Many of our studies have been of the canine parvovirus (CPV), a naturally derived host range variant of a long recognized virus of cats, feline panleukopenia virus (FPV)that emerged in 1978. We are also examining the canine influenza virus, which emerged in dogs around 2000 as a variant of the long known H3N8 equine influenza virus. 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. The canine influenza virus is very similar to the equine influenza virus, but there are a small number of changes that distinguish the viruses and we are examining some of the viral properties that distinguish them and which may control host specificity.
More about Host Range
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 varity of endocytic pathways within the cells.
More about Virus-Cell Interactions
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. The more 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.
More about the TfR receptor
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.
More about Virus Evolution
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 crystllize 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.
More about Virus Structure
