Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus

Abstract

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Fig 1

ML phylogeny of carnivore parvoviruses from various hosts based on 460 partial sequences of the VP2 gene. Red indicates that the sequence is from a raccoon, except for the marked (●) bobcat isolate (inset) that was sequenced during this study. Other sequences are colored by virus strain, regardless of host, with FPV-like sequences in green, CPV-2 sequences in black, and CPV-2a, -b, and -c sequences in blue. The phylogenetic positions of three leopard cat sequences from Southeast Asia (♦) and of one canine sequence from South Korea (∧) are also marked. The tree is rooted using the oldest sequence in the data set, FPV/Cat/US/FPV-d/64 (GenBank accession number U22189). Bootstrap values of >0.90 are marked by asterisks. Branch lengths are drawn to a scale of nucleotide substitutions per site. (Inset) Expansion of the phylogeny section containing the main group of virus sequences from raccoons, highlighting the amino acid substitutions that have occurred on each branch. All raccoon and bobcat viruses, including CPV/Raccoon/NJ/76836/10 (▿), have the 300Asp mutation.

Fig 2

ML tree of 48 NS1 gene sequences. Branches are color coded as explained in the legend to Fig. 1. The tree is rooted using the oldest sequence in the data set, FPV/Cat/US/FPV-3/67 (GenBank accession number EU659111). Bootstrap values of >0.90 are marked by asterisks. Branch lengths are drawn to a scale of nucleotide substitutions per site, and sample names are given for the raccoon isolates.

Fig 3

Capsid amino acid residues associated with viruses isolated from raccoons in different parts of the United States between 1978 and 2010, as well as viruses isolated from leopard cats in Vietnam in 1997, a mink isolate from Wisconsin recovered in 1973, and a bobcat virus from Kansas recovered in 2010. Also included are the sequences for the FPV, CPV-2, and CPV-2a-related prototypes. FPV-like residues are highlighted in black. CPV-like residues are highlighted in gray or yellow. Mutations unique to the CPV-derived isolates from raccoons are highlighted in blue. The 426E in CPV-2c strains is highlighted in yellow.

Fig 4

Relative infectivities of the different viruses for feline NLFK cells (filled bars), canine A72 cells (shaded bars), or Cf2Th cells (open bars). Three different raccoon isolates (Rac) were tested, along with prototype FPV, CPV-2, and CPV-2b sequences (FPV/Cat/NY/292/68, CPV-2/Dog/NY/CPV-d/79, and CPV-2b/Dog/TX/CPV-39/84) and the 300Asp mutant of CPV-2 (CPV-2/Dog/NY/CPV-d/79:300D). Virus titers were determined by TCID₅₀ assays in each cell line. Error bars indicate standard deviations based on three independent experiments.

Fig 5

Binding of capsids and canine transferrin (Tf) to the domestic cat, raccoon, and canine TfRs expressed on TRVb cells, as measured by flow cytometry. Cells were incubated with Cy5-conjugated Tf (to detect TfR-expressing cells) and with virus capsids. The cell-associated capsids were detected using Alexa 488-conjugated MAb 2. Binding is expressed as fluorescence intensity (FI) (A through D) or mean fluorescence intensity (MFI) (E). (A through D) Binding of Tf and CPV-2 (CPV-2/Dog/NY/CPV-d/79) (A and C) and of Tf and CPV/Rac/VA/118-A/07 (B and D) to feline and raccoon TfRs (A and B) or to canine TfR (C and D). (D) Tf binds the canine TfR, but CPV/Rac/VA/118-A/07 does not. (E) Comparison of the binding of viruses to all three TfRs. Error bars, standard deviations of the mean. Three independent experiments were performed. Asterisks indicate binding significantly higher than background levels (P < 0.05).

Fig 6

Antigenic analysis of viral variants using the hemagglutination inhibition (HI) assay and MAbs prepared against capsids of FPV (red), CPV-2 (blue), or CPV-2b (black). The results are expressed as comparisons of HI titers to those with the original capsid. Four different viruses from raccoons were tested, along with the FPV, CPV-2, and CPV-2b prototypes (FPV/Cat/NY/292/68, CPV-2/Dog/NY/CPV-d/79, CPV-2b/Dog/TX/CPV-39/84) and the 300Asp mutant of CPV-2 (CPV-2/Dog/NY/CPV-d/79:300D). Filled rectangles indicate binding within ±2-fold of the original titer; shaded rectangles, 4- to 16-fold reductions in titer; open rectangles, >16-fold reductions in titer.