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Control of Equine Infectious Anemia (EIA)
C.J. Issel, R.F. Cook, S.J. Cook
Department of Veterinary Sciences
Equine infectious anemia virus (EIAV) is a lentivirus and a close relative of the human immunodeficiency virus. Many isolates of EIAV have high disease-inducing potential even though they have been isolated from inapparent carriers of the virus. The ability of the virus to induce disease is directly related to viral and host factors. Our research is directed at defining the viral determinants of disease pathogenesis, as they are amenable to analysis and modification. Once the key genetic determinants are identified, rational approaches to removal of these genes may lead to effective immunization regimens.
By the same token, some viral genes may code for structural proteins parts of which may be recognized as dominant by equid hosts. The result may be a massive response to the dominant epitopes that are not protective while precluding or preventing effective immune responses to subdominant epitopes, which by themselves could be protective.
These research priorities are important for their own merit in horses, but also for their comparative value as a model for HIV. Control of equine infectious anemia requires an understanding of basic virus and host factors in protective immunity.
2011 Project Description
We have collaborated with scientists in Brazil, Argentina, Japan, Italy and Romania to develop a better understanding of EIA virus (EIAV) strain diversity/divergence and to improve where possible the genetic and serologic diagnosis of the infection.
Collaboration with scientists in Rome indicates a need for adoption of a three-tiered system for laboratory diagnosis of EIA as originally promulgated by the Committee on Infectious Diseases of Horses of the US Animal Health Assoc. In this system all serum samples are tested first by an EIA-ELISA format, positives confirmed in the AGID test (the current gold standard method of diagnosis), and further in an immunoblot test if needed. Although we have observed up to 20 percent of false-negative AGID test reactors in horses infected with genetically weakened variants, similar data from Italy with field samples compel international adoption of the three-tiered system. A jointly authored manuscript is in preparation.
In addition to contributing towards improvements in serological diagnosis of EIA, we have continued to refine methods to detect viral nucleic acids and have developed a rapid real-time PCR technique that has proven successful with field strains of EIAV from both the United States and Italy.
As genetic variation can have significant consequences for the design of nucleic acid-based diagnostic assays and vaccine development we have investigated variation with time in gag gene sequences from horses infected with both laboratory-adapted and field strains of EIAV. This has shown that while in most animals these sequences are as expected generally conserved with nucleotide substitution rates of 0.14 substitutions per year per 100 base-pairs in some they can be as high as 2.1 substitutions per 100 base-pairs per year which is similar to the level previously reported for known highly variable EIAV genes such as env. Therefore, the rate of evolution in the EIAV gag gene is host and or virus strain dependent.
The immunogenicity of DNA vaccines in horses can be improved 4-fold by the incorporation of fully deacylated polyethyleneimine (PEI) and 2-fold further by the concurrent injection of plasmid DNA encoding equine IL-15.
Progress toward understanding the immune correlates of protection in EIA have been aided by development of an in-vivo assessment of responses to defined envelope specific peptides. These studies suggest that the breadth of peptide recognition does not necessarily correlate with protection but may be more reflective of in vivo exposure to the virus.
This potential role of virus exposure in vivo driving the immune response was also seen in the clonal diversity of the responding T cells. Analysis of T cell receptor expression showed that an inapparent carrier's peptide response involved multiple T cell clones. By contrast, the breadth of the peptide-specific response of recently infected ponies was much narrower with only limited families involved. While the mechanism responsible for the more diverse T cell receptor utilization in the long-term infected inapparent carrier is unknown, frequent re-exposure to the virus in vivo could play a role.
Since 2006 this laboratory has been collaborating with Italian scientists to improve diagnostic methods. Our findings using experimental and natural cases of EIA have demonstrated the effectiveness of the three-tiered system for laboratory diagnosis of EIA in which samples are first screened by EIA-ELISA, with those samples producing positive reactions confirmed by AGID and finally EIA-immunoblot analysis employed to resolve those cases that are seropositive in EIA-ELISA but interpreted as negative in AGID.
It is anticipated that the three-tiered system will become the standard model for the control of EIA in other countries. It was predicted that it should be possible to develop PCR-based tests capable of detecting most if not all EIAV strains currently in circulation. Studies conducted during this reporting period support this conclusion suggesting that both conventional nested and real-time PCR-assays are likely to become extremely important adjuncts to serological diagnosis particularly in cases where recent exposure is suspected.
Studies conducted in the 1980's using T1 mapping suggested that gag gene sequences are extensively conserved over time in equids infected with a laboratory adapted EIAV strain. We have confirmed this finding for most cases using a more definitive direct nucleotide sequence analysis. However in a horse infected with an EIAV field strain nucleotide substitution rates in gag were 14-fold higher than in the other virus/host systems examined. This demonstrates the rate of evolution in EIAV gag gene sequences is highly dependent on as yet unidentified host and/or virus strain factors.
Although the frequency of nucleotide substitutions in the other genes of this virus have yet to be determined this result raises that possibility that mutation rates in some EIAV strain/host combinations are significantly higher than observed previously. Immune responses to DNA vaccines in animals other than small rodents are usually low level and short-lived. We have demonstrated both the level and the duration of these responses can be significantly improved in the horse by combining DNA with PEI prior to inoculation and by the simultaneous injection of a mammalian expression vector encoding equine IL-15. These results are currently being prepared for publication and have generated commercial interest.
The identification of immune correlates of protection in the EIAV system remains a high priority for our group. The discovery that in vivo cellular immune responses can be effectively monitored by injection of defined viral determinants provides opportunities to better measure relevant cellular and humoral responses to the variety of defined antigens of EIAV. This animal model for HIV has provided unique opportunities to expand our basic knowledge of immunity in the horse.
Capomaccio,S., K. Cappelli, R. F. Cook, F. Nardi, R. Gifford, M. L. Marenzoni, and F. Passamonti. 2012. Geographic structuring of global EIAV isolates: a single origin for New World strains Virus Research (in press)
Capomaccio,S., Z. A. Willand, S. J. Cook, C. J. Issel, E. M. Santos, J. K. P. Reis and R. F. Cook. 2012. Detection, Molecular Characterization and Phylogenetic Analysis of Full-Length Equine Infectious Anemia (EIAV) Gag Genes isolated from Shackleford Banks Wild Horses. Veterinary Microbiology (in press).