Avian paramyoxvirus-8 immunization reduces viral shedding after homologous APMV-8 challenge but fails to protect against Newcastle disease
© Grund et al.; licensee BioMed Central Ltd. 2014
Received: 19 June 2014
Accepted: 27 September 2014
Published: 8 October 2014
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 One 8: e72530, 2013]. However, it is unclear whether avirulent APMV-subtypes, like APMV-8 can induce subtype-specific immunity and protect from a homologous challenge.
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.
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.
Avian paramyxoviruses (APMV) replicate within the respiratory tract and intestine of their natural avian host. They belong to the genus Avulavirus in the family Paramyxoviridae within the order Mononegavirales. Currently, 12 subtypes have been identified with APMV-1 to -9 known as ‘classical strains’ and APMV-10 to 12 recently described[3–5]. The prototypic virus, APMV-1 or Newcastle Disease virus (NDV) causes a devastating disease in poultry and represents a major threat for poultry production in the world. In contrast, the other APMV-subtypes are not clinically relevant for poultry and circulate largely unnoticed in wild birds. Also for APMV-1, strains of low virulence are well known. They do not induce clinical signs in immune competent birds but confer protection against ND[7–9]. The generation of recombinant NDV (rNDV) containing specific alterations in the genome decreased residual virulence and were also used as vector system to express genes of other pathogens, e.g. highly pathogenic avian influenza virus (HPAIV)[11, 12]. To avoid interference by maternal NDV antibodies with vaccine vector performance, we created a chimeric virus (ch NDVFHN PMV8H5) by substituting the envelope glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F) of NDV by those of APMV-8 and expressing H5 of HPAIV. APMV-8 was chosen as donor of HN and F because of its apathogenicity for poultry, description of only weak cross-reactivity between APMV-1 and APMV-8[15, 16] and low prevalence[17–19]. Vaccination with this chimeric vector HPAIV-H5-vaccine resulted in efficient protection against HPAIV H5N1 infection in chickens with NDV specific maternal antibodies (MDA). However, evasion from maternal NDV antibodies was accompanied by a lack of protection against ND. This observation corresponds to investigations by Nayak et al. describing absence of protection against NDV challenge after APMV-8 infection. The serological data of the vaccination experiments with ch NDVFHN PMV8H5 as well as the APMV-8 infection suggested that APMV-8 glycoproteins were immunogenic in the host. However, it remained unclear whether an APMV-8 immune response was sufficient to induce protection against homologous APMV-8 challenge. Here we describe a set of animal experiments demonstrating, that APMV-8 infection does not prevent subsequent reinfection with homologous APMV-8 but limits viral shedding.
Materials and methods
Chickens from SPF-eggs (LAH, Cuxhaven), hatched at the FLI were infected oculo-nasally at 3 weeks of age with 0.1 ml of APMV-8/goose/Delaware/1053/76 containing 106 TCID50 (n = 18). Two days post infection (dpi) two naive chickens were introduced as sentinel birds. On each of day 2, 4, 6 and 21 dpi two inoculated chickens were sacrificed and indicated internal organs were tested for APMV-8 by RT-qPCR (primer probe sequence see Additional file1: Table S1). Three weeks after initial infection, groups were divided, one being challenged oculo-nasally with NDV/Herts33/56 (106 EID50/animal) and the other re-infected oculo-nasally with APMV-8/goose/Delaware/1053/76 (106 TCID50/animal). In addition, each of the two viruses at the same dose and route was administered to 6 naive chickens from the same hatch. Animals were scored daily according their clinical condition (0 = healthy; 1 = sick; 2 = dead) and clinical index was calculated analogous to determining intracerebral pathogenicity index (ICPI). At indicated time intervals post infection either oropharyngeal and cloacal swabs (primary infection) or combined oropharyngeal and cloacal swabs were taken for virus detection. Heparinised blood samples were obtained from all animals before vaccination, before challenge- or reinfection as well as from all surviving birds at the end of the observation period, and were tested for NDV- and APMV-8-specific antibodies using the hemagglutination inhibition (HI) assay. All animal experiments were carried out in BSL3 experimental animal facilities and had been approved by the animal welfare committee (LALLF M-V/TSD/7221.3-1.1-053/10). See Additional file for specification of viruses (Additional file2: Table S2) and APMV-subtype specific sera with their degree of cross-reactivity (Additional file3: Table S3).
Results and discussion
Three weeks after the initial experiment, five APMV-8 infected chickens and one sentinel animal, now six weeks old, were reinfected with 106 TCID50 of homologous APMV-8 strain. In addition, a group of six chickens from the same hatch but kept separately, were APMV-8 infected. As anticipated, birds inoculated with APMV-8 did not develop clinical signs but virus shedding was detected in naive 6-weeks-old chickens from day 2 pi by RT-qPCR as well as by virus isolation comparable to shedding observed before in the 3-weeks-old animals. In contrast, in animals with previous exposure to APMV-8, virus was only detected in 3 out of 6 (47, 89 and 12087 infectivity equivalents/ml) inoculated chickens on day 2 pi and only by RT-qPCR (Figure 3). The chickens that served as sentinel for the first infection remained virus negative in all tested samples. After APMV-8 reinfection, a slight increase in antibody titer was observed, supporting the notion of limited virus-replication. It is interesting to note, that age of the animal has apparently little effect on virus replication, since virus shedding (Figure 3, A and B) and antibody response (Figure 1, A and B control) in 3- and 6-weeks-old chickens were comparable.
Active local replication of APMV-8 induced an immune response efficacious to limit APMV-8 reinfection but unable to protect against heterologous APMV-1 subtype.
We like to thank Cornelia Illing for excellent technical assistance, Dr. Mario Ziller for sophisticated statistical analysis and our colleagues from the animal core facility (ATB) for diligent taking care of the animals.
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