3.6 Characterization of recombination detected in the genome of FPV HNZZ-2
Genetic recombination is generally considered as a key mechanism in virus evolution. Several studies have reported multiple infections of autonomous parvovirus, revealing that parvoviruses have a considerably higher degree of variability (João Vieira et al., 2008; Liu et al., 2015). The putative recombinant sequence and its parents were identified using two different recombination detection programs, namely RDP4 and Simplot software packages. Interestingly, both Simplot and RDP4 programs detected recombination signals in the nucleotide sequence of FPV HN-ZZ2(Figure 7). RDP4 identified nucleotide position 2,841 as an ending breakpoint and predicted a recombination region located at nucleotide positions 1,956 and 2,841 of the alignment (Figure 7A and 7B). Among the nucleotide sequence data for all the 40 strains in the RDP4 program, nucleotides 1,956-2,481of the FPV HN-ZZ2 possessed the greatest similarity (99.7%) to the corresponding strain CPV-L, meanwhile, other parts have the highest homology with the FPV Raccoon/RC9/BC2010 strain (99.3%). Further to confirm whether reorganization existed in FPV HN-ZZ2, Simplot was used, and nt position 2580 was identified as a putative breakpoint for recombination (Figure 7C). This indicated that the FPV HN-ZZ2 strain was likely to be a new recombinant virus between CPV and FPV.
A phylogenetic tree based on the full-length NS1 and VP2 nucleotide sequence was constructed using the isolated strain and 39 reference strains (Figure 8). Phylogenetic analysis showed that the VP2 gene of FPV HN-ZZ2 was in the same branch as CPV (Figure 8A), and the sequence of the NS1 gene is closely related to FPV and MEV (Figure 7B). The results are consistent with previous recombination analysis results, further indicating that the isolate may be a recombinant virus.
In this study, the amino acids of NS1 at positions 23, 247, 443, 545, 595, and nucleotide at positions 309, 492, 621, 1543, 1959,1990 were found to be identical to those of CPV reference strains. Interestingly, the 60th amino acid of NS1 showed a mutation (I→V), which was different from the three control strains (Table 3). Analysis of the VP2 protein found that the amino acids at the host-specific sites at positions 80, 87, 93, 103, 300, 305, 323, 564, and 568 were identical to the amino acid of the FPV reference strain. The 217th amino acid of VP2 showed a mutation (T→R), which was different from the three control strains (Table 4). The 300th amino acid was A (Ala) and the 562nd amino acid was L (Leu), which also confirmed that the isolate FPV HN-ZZ2 belonged to FPV rather than MEV (Shackelton et al., 2007). Whether new variants of NS1 and VP2 causes the clinical symptoms to be different from the classic strains remains unclear. Besides, compared with the FPV HN-ZZ1 strain, a unique substitution in NS1 and VP2 protein sequence was observed (Table 5). Whether these amino acid differences are evidence of host differences between FPV HN-ZZ2 and HN-ZZ1 remains to be studied.
DISCUSSION
Clinical disease associated with FPV in adult cats is usually progressive and fatal, leading to fever, vomiting, and diarrhea, associated with pancytopenia and necrotizing enteritis (Lamm et al., 2008). Fetal infection by FPV most commonly leads to abortion or stillbirths, whereas Late embryonic infection is more often accompanied by brain lesions, including cerebellar hypoplasia, hydrocephalus and hydranencephaly. In young wild and domestic carnivores, the infection usually causes severe gastroenteritis, which is in most cases hemorrhagic and there have been no previous reports of lions contracting brain lesions (Stuetzer et al., 2014; Sharp et al., 1999). In this study, the young lion mainly developed brain tissue changes. This is similar to previous reports related to the pathological changes of FPV infections in kittens such as lymphoplasma meningoencephalitis and neuronal necrosis, the combined CNS malformations of hydranencephaly and cerebellar hypoplasia (Stuetzer et al., 2014; Allison et al., 2013). This study further conducted a comprehensive analysis of the full sequence of FPV HN-ZZ2 and found that the NS1 gene had higher similarity to CPV and VP2 gene has a high similarity with FPV.
In summary, combined with clinical manifestations and pathogen detection results of the infected cub, it should be determined that the dead cub have resulted from an FPV embryonic infection. This study speculated that gene recombination forms a new virus. As a result of mutations, FPV continues to expand its host range and change its tissue tropism to become the main pathogen causing infections in many wild animals. FPV has continuously evolved via the accumulation of point mutations in the viral genome, resulting in two important implications. Following the emergence of new antigen variants with different biological and antigenic characteristics, the vaccine program may need to be adjusted accordingly and the virus strains in commercial products are updated. This study supports the need for continued epidemiological surveillance to detect new FPV variants that may escape the host immune system and detection methods. Besides, mixed breeding should be avoided for captive animals to reduce the risk of disease transmission. Therefore, this study not only provides new gene sequences for the global study of FPV-infected lions but also helps to further study the incidence and genetic diversity of FPV.