Parvovirus Host Range Variation and Control
A key area of the laboratory work is studying the origins and evolution of canine parvovirus (CPV), a parvovirus of dogs that was first recognized in 1978 when the virus spread world-wide in dogs, causing serious disease and killing many millions of dogs. CPV appears to be a naturally derived host range variant of a virus that is similar to the feline panleukopenia virus (FPV) which infects cats and related animals. CPV differs from FPV in only a small number of sequence differences, and we have shown that the new canine host range of CPV resulted from only two changes in its capsid protein. We are now seeking to understand the mechanisms of virus infection, and to determine how viral host range is controlled by cells from different hosts. We are examining the atomic structures of the parvovirus capsids and defining point mutants that affect host range, and are also studying the flexibility of the capsid using structure -specific probes and antibodies. Studies of a series of mutants indicate that the structure of the capsid is very precisely designed for canine cell infection, and that most modifications of that optimal structure result in viruses that cannot infect dog cells. This points to a very close interaction between the virus capsid and the uptake pathway of the host cell.
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Influenza Viruses in Horses and Transfer to Dogs
A second model system that we are examining is the emergence of the canine influenza virus (CIV) in dogs after transfer of the H3N8 equine influenza virus to greyhounds around 2000. Those studies are examining the evolution of the virus in dogs to determine how the virus has changed during the 14 years of continuous passage in dogs. We are also examining the host barriers to infection that normally prevent the infection of dogs by influenza viruses. The viruses in dogs are confined to only a small number of large animal shelters, and they appear not to be able to spread readily into more dispersed populations of dogs.
Parvovirus Host Range Variation and Control
Our studies of host range are examining the sequence of events which lead to infection of restriction of the different virus capsids – i.e. transport into the correct pathway for infection; disassembly of the virus capsid in the appropriate cellular compartment; and the transport of the capsid or viral DNA to the nucleus in a form which allows productive infection. These studies will include definition of the endocytic uptake pathways which are used by the viruses during the process of cell entry and infection (as described above), but characterizing the cellular molecules which act on the virus. We find that the capsids follow a variety of endocytic pathways within the cells.
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Transferrin Receptor and Interactions with Viral Capsids
Mutational analysis of the feline TfR and mapping using canine and feline TfR chimeras shows that the changes in the apical domain of the receptor determine the interaction with the capsid. Our recent work looking at the specific interactions of the feline and canine TfRs with the capsids of the different viruses shows that the interactions differ between different viruses and mutant forms of the TfR. The apical domain of the feline TfR interacts with CPV and FPV capsids.
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Evolution of Parvovirus and Influenza Virus
Viral evolution is being examined in order to compare the variation of the virus during periods of endemicity, as well as during the periods of host adaptation and epidemic spread. The studies to date indicate that although the viral DNA sequences are not hypermutable, there are consistently high levels of variation observed in both the endemic and epidemic situations. We will continue to examine the natural variation of virus sequences, and are undertaking laboratory studies of the types of variation which are allowed by the virus genome. Those studies include using mutagenesis and selection for various types of change, as well as examining the effects of altered nucleotide compositions (particularly changes in CpG content), and testing for experimental recombination.
In related studies we are examining the variation of the canine influenza virus in tissue culture and in dogs to examine how that virus varies and the relationships to the variation seen in horses.
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Virus Structure – Receptor and Antibody Interactions
Our studies of virus capsid structure are looking at the flexibility and structural variation of the capsid using a variety of approaches – such as sequence-specific antibodies, proteases, and isoelectric focusing. We are also using X-ray crystallography in close collaboration with Drs. Michael Rossmann and Susan Hafenstein at Purdue University. We prepare capsids at Cornell and crystallize those under a variety of conditions (low pH, with EGTA, etc.) and then those are frozen, and the diffreaction data is collected at the Cornell High Energy Synchrotron Source (CHESS) F1 beam line, or at the Advanced Photon Source. Other studies include cryoEM analysis of virus bound to different antibodies, as well as viral capsids bound to purified transferrin receptors.
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