Retrospective Differentiation of Canine Distemper Virus and Phocine Distemper Virus in Phocids

Abstract

Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay.

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“…The F -, H -, and P -gene sequence analyses (figures 4,5,6) indicate that the 1998 viruses stem from a different CDV lineage that includes American CDV strains Lederle and Snyder Hill. A recent phylogenetic analysis of the P -gene by an independent laboratory that utilized some of our P -gene data generated similar results [42]. Because they were isolated before CDV Lederle and Snyder Hill were acquired from the ATCC for this study and have distinguishable F - and H -gene sequences [22], it is certain that the 1998 CDV isolates are not due to laboratory contamination.…”

Phocine Distemper Virus: Current Knowledge and Future Directions

“…Immunohistochemistry is also extremely useful if the classic histological lesions or inclusions are no longer visible or equivocal as can occur in more chronic cases and in animals where the viral lesions are masked by severe inflammation or necrosis caused by secondary infections. Currently, most laboratories use an avidin-biotin complex (ABC) technique and either a primary monoclonal antibody against the nucleocapsid (N) of CDV (VMRD, Inc., Pullman, WA, USA) or polyclonal rabbit anti-CDV nucleoprotein [ 112 , 113 ].…”

“…This assay was used to test tissue samples from the 2002 European harbor seal epidemic [ 116 ] and more recently from nasal swabs and archived tissues from Northern sea otters ( Enhydra lutris kenyoni ) in Alaska [ 117 ]. Stanton et al [ 113 ] modified the P gene primers to shorten the amplicon to a 149 bp fragment for use in formalin-fixed paraffin-embedded tissues. The assay maintained the sensitivity to differentiate CDV and PDV, and was useful for determining that CDV was present in formalin-fixed paraffin-embedded tissues from a Caspian seal; and PDV in harp, hooded and European harbor seal tissues.…”

Use of a Slam Transfected Vero Cell Line to Isolate and Characterize Marine Mammal Morbilliviruses Using an Experimental Ferret Model

“…Isolation of the morbillivirus responsible for the mass mortality of Mediterranean monk seals (Monachus monachus) in 1997 was accomplished using Vero cells and standard cell culture isolation methods, but first required several ''blind passages'' (Osterhaus et al, 1997). Although there are now commercial polyclonal and monoclonal antibodies available that can differentiate between CDV and PDV immunohistochemically (Stanton et al, 2004), there is a very limited number of molecular-based methods of species differentiation. Restriction fragment length polymorphism, combined with conventional RT-PCR analysis, can identify species of morbilliviruses (Saliki et al, 2002); however, since some genes are not conserved, and extra manipulations of RT-PCR products are also required, this limits the usefulness of this approach.…”