Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Lineage diversification of pigeon paramyxovirus effect re-emergence potential in chickens.

Genotype VI-paramyxovirus (GVI-PMV1) is a major cause of epidemic Newcastle-like disease in Columbiformes. This genotype of avian paramyxovirus type 1 has diversified rapidly since its introduction into the US in 1982 resulting in two extant lineages, which have different population growth properties. Although some GVI-PMV1s replicate poorly in chickens, it is possible that variants with different replicative or pathogenic potential in chickens exist among the genetically-diverse GVI-PMV1s strains. To determine if variants of Columbiform GVI-PMV1 with different phylogenetic affiliations have distinct phenotypic properties in chickens, we investigated the replicative properties of 10 naturally circulating pigeon-derived isolates representing four subgroups of GVI-PMV1 in primary chicken lung epithelial cells and in chicken embryos. Our data demonstrate that GVI-PMV1 variants have different infection phenotypes in their chicken source host and that properties reflect subgroup affiliation. These subgroup replicative properties are consistent with observed dynamics of viral population growth.

Characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (Calidris ruficollis).

A hemagglutinating virus (8KS0813) was isolated from a red-necked stint. Hemagglutination inhibition and neutralization tests indicated that 8KS0813 was antigenically related to a prototype strain, APMV-6/duck/Hong Kong/18/199/77, but with an 8- and 16-fold difference, respectively, in their titers. The full genome sequence of 8KS0813 showed 98.6 % nucleotide sequence identity to that of APMV-6/duck/Italy/4524-2/07, which has been reported to belong to an APMV-6 subgroup, and showed less similarity to that of the prototype strain (70.6 % similarity). The growth of 8KS0813 and the prototype strain in four different cell cultures was greatly enhanced by adding trypsin. Interestingly, this virus induced syncytia only in Vero cells. 8KS0813 was identified as APMV-6/red-necked stint/Japan/8KS0813/08, but it is antigenically and genetically distinguishable from the prototype strain, suggesting that variant APMV-6 is circulating in migratory birds.

Evaluation of the genetic diversity of avian paramyxovirus type 4.

Avian paramyxoviruses (APMVs) belong to the genus Avulavirus in the family Paramyxoviridae and include at least nine serotypes, APMV-1 to -9, as well as two additional provisional serotypes. Newcastle disease virus (NDV), which comprises APMV-1, is the most extensively studied APMV because it is an important poultry pathogen. A moderate level of antigenic and genetic diversity is recognized for APMV-1 isolates, but our knowledge of the antigenic and genetic diversity of the other APMV serotypes is limited. APMV-4 is frequently isolated from waterfowl around the world. To date complete genome sequences of APMV-4 are available for only strains, which were isolated from ducks in Hong Kong, Korea and Belgium over a period of 37 years. We have carried out genome sequencing from the nucleocapsid (N) gene-end signal to the polymerase (L) gene-start signal of five APMV-4 strains recently isolated from Italy. Each of the eight APMV-4 strains has the same F protein cleavage site, DIQPR↓F. They also share a high level of nucleotide and amino acid sequence identity: for example, the F and HN glycoproteins have greater than 97% sequence identity between the various strains. Thus, comparison of these eight strains of APMV-4 did not provide evidence of substantial diversity, in contrast to similar studies with APMV-2, -3, and -6, in which the F and HN glycoproteins exhibited up to 20-30% amino acid sequence variation within a subgroup. Reciprocal cross-HI assay using post infection chicken sera also failed to detect significant antigenic variation among the available APMV-4 strains.

Complete sequence of the genome of avian paramyxovirus type 2 (strain Yucaipa) and comparison with other paramyxoviruses.

The complete RNA genome sequence of avian paramyxovirus (APMV) serotype 2, strain Yucaipa isolated from chicken has been determined. With genome size of 14,904 nucleotides (nt), strain Yucaipa is consistent with the "rule of six" and is the smallest virus reported to date among the members of subfamily Paramyxovirinae. The genome contains six non-overlapping genes in the order 3'-N-P/V-M-F-HN-L-5'. The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 3 to 23nt. The genome contains a 55nt leader sequence at 3' end and a 154nt trailer sequence at 5' end. Alignment and phylogenetic analysis of the predicted amino acid sequences of strain Yucaipa proteins with the cognate proteins of viruses of all of the five genera of family Paramyxoviridae showed that APMV-2 strain Yucaipa is more closely related to APMV-6 than APMV-1.

Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses.

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.

Inhibition of replication of goose paramyxovirus SF02 by hammerhead ribozyme targeting to the SF02 F mRNA in chicken embryo fibroblasts.

Hammerhead ribozyme RzF598 and its dysfunctional mutant dRzF598 targeting to the (F) gene of goose paramyxovirus SF02 have been designed. The transgenic plasmids pcDNA-RzF598 and pcDNA-dRzF598 were constructed by inserting ribozyme genes into eukaryotic expression vector pcDNA3. The plasmid pcDNA3 that lacks full ribozyme gene was used as a control. Plasmids pcDNA-RzF598, pcDNA-dRzF598 and pcDNA3 were transfected into chicken embryo fibroblasts (CEFs). The concentration of virus released by infected CEFs and the survival percentages of CEFs were identified. The results indicated that RzF598 successfully suppressed the replication of SF02 in CEFs. Survival percentage of CEFs being transfected with pcDNA-RzF598 and infected SF02 was up to 78.8%, while the survival percentages of untransfected CEFs and CEFs transfected with pcDNA3 after infection with SF02 were only about 5%.