Pathotyping of Newcastle disease virus isolates from pet birds

Four Newcastle disease virus (NDV) isolates obtained from a pigeon, lory, parrot, and love bird were subjected to biological and molecular characterization. All the isolates were identified as velogenic with intracerebral pathogenicity indices (ICPI) of 1.9-2.0. All the isolates had a 112RRQKRF117 motif in the fusion protein cleavage site (FPCS), typical for pathogenic NDV. Phylogenetic analysis placed the isolates along with a velogenic Indian isolate of Cl group recovered during 1987.     

Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran

The pathogenicity and genetic characterizations of six Newcastle disease virus (NDV) isolates obtained from chicken farms in six different regions in Iran were carried out using conventional and molecular techniques. Based on the pathogenicity indices (MDT, ICPI, and IVPI), all of these isolates were found to be velogenic (highly virulent) strains. A sequence analysis of the full-length mRNA encoding the fusion glycoprotein precursor (F₀) of the NDV’s fusion proteins F₁ and F₂ in these six isolates showed the presence of point mutations in form of nucleic acid substitutions at positions 82^(C→T), 83^(T→C), 736^(A→G), and 1,633^(G→A). However, the nucleic acid residues at positions 330–347 of the precursor F₀ gene, corresponding to the cleavage site of the F₀ protein, were found to have remained conserved among the six NDV isolates. A phylogenetic comparison between the six Iranian isolates and the NDVs whose F₀ gene sequences were previously deposited in GenBank Database showed that all of the newly characterized Iranian NDV isolates belonged to genotype VII.     

Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003 outbreak in California and other recent outbreaks in North America

Isolates from the 2002-2003 virulent Newcastle disease virus (v-NDV) outbreak in southern California, Nevada, Arizona, and Texas in the United States were compared to each other along with recent v-NDV isolates from Mexico and Central America and reference avian paramyxovirus type 1 strains. Nucleotide sequencing and phylogenetic analyses were conducted on a 1,195-base genomic segment composing the 3' region of the matrix (M) protein gene and a 5' portion of the fusion (F) protein gene including the M-F intergenic region. This encompasses coding sequences for the nuclear localization signal of the M protein and the F protein cleavage activation site. A dibasic amino acid motif was present at the predicted F protein cleavage activation site in all v-NDVs, including the California 2002-2003, Arizona, Nevada, Texas, Mexico, and Central America isolates. Phylogenetic analyses demonstrated that the California 2002-2003, Arizona, Nevada, and Texas viruses were most closely related to isolates from Mexico and Central America. An isolate from Texas obtained during 2003 appeared to represent a separate introduction of v-NDV into the United States, as this virus was even more closely related to the Mexico 2000 isolates than the California, Arizona, and Nevada viruses. The close phylogenetic relationship between the recent 2002-2003 U.S. v-NDV isolates and those viruses from countries geographically close to the United States warrants continued surveillance of commercial and noncommercial poultry for early detection of highly virulent NDV.     

Analysis of matrix protein gene nucleotide sequence diversity among Newcastle disease virus isolates demonstrates that recent disease outbreaks are caused by viruses of psittacine origin

Nucleotide sequence analysis was completed for isolates of Newcastle disease virus (NDV; avian paramyxovirus 1) from 1992 outbreaks in cormorants and turkeys. These isolates were of the neurotropic velogenic type. The cormorant and turkey NDV isolates had the fusion protein cleavage sequence¹⁰⁹SRGRRQKR/FVG¹¹⁹, as opposed to the consensus sequence¹⁰⁹SGGRRQKR/FIG¹¹⁹ of most known velogenic NDV isolates. The R for G substitution at position 110 may be unique for the cormorant and turkey isolates. For comparative purposes, nucleotide sequencing and analysis of the conserved matrix protein gene coding region were completed for isolates representing all pathotypes. Phylogenetic relationships demonstrated that there are two major groups of NDV isolates. One group includes viruses found in North America and worldwide, such as B1, LaSota, Texas/GB, and Beaudette/C. The second group contains isolates, such as Ulster/2C, Australia/Victoria, and Herts/33, considered exotic to North America. Within this second group are viruses of psittacine origin. The viruses from 1992 outbreaks of Newcastle disease in North America, and an isolate thought to have caused the major outbreak in southern California during the 1970s, are most closely related to an NDV isolate of psittacine origin.     

Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae

Matrix (M) gene sequences for recent field isolates and older reference Newcastle disease viruses (NDV) were examined to determine phylogenetic relationships and population trends among these viruses. Overall, the M gene has a majority of synonymous nucleotide sequence substitutions occurring among NDV isolates. However, several predicted amino acid changes in the M protein of specific NDV isolates have occurred that correlate to phylogenetic relationships. Nucleotide substitutions in these codons have a greater number of nonsynonymous base changes. The NDV isolates arising since the 1970s belong to a population of viruses that expanded worldwide at an exponential rate. These viruses may have their origins in free-living birds, are present worldwide, and continue to circulate causing disease in poultry. A specific NDV lineage composed of virulent isolates obtained in the US prior to 1970 appears to no longer exists among free-living birds or commercial poultry. However, "vaccine-like" viruses are common in the US and continue to circulate among commercial poultry. Based on M protein amino acid sequences, NDV separates as a clade most closely related to morbilliviruses and not with their current designated category, the rubulaviruses among the Paramyxoviridae. Consequently, avian paramyxoviruses should have their own taxonomic subfamily among the Paramyxovirinae.     

Neurological lesions in chickens experimentally infected with virulent Newcastle disease virus isolates

Distribution, character, and severity of lesions were evaluated in tissues from the central nervous system of chickens inoculated with 10 different Newcastle disease virus (NDV) isolates: CA 1083, Korea 97-147, Australia (all velogenic viscerotropic), Texas GB and Turkey North Dakota (both velogenic neurotropic), Nevada cormorant, Anhinga and Roakin (all mesogenic), and B1 and QV4 (lentogenic). Tissues for the present study included archived formalin-fixed, paraffin-embedded brain (all strains) plus spinal cord (two strains). Encephalitis was observed in all velogenic viscerotropic and velogenic neurotropic strains, and in some mesogenic strains. In general, the encephalitic lesions began at 5 days post infection, with more severe lesions occurring around 10 days post infection. At this time point, especially in the grey matter of the brain, cerebellum and spinal cord, there were neuronal necrosis, neuronal phagocytosis, and clusters of cells with microglial morphology. Axonal degeneration and demyelination was also observed. Immunohistochemistry (IHC) for viral nucleoprotein confirmed the presence of virus. In the areas of encephalomyelitis, IHC for CD3 revealed that many of the inflammatory cells were T lymphocytes. IHC using an antibody for glial fibrillar acid protein showed reactive astrogliosis, which was most pronounced at the later time points.     

Biological and molecular characterization of Indian isolates of Newcastle disease virus from pigeons

Five Newcastle disease virus (NDV) isolates from pigeons were characterized by biological and molecular methods. Four of the five isolates were found to be velogenic with high intracerebral pathogenicity indices (ICPI). The fusion protein cleavage site (FPCS) sequences of these isolates had multiple basic amino acids RRQKRF at positions 112-116 and a phenyl alanine at position 117 characteristic of velogenic isolates. Three of these velogenic isolates were phylogenetically related to mesogenic vaccine virus strain and the fourth one to a few exotic velogenic isolates. The lentogenic isolate obtained in this study was identical with the LaSota strain.     

Influenza surveillance during the 2009-2010, 2010-2011, 2011-2012, and 2012-2013 seasons in Algeria

We report the activity and circulation of influenza viruses in Algeria during four influenza seasons, from a national surveillance study carried out from 2009-2010 to 2012-2013. A total of 2766 samples from in- and outpatients, with no age restriction, were collected. The overall proportion of specimens that tested influenza positive was 46.0%. Overall, 96.6% of influenza A viruses were subtyped, and A/H1 subtypes accounted for 57.3% of influenza A viruses. Influenza A/H1 and A/H3 virus subtypes cocirculated in 2009-2010. In 2010-2011, a high proportion of type B viruses (66.2%) was observed. The subtype H3N2 was identified in 99% of cases typed in 2011-2012. Influenza A/H3N2 and B virus cocirculated in 2012-2013. A remarkably low influenza vaccination rate of 2.4% was observed among all age groups. Antibiotics were prescribed for 926 (41.3%) patients, and no difference was observed between patients with confirmed influenza and patients with influenza-like illness not related to influenza. The burden of influenza is largely undocumented in Algeria and strategies to expand this surveillance across the country are needed. Strategies to increase vaccination coverage are warranted to control and prevent influenza in individuals at risk of complications as well as in the general population.     

Effects of the HN gene C-terminal extensions on the Newcastle disease virus virulence

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase, and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open-reading frame (616 amino acids, aa) with additional 45 aa at its C-terminus when compared with that (571 aa) of virulent NDV strains. Therefore, it has been suspected that the 45 aa extension at the C-terminus of the HN may affect the NDV virulence. In this study, we generated an NDV mesogenic strain Anhinga-based recombinant virus with an HN C-terminal extension of 45 aa (rAnh-HN-ex virus) using reverse genetics technology. The biological characterization of the recombinant virus showed that the rAnh-HN-ex virus had similar growth ability to its parental virus rAnh-wt both in embryonating chicken eggs and DF-1 cells. However, the pathogenicity of this recombinant virus in embryonating chicken eggs and day-old chickens decreased, as evidenced by a longer mean death time and lower intracerebral pathogenicity index when compared with the parental virus. This is consistent with our previous finding that the recombinant LaSota virus with a 45-aa extension at its HN C-terminal was attenuated in chickens and embryonating eggs. These results suggest that the HN protein C-terminal extension may contribute to the reduced virulence in some low virulence NDV strains.     

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.     

F gene recombination between genotype II and VII Newcastle disease virus

A velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense RNA viruses.     

Characterization of signal sequences determining the nuclear export of Newcastle disease virus matrix protein

The Newcastle disease virus (NDV) matrix (M) protein has been demonstrated to be a nuclear-cytoplasmic trafficking protein. Previous studies have shown that the M protein localizes in the nucleus through a bipartite nuclear localization signal. Here, we report that the ability of the M protein to shuttle to the cytoplasm is mediated by three nuclear export signal sequences (NESs). Using leptomycin B (LMB), a specific inhibitor of CRM1, we found that the nuclear export of the three NESs was LMB insensitive and thus was CRM1 independent. In addition, inactivation of these NESs led to nuclear accumulation of the M protein. Our results highlight the significance of these NESs to the nuclear export of the NDV M protein.     

Physicochemical studies of Newcastle disease virus

Summary Chemical analysis was performed on the highly purified Newcastle disease virus (NDV) obtained from infected allantoic fluid. Mean values of RNA content of the virus based on optical density at 260 m, phosphorus content and orcinol reaction which were carried out after the fractionation bySchmidt-Thannhauser-Schneider procedure modified byFleck andMunro were 0.72%, 0.99% and 0.92%, respectively. Contents of protein and lipid in the virus were also determined.Isolation of the RNA from NDV was performed by hot-SDS-phenol extraction from³²P-labeled virus and sedimentation patterns of the RNA in sucrose density gradients were examined. The sedimentation constant of NDV-RNA was found to be 55S, and this component was completely digested with ribonuclease. By this observation and the base composition previously determined, the RNA of NDV must be single stranded. The molecular weight of the RNA was estimated 5.8×10⁶ at least from the weight and RNA content of the virus particle.Results of this study were presented at the 13th General Meeting of the Society of Japanese Virologists, in Nagasaki, October 1965.     

Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutinin-neuraminidase gene

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) strain Texas was generated. Immunoprecipitation with chicken anti-NDV serum confirmed authentic expression of the HN protein. Protection of chickens from infection with NDV was observed when birds were immunized with the recombinant HN fowlpox virus by the intramuscular route after one or two inoculations. Vaccination by the ocular route with a mixture of fowlpox recombinants expressing the fusion and HN proteins did not show added protection over that seen with the individual viruses.     

A study of Newcastle disease vaccine virus in sprays and aerosols

The administration of two commercial Newcastle disease (ND) vaccines to chickens by aerosol was studied. The size distribution of non-evaporating droplets, produced by different aerosol generators, was measured. Using one of the generators - the Atomist - the size distribution of particles evaporating to equilibrium was determined. The sedimentation of the dry particles was judged by repeating the measurement after 10 and 30 min. This was done with distilled water, tap water, saline, and 1% and 2% solutions of Casitone, with and without vaccine. The stability of the vaccine virus at 20 degrees C in an aerosol of distilled water was minimal at high relative humidities. Measurement of viral stability in aerosols of different diluents produced with the Atomist in small pens showed an initial loss of infectivity of between 1 and 3 log(10) median embryo infectious doses (EID50). Further loss of infectivity was between 0.2 and 3.4 log(10) EID50/ hour. Distilled water was the optimal diluent for these commercial ND vaccines.