Regional adaptations and parallel mutations in Feline panleukopenia virus strains from China revealed by nearly-full length genome analysis

Abstract

Protoparvoviruses, widespread among cats and wild animals, are responsible for leukopenia. Feline panleukopenia virus (FPLV) in domestic cats is genetically diverse and some strains may differ from those used for vaccination. The presence of FPLV in two domestic cats from Hebei Province in China was identified by polymerase chain reaction. Samples from these animals were used to isolate FPLV strains in CRFK cells for genome sequencing. Phylogenetic analysis was performed to compare our isolates with available sequences of FPLV, mink parvovirus (MEV) and canine parvovirus (CPV). The isolated strains were closely related to strains of FPLV/MEV isolated in the 1960s. Our analysis also revealed that the evolutionary history of FPLV and MEV is characterized by local adaptations in the Vp2 gene. Thus, it is likely that new FPLV strains are emerging to evade the anti-FPLV immune response.

Fig 1. FPLV detection by colloidal gold test strip and PCR assay.

A Detection of the FPLV antigen by colloidal gold test strip. A1 negative control, A2 positive sample. B Detection of FPLV in samples by PCR. Lanes 1 and 5 DNA marker; Lane 2 positive control; Lane 3 positive sample Lane 4 negative control.

Fig 2. Cytopathic effect of feline panleukopenia virus on CRFK cells.

Panel A shows the cytopathic effect induced by FPLV in CRFK cells visualized by inverted microscopy at magnification of 200 μm 72 hours post infection. Panel B is the negative control of uninfected CRFK cells.

Fig 3. Indirect immunofluorescence assay of FPLV in CRFK cells.

Immunofluorescence staining of CRFK cells after infection with FPLV. Panels A and B indicate nucleous stained with DAPI. C) Mock-infected controls. Uninfected CRFK cells served as a negative control (D). A green color indicates positive Fluorescein isothiocyanate (FITC) staining and blue indicated the nuclear stained with DAPI of FPLV infected cells. Scaled indicate of 200 μm of magnification.

Fig 4. Phylogenetic tree constructed using near-full genomes of carnivorous parvoviruses.

The tree was inferred using a maximum likelihood (ML) approach assuming HKY model plus a discrete Gamma correction and the rate of invariable sites. The FPLV strains isolated in this study are indicated by red arrow in the tree. Branch support is indicated by a colored scale and were obtained by the approximate likelihood ratio test (aLRT) where one is the highest score. Black diamonds indicate the position of the historical FPLV strains; those that were isolated prior or near the spread of CPV in domestic dogs. Some clusters of FPLV or MEV that are discussed in the manuscript are indicated by the symbol #. Values inside the gray areas indicate the genetic distances of each clade. The Genbank identification of all sequences used in the analyses are listed in Material and Methods. Phylogenetic analyses and estimation of genetic distances were conducted with MEGAX software.

Fig 5. Phylogenetic tree constructed using NS1, VP1 and VP2 of carnivorous parvoviruses.

Partitions corresponding to the genes Ns1, Vp1 and Vp2 were used to construct trees and show the clustering pattern of sequences. The upper panel shows the tree inferred using the partitions corresponding to the Ns1 gene of parvoviruses (2004 nucleotides long). The lower panels in the figure show the trees inferred using the Vp1 gene region with 2182 nucleotides and the Vp2 gene with 1753 nucleotides. All trees were inferred using a maximum likelihood (ML) approach assuming the HKY model plus a discrete Gamma correction and the rate of invariable sites. The FPLV strains isolated in this study are indicated by blue arrows in the tree. Strains KP769859, KX434461 and KX434462 are indicated by green arrows in the trees. Branch support are indicated in a colored scale and were obtained by the approximate likelihood ratio test (aLRT) where one is the highest score. Black diamonds indicate the position of historical FPLV strains, those that were isolated prior or near the spread of CPV in domestic dogs. Some clusters of FPLV or MEV discussed in the manuscript are indicated by numbers. The Genbank identification of all sequences used in the analyses are listed in Material and Methods. Phylogenetic analyses and estimation of genetic distances were conducted in MEGAX software.

Fig 6. Ancestral reconstruction of amino acids of VP2.

The tree was inferred using a maximum likelihood (ML) approach assuming the HKY model plus a discrete Gamma correction and the rate of invariable sites. The FPLV strains isolated in the present study are indicated by red arrow in the tree. Branch support are indicated by a color scale and were obtained by the approximate likelihood ratio test (aLRT) where one is the highest score. Some clusters of FPLV or MEV that are discussed in the manuscript are indicated by hashtag symbols. Circles with distinct colors indicate strains isolated in different species according to the color code in the figures in the right portion of the tree. Grey rectangles indicate amino acid changes that occurred in some clusters, numbered rectangles indicate the position in the VP2 protein. The letters indicate the one letter amino acid code as follows: A = Alanine; C = Cysteine; D = Aspartic Acid; E = Glutamic Acid; F = Phenylalanine; G = Glycine; H = Histidine; I = Isoleucine; K = Lysine; L = Leucine; M = Methionine;N = Asparagine; P = Proline; Q = Glutamine; R = Arginine; S = Serine; T = Threonine; V = Valine; W = Tryptophan; Y = Tyrosine. The Genbank identification of all sequences used in the analyses are listed in Material and Methods. Sequences used to infer the VP2 tree were absent from any signal of recombination according to the analysis based on distinct methods as implemented in the RDP v.4 software.