Intracerebral Pathogenicity Index Tests Research Articles

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.

Characterization of avian paramyxovirus serotype 14, a novel serotype, isolated from a duck fecal sample in Japan

A hemagglutinating virus isolate designated 11OG0352, was obtained from a duck fecal sample. Genetic and virological analyses indicated that it might represent a novel serotype of avian paramyxovirus (APMV). Electron micrographs showed that the morphology of the virus particle was similar to that of APMV. The complete genome of this virus comprised 15,444 nucleotides complying with the paramyxovirus “rule of six” and contains six open reading frames (3′-N-P-M-F-HN-L-5′). The phylogenetic analysis of the whole genome revealed that the virus was a member of the genus Avulavirus, but that it was distinct from APMV-1 to APMV-13. Although the F-protein cleavage site was TREGK↓L, which resembles a lentogenic strain of APMV-1, the K residue at position -1 of the cleavage site was first discovered in APMV members. The phosphoprotein gene of isolate 11OG0352 contains a putative RNA editing site, 3′-AUUUUCCC-5′ (negative sense) which sequence differs from that of other APMVs. The intracerebral pathogenicity index test did not detect virulence in infected chicks. In hemagglutination inhibition (HI) tests, an antiserum against this virus did not detectably react with other APMVs (serotypes 1–4, 6–9) except for low reciprocal cross-reactivity with APMV-6. We designated this isolate, as APMV-14/duck/Japan/11OG0352/2011 and propose that it is a novel APMV serotype. The HI test may not be widely applicable for the classification of a new serotype because of the limited availability of reference antisera against all serotypes and cross-reactivity data. The nucleotide sequence identities of the whole genome of 11OG0352 and other APMVs ranged from 46.3% to 56.1%. Such comparison may provide a useful tool for classifying new APMV isolates. However, the nucleotide sequence identity between APMV-12 and APMV-13 was higher (64%), which was nearly identical to the lowest nucleotide identity (67%) reported in subgroups within the serotype. Therefore, consensus criteria for using whole genome analysis should be established.

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein--a well known virulence determinant of NDV--but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

Surveillance for avirulent Newcastle disease viruses in domestic ducks (Anas platyrhynchos and Cairina moschata) at live bird markets in Eastern China and characterization of the viruses isolated

We isolated and identified 201 Newcastle disease viruses (NDVs) from domestic ducks in a 5-year surveillance study at live bird markets in Eastern China. Seventy-three of these isolates were characterized biologically and genetically. Fusion protein (F) genes of these isolates were amplified by reverse transcription-polymerase chain reaction and sequenced. Intracerebral pathogenicity index tests in 1-day-old specific-pathogen-free chickens and the mean death time of embryonated fowl eggs in addition to the cleavage site analysis of the F-protein precursor for these viruses showed that they were all avirulent NDVs. Phylogenetic analysis based on partial sequences of the F gene showed that 30 isolates clustered into the class I clade and the other 43 isolates clustered into genotype I of class II, but diverged from the vaccine virus Queensland V4, which is extensively used in China. Most class I viruses (18/30) formed a separate branch closest to the Hong Kong live bird market strains that have been recently designated as genotype 3, while the rest (12/30) were closely related to some European viruses within genotype 2. All of the 43 class II genotype I viruses diverged from viruses originally assigned to genotype Ia and formed a separate sublineage designated as Ib with water bird isolates from the Far East, suggesting the possible transmission between the wild and domestic waterfowl. The results in the present study clearly showed that the domestic duck population carries avirulent NDVs with genetic divergence regularly and may act as one of the important reservoirs.

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.

Molecular Characterization of an Unassigned Israeli Newcastle Disease Virus Isolate

Detection of Newcastle disease virus (NDV) and avian pathotyping of NDV isolates are extremely important because the appearance of virulent virus has significant economic consequences in terms of vaccination, eradication, and the ability to export poultry products. By using nucleotide and amino acid (aa) homology analysis, we could demonstrate that a NDV broiler isolate is a velogenic virus. This analysis was done after mean death time and intracerebral pathogenicity index tests gave inconsistent results. By establishing a nucleotide sequence dendrogram, we found that the disputed Ber-Tuvia was clearly in the same group as the known Herev-Laet, a velogenic isolate. The difference between Ber-Tuvia 92 and the Herev-Laet velogenic isolate was 6% as opposed to > 16% of the meso- and lentogenic isolates. The Ber-Tuvia isolate contains the Arg/Arg and Lys/Arg aa at positions 112, 113 and 115, 116, respectively, in the fusion protein cleavage aa sequence, which is typical for virulent NDV isolates.

A study on the characteristics of La Sota strain of Newcastle disease virus adapted to grow in a cell line from bovine embryo kidney (BS/BEK)

The lentogenic La Sota strain of Newcastle disease virus (NDV) was adapted to grow in the BS/BEK cell line of bovine embryo kidney origin with a infectious titre similar to that in chicken embryos. No modification in the biological properties was detected after serial passages in the cell line, as indicated by CPE and by size and shape of the plaques. By contrast, the intracerebral pathogenicity index test, determined in 1-day-old chicks, was lower than for La Sota grown in chicken embryos. However, the immunogenicity of the cell culture adapted virus did not show any variation as demonstrated by serological response and by protection following challenge with virulent NDV. Accordingly, it appears that La Sota grown in cell cultures has retained its biological and immunological characteristics and if these results are confirmed by field trials and long term protection tests, the use of the BS/BEK cell line could be an alternative to chicken embryos for the cultivation of NDV.

Intracerebral pathogenicity of influenza A viruses for chickens.

The virulence of influenza A viruses was determined using the intracerebral pathogenicity index test for chickens. The viruses were divided into high virulent, low virulent and avirulent strains. A low virulent strain was recovered from the jejunum and faeces of infected chickens.

Use of Virulence Index Tests for Avian Influenza Viruses

The intravenous and intracerebral pathogenicity index tests normally used for Newcastle disease virus isolates were used to measure the virulence of 13 avian influenza viruses. The tests allowed quantitative measurements of the virulence of the avian influenza viruses, and the results confirmed the range in virulence, between the two extremes, of the avian influenza viruses and demonstrated the lack of correlation between virulence and antigenic type.