A proper vaccination program can play a critical role in prevention and control of
avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses
(LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry
industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to
trade implications, differentiation of infected from vaccinated animals (DIVA) is an
important issue in the control of AI. Therefore, the development and characterization of
vaccine candidates with DIVA properties is critical in improving vaccination programs.
Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a
potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a
major role in blocking the host's antiviral response. Using an eight-plasmid reverse
genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant
(H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3
and H5N3/NS1/144 were compared in cell culture and in different age embryonated
chicken eggs. Our results confirmed that NS1 is involved in down regulation of
interferon as shown by IFN-beta mRNA expression analysis and by the inability of
H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos.
However with regards to safety the virus reverted to virulence within five back passages
in chickens and was therefore not a safe vaccine candidate. However the killed form of
H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection
not significantly different from the parental H5N3 as vaccine. To further understand the
reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of
H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1
gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all
three lineages showed the presence of a 54 amino acid deletion resulting in the
generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of
deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point
mutations in the RNA binding domain (Q40R and T73M). To determine if these
mutations played a role in increased virulence, recombinant viruses expressing these
mutant NS1 proteins in the background of parental virus were generated by reverse
genetics and their replication properties and pathogenicity was examined in vitro, in ovo
and in vivo systems.
Our results showed that the 87 amino acid long NS1 protein clearly increased
virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein
slightly increased the virus virulence.
Overall this study reinforces the role of NS1 in influenza virus pathogenicity and
supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA
vaccines.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-08-6992 |
| Date | 2009 August 1900 |
| Creators | Brahmakshatriya, Vinayak |
| Contributors | Reddy, Sanjay, Lupiani, Blanca |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
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