Avian paramyoxvirus-8 immunization reduces viral shedding after homologous APMV-8 challenge but fails to protect against Newcastle disease

BACKGROUND: Protection against infection by Newcastle disease virus (NDV), also designated as avian paramyxovirus subtype-1 (APMV-1), is mediated by immune responses to the two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Thus, a chimeric APMV-1 based vaccine that encodes APMV-8 HN- and F-proteins and expresses the hemagglutinin of avian influenza virus (AIV) H5N1, is able to protect against HPAIV H5N1 but fails to protect against NDV [PLoS One8:e72530, 2013]. However, it is unclear whether avirulent APMV-subtypes, like APMV-8 can induce subtype-specific immunity and protect from a homologous challenge. FINDINGS: APMV-8 infections of 3- and 6-weeks-old specific pathogen free (SPF)-chickens did not induce any clinical signs but was associated with virus shedding for up to 6 days. Viral replication was only detected in oropharyngeal- and never in cloacal swabs. Upon reinfection with homologous APMV-8, viral shedding was restricted to day 2 and in contrast to naive SPF-chickens, only RNA but no infectious virus was recovered. No protection was induced against virulent NDV challenge, although morbidity and mortality was delayed in APMV-8 primed chickens. This lack of protection is in line with a lack of reactivity of APMV-8 specific sera to APMV-1 HN-protein: Neither by hemagglutin-inhibition (HI) test nor immunoblot analyses, cross-reactivity was detected, despite reactivity to internal proteins. CONCLUSIONS: Immune responses mounted during asymptomatic APMV-8 infection limit secondary infection against homologues reinfection and facilitates a delay in the onset of disease in a subtype independent manner but is unable to protect against Newcastle disease, a heterologous APMV-subtype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1743-422X-11-179) contains supplementary material, which is available to authorized users

Different regions of the newcastle disease virus fusion protein modulate pathogenicity.

Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site

Chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived NDV immunity.

Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination

Syncytia size.

<p>BSR-T7 cells were infected at an moi of 0.01 with the respective viruses. At 24 h p.i. cells were fixed and syncytia of the recombinant viruses were visualized by immunostaining using a hyperimmune serum against NDV. The syncytia sizes were determined by measuring the areas of 20 syncytia per virus in triplicate assays with NIS-Elements BR imaging software. All values are expressed relative to R75/98 which had been set as 100%. Error bars represent standard deviations. Significant differences (P<0.05, Bonferroni correction) between R75/98 and recombinant viruses are indicated (*).</p

Kinetics of replication in ECE (A) and QM9 cells (B).

<p>SPF eggs were infected with 200 µl of 10³ TCID50 ml⁻¹ and QM9 cells were infected at an moi of 0.01 with the respective viruses. Allantoic fluids of the eggs and cell culture supernatants were harvested at indicated time points and titers were determined by titration on QM9 cells, followed by immunofluorescence.</p

Construction of full-length plasmids.

<p>Schematic representation of genome organization of new generated recombinant NDV based on pflRFL60. Sites of restriction enzymes used for different cloning steps are given. Sequence regions raised from R75/98 are in dark grey, sequence regions raised from rNDV are in light grey. ICPI values for each virus are given on the right.</p

Western blot analysis of NDV recombinants.

<p>Purified virions with normalized protein levels (10 µg/lane) were separated by SDS-PAGE and blotted onto nitrocellulose. Membranes were incubated with polyclonal monospecific rabbit anti-NDV F serum (A) and monoclonal NDV-NP antibodies (B). Proteins were detected by chemiluminescence substrate (Pierce) after incubation with peroxidase-conjugated species-specific secondary antibodies.</p