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. 2011 Sep;85(17):8582-96.
doi: 10.1128/JVI.00652-11. Epub 2011 Jun 15.

Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses

Shin-Hee Kim  1 , Madhuri Subbiah, Arthur S Samuel, Peter L Collins, Siba K Samal
Affiliations

Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses

Shin-Hee Kim et al. J Virol. 2011 Sep.

Abstract

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.

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Figures

Fig. 1.
Fig. 1.
Construction of full-length antigenomic cDNAs of NDV (A) or APMV-2 (B) in which the complete F and/or HN glycoprotein or its ectodomain were swapped with that of the other virus, and successful recovery of three of these viruses. NDV and APMV-2 sequences are indicated with shaded and open boxes, respectively. The amino acid lengths of individual proteins or protein domains are indicated. The three chimeric viruses that could be successfully recovered are indicated with asterisks. (A) The coding sequences for the complete ORFs (top three constructs) or ectodomains (bottom three constructs) of F and HN of APMV-2 were inserted individually or together into a full-length antigenomic NDV cDNA in place of the corresponding NDV sequence. Each insert was flanked by 5′ UTR and 3′ UTR and transcription signals of the respective NDV F or HN gene: these flanking sequences are not shown. (B) A parallel set of constructs was made in the APMV-2 backbone. (C) Western blot analysis of virion proteins using polyclonal antibodies raised against the NDV F or HN protein. The parental NDV and APMV-2 viruses and the three chimeric viruses that were successfully recovered were grown in embryonated eggs and subjected to discontinuous sucrose gradients (9) to obtain partially purified stocks. The levels of the F and HN proteins incorporated into virions were quantitated by Western blotting using the sera specific to the individual NDV and APMV-2 glycoproteins, as indicated. Values for all proteins were expressed as percentages of these values for the proteins of respective parental viruses, which were considered to be 100%. Each bar represents the mean and standard error of the mean of three independent experiments. The detected blots by the sera for NDV F protein are F0 protein, corresponding to approximately 50 kDa. Lanes: 1, rNDV; 2, rNDV/2-F-HN; 3, rAPMV-2; 4, rAPMV-2/NDV- F-HN; 5, rAPMV-2/NDV-F.
Fig. 2.
Fig. 2.
Cytopathic effect in Vero cells infected with chimeric NDV and APMV-2 viruses. (A) Vero cells in six-well plates were infected with each of the parental and chimeric viruses at a multiplicity of infection (MOI) of 0.01 PFU/cell and incubated for 48 h. (B) The viral plaques in the infected cells were visualized by immunoperoxidase staining using polyclonal antibody raised against the N protein of NDV (top row) or APMV-2 (bottom row).
Fig. 3.
Fig. 3.
Biological activities of parental and chimeric viruses. The fusion index (A), HAd (B), and NA (C) were determined in virus-infected Vero cells at an MOI of 0.1 PFU/cell. All values are expressed relative to rNDV as 100%. Each bar represents the mean and standard error of the mean of duplicate samples.
Fig. 4.
Fig. 4.
In vitro growth characterization of chimeric viruses in DF1 cells. The growth characteristics of parental and chimeric viruses were determined by multicycle growth curve in DF1 cells infected with an MOI of 0.01 PFU/cell. The viral titers were determined by limiting dilution on DF1 cells and immunostaining with a polyclonal antibody raised against the respective N protein. Each bar represents the mean and standard error of the mean of duplicate samples.
Fig. 5.
Fig. 5.
Growth kinetics of parental and chimeric viruses in the brains of 1-day-old chicks. Ten 1-day-old SPF chicks were inoculated with 256 HA units (approximately 1.28 × 106 PFU) of parental or chimeric virus via the intracerebral route. Two birds in each group were sacrificed daily until 5 dpi. The virus titers in the collected samples were determined by limiting dilution in DF1 cells and immunostaining with a polyclonal antibody raised against the respective N protein.
Fig. 6.
Fig. 6.
Detection of viral antigens of parental and chimeric viruses in the brains of 1-day-old chicks infected by the intracerebral or intranasal route. As described in Table 2, footnote a, chicks were inoculated with each virus (1 × 105 TCID50/bird) by the intracerebral (A and C) or intranasal (B) route, and brain tissue was harvested for immunohistopathology at 3 dpi. The tissues were fixed in phosphate-buffered formalin and sections were prepared and stained using an antibody against the respective N protein.
Fig. 7.
Fig. 7.
Replication of parental and chimeric viruses in 2-week-old chickens. Groups of 10 2-week-old chickens were inoculated with 1 × 105 TCID50 of parental or chimeric virus by the intranasal route. Three birds from each group were sacrificed at 3 (shown) and 5 (not shown) dpi, and brain, trachea, lung, and spleen tissue was collected and virus titers determined by limiting dilution and immunoperoxidase staining. The day 3 results are shown because the titers were higher; the day 5 results were of lower titer but otherwise similar.
Fig. 8.
Fig. 8.
Replacement of the globular head (GH) domain of the NDV HN protein to create the chimeric virus rNDV/HN-2GH. The globular head domain of NDV was replaced with that of APMV-2 (A). Fusion activity of the parental rNDV or chimeric rNDV/HN-2GH virus was determined in Vero cells, visualized at 48 hpi (B).
Fig. 9.
Fig. 9.
Construction of HN stalk domain mutant NDVs. Four amino acids (alanine, leucine, proline, and leucine at amino acid positions 89, 90, 93, and 94, respectively) in the HN stalk domain were individually mutated into glutamine or alanine. Among the four constructs, one rNDV with substitution of glutamine for alanine (rNDV/HN-A89Q) was recovered.
Fig. 10.
Fig. 10.
Biological activities of HN mutant NDVs. The NA, HAd, and fusion index of the mutant viruses were determined in virus-infected Vero cells at an MOI of 0.1. Values for all viruses were expressed as percentages of these values for rNDV, which were considered to be 100%. Each bar represents the mean and standard error of the mean of duplicate samples.
Fig. 11.
Fig. 11.
In vitro growth characterization of HN mutant NDVs in DF1 cells. The growth characteristics of rNDV and HN mutant viruses were examined by multicycle growth curve in DF1 cells (MOI of 0.01 PFU/cells). The viral titers were determined by immunoperoxidase assay. Each bar represents the mean and standard error of the mean of duplicate samples.
Fig. 12.
Fig. 12.
Growth kinetics of the HN mutant NDVs in the brains of 1-day-old chicks. One-day-old SPF chicks were inoculated with each virus via the intracerebral route. Virus titers in the brain samples were determined by immunoperoxidase assay in DF1 cells.
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