In this study, we document and compare the genotypes and phenotypes of 73 PsHVs originating from Europe and the United States. Comparing the sequences of these viruses, we identified four major genotypes comprising 12 variants. Naming the specific genotypes is problematic as previous PsHV designations were based on serotype and the two most closely related genotypes share the same serotype (serotype 1). To keep the names of the genotypes and serotypes as consistent as possible, we propose that the genotype of the original PsHV1 reference strain KS144/79 be called genotype 1 and that genotype 2 and genotype 3 be those of the reference strains for serotype 2 (6840/87) and serotype 3 (3115/88), respectively. Genotype 4 corresponds to viruses that are most closely related to those of genotype 1 but can be distinguished from genotype 1 by both point mutations and a 6-bp deletion. All the viruses of genotype 4 are serotype 1, with the exception of the unique serotype 4 virus (1483/92).
Subdivisions of the four PsHV genotypes allowed resolution of all of the previously reported RFLP patterns with the exception of those of three viruses (15
). A virus classified as AA3A3 cannot be differentiated from an AA2A2 viruses, the BB2B2 virus cannot be differentiated from a BB1B1 virus, and the FFF virus cannot be differentiated from the GGG virus. Additional sequence data are needed to further differentiate these viruses and will undoubtedly define additional branches within each genotype.
The correlation between genotype and serotype is excellent. All but one serotype 1 virus map to either genotype 1 or 4, all but one serotype 3 virus map to genotype 3, and all serotype 2 viruses map to genotype 2. The serotype 4 virus maps to genotype 4 and cannot be differentiated from several serotype 1 viruses with our data. However, it has a unique RFLP pattern, indicating that it represents a subpopulation of the genotype 4 viruses and contains mutations that resulted in its becoming a unique serotype. The two viruses that were neutralized by antiserum to both serotype 2 and 3 viruses should be considered a genetically distinct variant of genotype 3 as they are identical to each other but represent unique variants of this genotype. Isolate 8326/87 (serotype 5) was identified as a herpesvirus based on its cytopathic effects and sensitivity to chloroform, inhibition of replication by 5-iodo-2′-deoxyuridine, and electron microscopic morphology. However, it was difficult to grow in CEFs (E. F. Kaleta, unpublished data), had a unique RFLP pattern, and was serologically distinct from all of the other PsHVs (6
). We were unable to amplify its DNA from three separate samples by using six different PsHV primer sets and additional degenerate primer sets that have successfully amplified DNA from highly divergent herpesviruses (21
). We must therefore conclude that the so-called PsHV serotype 5 is either a highly divergent virus and should not be considered a PsHV or was not present in the samples we analyzed.
Two European viruses, 1070/93 and 132/91, are problematic. Virus 1070/93 mapped to genotype 2 but was reported to be serotype 1 and virus 132/91 mapped to genotype 4 but was reported to be serotype 3 (5
). Both viruses also had sequences and RFLP patterns identical to those of another virus within each genotype, viruses that have the predicted serotypes. It is possible that these two viruses have mutations that have resulted in a change of serotype. It is also possible that these viruses represent a recombination event that occurred in a bird infected simultaneously with two serotypes of the PsHV. We cannot rule out this possibility; however, coinfection of parrots with multiple serotypes has yet to be demonstrated and the RFLP patterns of these viruses do not support this possibility. Finally, we cannot completely rule out the possibility of laboratory error during the isolation or serologic typing process.
Previously reported PCR amplification patterns of the PsHVs are only moderately predictive of genotypes. All viruses with PCR amplification pattern 6 are genotype 3. However, not all genotype 3 viruses have the same amplification pattern. The amplification pattern 1 viruses are predominately those of genotypes 1 and 4, but three (13.6%) are of other genotypes.
A major objective of this study was to determine whether the viral serotypes and genotypes found in Europe are representative of those found in the United States. Largely they are. Representatives of all genotypes are present in both the United States and Europe. Variants of some of the genotypes, however, are not always present in both Europe and the United States. Whether these finding are the result of a small sample size or an actual difference in prevalences of these viruses will require additional investigation. Some differences in the prevalences of the genotypes observed among the sequenced U.S. and European viruses are noted. In particular, genotype 2 viruses made up 27% of the European viruses sequenced and only 9% of the U.S. viruses sequenced. However, conclusions cannot be drawn as to the actual prevalences of these viruses in birds dying from Pacheco's disease as all European viruses are of cell culture origin and the ability to isolate each genotype in CEFs may vary. Additionally, neither the European isolates nor U.S. viruses examined in this study are selected randomly. The likelihood of the presence of all three PsHV serotypes in the United States is significant, as it is thought that a polyvalent vaccine may be necessary to protect against these three serotypes (10
) and the only vaccine currently available is monovalent.
Our data suggest that different genotypes are more pathogenic to some species of parrots than others. Specifically, genotype 4 appears to be pathogenic to both macaws and conures, genotype 3 rarely is, and genotypes 1 and 2 may not cause Pacheco's disease in these species at all. Similarly, genotype 4 is not found to cause Pacheco's disease in Amazon parrots in the United States, and no African grey deaths are attributed to infections with genotype 1. The Pacific species appear to be equally susceptible to all genotypes. Our data also suggest that genotype 4 viruses have a reduced ability to grow in CEFs derived from SPAFAS eggs. Alternately, this virus may grow to lower titers in the infected host. Given the close correlation between genotype and serotype and the different pathogenicities of these genotypes for different species and CEFs, we suggest that each genotype can also be considered a specific pathotype.
Herpesviruses are highly host adapted, causing little or no disease but lifelong infections in the species that they typically infect (4
). Disease results when infection occurs in nonadapted species. It is thought that Pacheco's disease is the manifestation of persistently infected, virus-adapted parrots' infecting naïve species (5
). Our data do not define which species may ultimately be carriers of the observed genotypes, but our data do have the potential to be used to address this question. Previously, it has been shown that PsHVs can be detected in swabs of mucous membranes from many persistently infected parrots by PCR (13
). However, this assay did not detect PsHVs in every bird in this study that was thought to be infected. Retrospectively, this may have been the result of the choice of primers and the inability of one or both primers to detect all genotypes (E. K. Tomaszewski, unpublished observations). In the present study, the primer set used in the initial amplification step appears to amplify DNA from all known PsHV genotypes. This primer set therefore has the potential to be used with PCR to screen swabs of mucosal surfaces from a range of parrot species to identify which ones are persistently infected with PsHVs. Point mutations and deletions within the four genotypes potentially allow for the design of PCR primer sets that can differentiate between the four genotypes, permitting the identification of the specific genotype infecting a bird. Alternately, genotypes can be defined by sequencing of amplicons. These tools could also be used to search for and identify genotypes of PsHVs associated with mucosal papillomas.