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Innate Immune Responses to Influenza Virus Infection
T. Chambers, D. Horohov
Department of Veterinary Sciences
The equine-1 (H7N7) influenza virus possesses the most characteristic molecular feature of the high-pathogenicity avian influenza viruses, i.e. the furin-cleavable connecting peptide between the subunits of the viral surface, protein, hemagglutinin. And indeed equine H7N7 viruses are lethal in chickens and mice-but not in horses. The reason for the difference in pathogenicity of these viruses in the different species is unclear.
Influenza pathogenesis is the outcome of interactions between the host innate immune responses-the first line of defense-and virus-coded factors, the properties of which are not uniform among influenza virus strains. Also, new knowledge is accumulating about the host genes and proteins that are collectively responsible for host innate immune responses.
The purpose of this proposed research is to decipher the basis for the differential pathogenicity of equine-1 influenza virus in different species, and to elucidate further the interaction of influenza factors with some newly recognized players in the innate response.
2009 Project Description
Progress on both specific aims was made in the third year of the project.
Specific Aim 1: Following up on our finding H7N7 equine influenza (EIV), but not H3N8 EIV, produced intense inflammatory cytokine responses in infected Balb/c mice but not in horses, we compared the resistance of H7N7 and H3N8 viruses to interferons (IFN) using a chicken embryo model. In the developing chicken embryo, the IFN system matures from an undeveloped state at 7 days, to a functional state at 11 days. Chicken embryos of 7, 9, and 11 days of age were inoculated with different doses (10e2, 10e4, and 10e6 EID50 units/egg) of H7N7 and H3N8 viruses, and cumulative mortality was recorded for 5 days. In 7-day-old embryos, both viruses induced >80% mortality at all virus doses by Day +3 post-infection. In 9-day-old embryos, H7N7 virus produced 100% mortality but H3N8 virus produced 60-70% mortality. In the 11-day-old embryos, H7N7 virus produced 30% mortality at the lowest virus dose and near-100% mortality at both higher doses, whereas H3N8 virus produced a maximum of only 10% mortality at any dose.
As pathogenicity of H7N7 virus is believed to be dependent upon the presence of multiple basic amino acids at the connecting peptide between the HA1 and HA2 subunits, we sequenced the HA of the strain used here (eq/NY/49/73 strain) and verified that its connecting peptide has the multiple basic amino acid sequence R-K-K-R-/G, in comparison with the connecting peptide of the H3N8 strain used (eq/KY/5/2002): K-Q-I-R-/G.
Specific Aim 2: Evasion of cellular innate immune responses at the level of dendritic cells (DC). We compared various measures of inhibition (spindle morphology and diameter, expression of surface markers CD11c, CD172a, CD1w2, antigen endocytosis using DQ-ovalbumin labeling), using UV-inactivated virus versus intact virus. Monocytes infected with UV-inactivated virus acquired distinctive DC morphology and exhibited DC-like antigen endocytosis. They had up-regulated surface markers although not to the same degree as mock-infected cells. This agrees with previous findings that TLR7/8 agonists inhibit surface expression of CD1-family markers. Significantly enhanced expression of CD86, MHC I and MHC II were observed on cells infected with both intact and UV-inactivated viruses, suggesting that both served to activate TLR.
Experiments are in progress to explore whether inhibition is dependent upon virus subtype, and whether the influenza NS protein, which possesses a variety of host-inhibitory functions, is also responsible for inhibition of monocyte differentiation. This work has been presented at seminars at Emory University and University of Kentucky, and submitted for publication in Veterinary Immunology and Immunopathology.
Specific Aim 1: The H7N7 mortality rate exceeded the H3N8 mortality rate under every study condition. This suggests that both viruses are pathogenic in chicken embryos in the absence of a functioning IFN system, but the H3N8 virus is more sensitive to IFN inhibition. Better understanding of the causes of viral pathogenicity in horses will lead to more effective practical therapies. Our experiments in comparative pathogenesis of H7N7 and H3N8 viruses point to their differing interaction with the IFN system as key to their differing lethality in mice and chicken embryos. Since that lethality is not manifest in the equine, this suggests that the equine IFN system must have unique features that can possibly be exploited. Specific Aim 2: As cytokines are largely responsible for the mediation of adjuvant responses, better understanding of influenza-induced cytokine responses will aid in development of more effective vaccines. We previously showed that equine monocyte-derived DC can be infected by both subtypes of EIV. Infection using the H7N7 virus was shown to inhibit monocyte differentiation into DC. We have now extended those results by showing that at least limited virus replication is necessary for complete inhibition of differentiation. Our findings on viral inhibition of monocyte maturation into DC identify a new strategy by influenza virus to block activation of immediate innate and subsequent adaptive immune responses and evade antiviral immunity; and once this viral function is localized in the viral genome/proteome, it too can become a target for future exploitation as anti-influenza therapy.
Chambers, T.M., Quinlivan, M., Sturgill, T., Cullinane, A., Horohov, D.W., Zamarin, D., Arkins, S., Garcia-Sastre, A., and Palese, P. (2009) Influenza A viruses with truncated NS1 as modified live virus vaccines: Safety and efficacy studies in horses. Equine Vet. J. 41:87-92.
Bryant, N.A., Rash, A.S., Russell, C.A., Ross, J., Cooke., Bowman, S., Griffiths, L., MacRae, S., Zanoni, R., Meier, H., Paillot, R., Daly, J.M., Chambers, T.M., Newton, J.R., and Elton, D.M. (2009) Equine influenza virus surveillance in North America and Europe from 2006 to 2007. Vet. Microbiol. 138:41-52.
Lu, Z., Chambers, T.M., Boliar, S., Timoney, P.J., Branscum, A.J., Reedy, S.E., Tudor, L., Dubovi, E.J., Vickers, M.L., Sells, S., and Balasuriya, U.B.R. (2009) Development and evaluation of one-step Taqman real-time reverse transcription-PCR assays targeting NP, M, and HA genes of equine influenza virus. J. Clin. Microbiol. 47:3907-3913.
McCormick, J.D., Collins, J.K., Holland, R.E., Barnett, C., Chambers, T.M., and Tudor, L.R. (2009) Comparison of single versus boosted vaccine protocols for a modified live and killed virus vaccine in inducing a serologic response against equine influenza in performance horses of different ages. AAEP Proceedings 55:302-303.
Chambers, T.M. (2009) Why take nasal swabs Equine Disease Quarterly, v.18 no.2.