The affected psittacine birds--yellow-headed Amazon parrots (Amazona ochrocephala oratrix), yellow-naped Amazon parrots (Amazona ochrocephala auropalliata), cockatiels (Nymphicus hollandicus), and conures (unknown species)--exhibited respiratory and/or central nervous system signs. The velogenic viscerotropic Newcastle disease virus (VVNDV) was isolated from cloacal and tracheal swabs and various tissues, such as the lung, trachea, distal intestine, and spleen. The origin of the birds could not be established. The disease in the six states was promptly controlled, with no evidence that domestic poultry had been exposed. Also, VVNDV was isolated from quarantined birds intended for importation into the United States. Included were 53 moustached parakeets (Psittacula alexandri fasciata), a mynah (Gracula religiosa), a drongo (Dicrurus sp.), and three partridges (family Phasianidae). Groups of birds that yielded VVNDV were denied entry into the United States. Birds that are illegally imported and therefore not tested for the presence of foreign animal pathogens are a potential source of VVNDV and a threat to domestic poultry and caged birds.

Comparative Efficacy of the B-1 and VG/GA Vaccine Strains against Velogenic Viscerotropic Newcastle Disease Virus in Chickens

Groups of eight 1-day-old white rock chickens were vaccinated with either B-1 or VG/GA strain of Newcastle disease virus (NDV) by eyedrop instillation. Some of the chickens were vaccinated a second time at 17 days of age. Eight groups of chickens vaccinated either once or twice were challenged with the California 1083 strain of velogenic viscerotropic Newcastle disease virus (VVNDV) at 30 days of age by either intramuscular injection or eyedrop instillation. One group of unvaccinated control chickens was challenged by eyedrop instillation. All eight unvaccinated controls, two of the 16 B-1 vaccinates, and none of the 16 VG/GA vaccinates died following challenge. There were no obvious differences in pre-challenge serum antibody levels among the vaccinates. Only the twice-vaccinated chickens that were challenged by eyedrop and the unchallenged vaccinates failed to show a marked rise in serum antibody titers. The VG/GA strain of NDV provided protection against the mortality associated with VVNDV challenge similar to that provided by the B-1 strain within the conditions of this experiment.

Studies of the pathology of velogenic Newcastle disease: Virus infection in non‐immune and immune birds

The pathology of velogenic viscerotropic Newcastle disease virus infection was compared in 7-and 20-week-old groups of non-immune birds and birds with two levels of immunity as determined by the haemagglutinin inhibition test. In non-immune birds the bursa at 7 and 20 weeks was the only lymphoreticular organ to show sustained reticular and lymphoid cell reactions until death took place. Caecal tonsil and spleen were extensively necrotized on day 4 after contact exposure, and similar changes occurred in lung and proventriculus. There was evidence of lymphoid recovery in birds which survived for 18 days. In immune birds the spleen showed two main responses. The first, acute reticular cell response around the ellipsoids indicated that renewed exposure to antigen was often associated with localized cell degeneration. The second, immunological, reaction was rapid formation of germinal centres which occurred somewhat earlier in 20-week-old birds (4-5 days). Especially from the second week, reticular (dendritic) cell and lymphoid hyperplasia occurred diffusely in the bursal medulla of both age groups although marked atrophy and cellular depletion, probably of physiological origin, was a feature of 20-week-old birds with high antibody levels. In the gastro-intestinal tract, germinal centre formation was most marked in the caecal tonsils at 20 weeks. With the Indonesian ITA strain of ND virus, degenerative and inflammatory changes in the brain were mild in all groups up to day 18 irrespective of immune status.

The Pathogenesis of Velogenic Newcastle Disease Virus Infection of Chickens of Different Ages and Different Levels of Immunity

Chickens of 7 weeks or 20 weeks of age were divided into three groups according to their antibody status (high, low, absent) and were infected with a velogenic viscerotropic Newcastle disease virus. To follow patterns of viral replication, birds were necropsied at regular intervals up to 22 days and organs were sampled from each bird. In non-immune birds, virus could be isolated from all organs examined. In birds with antibody, virus was most frequently isolated from the proventriculus, cecal tonsil, bursa, and brain. However, because no one organ could be recommended for all situations, all four should be sampled for field diagnosis. In immune birds, although clinical signs were either mild or absent, widespread virus replication occurred up to 19 days post-challenge.

Immune Response to Oil-Emulsion Vaccines with Single or Mixed Antigens of Newcastle Disease, Avian Influenza, and Infectious Bronchitis

Inactivated Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) antigens were evaluated for immunological efficacy in monovalent and polyvalent vaccines. Vaccinated broilers were bled for hemagglutination-inhibition (HI) tests at 1- or 2-week intervals. Half of the chickens were challenged with the Largo isolate of velogenic viscerotropic (VV) NDV at 8 weeks post-vaccination, and the remainder were challenged with the Massachusetts 41 strain IBV at 9 weeks post-vaccination. Newcastle disease HI titers were reduced significantly (P less than 0.05) from those of monovalent control vaccine groups when IBV antigen was emulsified in mixtures with low (1-3x) concentrated NDV or NDV and AIV antigens. Avian influenza HI titers were significantly (P less than 0.05) lower than those of the control monovalent groups when highly concentrated NDV was part of the polyvalent vaccine. Infectious bronchitis HI titers were higher than those of control monovalent groups in 13 of 15 vaccine groups when IBV antigen was in polyvalent formulations. VV NDV challenge killed all non-NDV vaccinates and induced increased HI titers in NDV vaccinates but no morbidity or mortality. Sixty of 80 IBV vaccinates experienced a fourfold or greater HI titer increase following challenge. All non-IBV vaccinates seroconverted at 1 week post-challenge.