The role of Newcastle disease virus internal proteins in pathogenesis

The internal proteins, nucleocaspid protein (NP), phosphoprotein (P) and large polymerase protein (L) of Newcastle disease virus (NDV), play an important role in transcription and replication of the viral genome. However, their role in NDV pathogenesis has not been explored. In this study, the importance of internal proteins in NDV virulence was evaluated through a chimeric approach using an established reverse genetics technique. The L gene between an avirulent NDV strain LaSota and a moderately virulent NDV strain Beaudette C (BC) was exchanged, recombinant chimeric viruses were recovered and studied for their pathogenicity in the natural host, chicken. The results obtained from in vivo studies indicated that the L gene of NDV modulate role in NDV virulence in chickens. The NP and P genes of NDV were exchanged between BC and LaSota individually as well as in combination; chimeric viruses were recovered, indicating that heterologous NP and P genes were functional. In vitro replication of chimeric NP and P recombinant viruses in DF-1 cells indicated that the exchange of NP or P gene in NDV did not affect the replication of the chimeric viruses. The in vivo studies in chickens showed that the change in pathogenicity of these chimeric viruses was minimal and homotypic interaction between NP and P proteins is necessary for optimum pathogenicity of the virus

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1 strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals

NDV- and HPAIV-specific serum antibody responses following immunization with rNDV-HA and rNDV-HAF and survival of chickens after homologous and heterologous HPAIV challenge.

1<p>Chickens in groups of 5 or 10, as indicated, were vaccinated on day 0 with wtNDV, rNDV-HA, or rNDV-HAF. On day 21, serum samples were taken and the birds were challenged with 100 CLD₅₀ of the indicated HPAIV. Groups are named according to the immunizing NDV vector and the HPAIV strain used in the HI assays and challenge. Viet = homologous A/Vietnam/1203/2004; Egypt = heterologous A/Egret/Egypt/01/2006.</p>2<p>Mean of the viruses in #1 and #2.</p

Frequency of shedding of the NDV-based vaccine viruses and HPAIV challenge viruses¹.

1<p>From the experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006509#pone-0006509-t002" target="_blank">Table 2</a>. Shedding was determined by collecting oral and cloacal swabs on 3 day post vaccination and 3 day post challenge and inoculation into embryonated chicken eggs followed by HA tests for virus growth.</p

Immunoelectron microscopy of purified virions of rNDV, rNDV-HA and rNDV-HAF, analyzed using mouse monoclonal antibodies against the NDV HN protein (upper panel) or chicken polyclonal antibodies against H5N1 AIV (lower panel).

<p>Immunoelectron microscopy of purified virions of rNDV, rNDV-HA and rNDV-HAF, analyzed using mouse monoclonal antibodies against the NDV HN protein (upper panel) or chicken polyclonal antibodies against H5N1 AIV (lower panel).</p

Multicycle growth kinetics (A) and plaque morphology (B) of the rNDV, rNDV-HA and rNDV-HAF viruses in DF-1 cells.

<p>Multicycle growth kinetics (A) and plaque morphology (B) of the rNDV, rNDV-HA and rNDV-HAF viruses in DF-1 cells.</p