A novel Chaphamaparvovirus is the etiological agent of hepatitis outbreaks in pheasants (Phasianus colchicus) characterized by high mortality

Abstract

In the present study, we report the occurrence of several outbreaks of hepatitis in flocks of young pheasants in France, between 2017 and 2021. The disease was characterized by prostration, apathy and a median cumulative mortality of 12%, with the birds presenting multifocal to coalescing necrotizing hepatitis on necropsy. Severe extensive areas of degeneration and necrosis were observed in the liver, with degenerative hepatocytes presenting large amphophilic to acidophilic intranuclear inclusion bodies. Transmission electron microscopy examination of liver samples showed the presence of parvovirus-like virions of 21-24 nm, a finding already reported decades ago. Further investigations by Next Generation Sequencing and PCR revealed the complete genome of a novel species of parvovirus, here designated Phasianus chaphamaparvovirus 1 (PhChPV-1), that belongs to the new genus Chaphamaparvovirus in the Hamaparvovirinae subfamily. In situ hybridization and real-time PCR confirmed the etiology of the outbreaks, demonstrating the viral genome in the lesions. The findings establish the etiology of a pathology first described in pheasants 50 years ago and pave the way for a targeted protection strategy.

Keywords: Chaphamaparvovirus; hepatitis; inclusion bodies; outbreaks; pheasants.

FIGURE 1

Post‐mortem and histopathology investigations. Diseased birds presented severe hepatitis that varied in appearance: (a) swollen, mottled, yellowish‐brown livers and (b) haemorrhagic hepatitis with multiple ecchymotic lesions on the liver surface. The histopathology investigation revealed (c) extensive areas of necrosis in liver, with infiltration of mononuclear inflammatory cells and heterophils (*) and degenerated hepatocytes presenting eosinophilic intranuclear inclusion bodies (→) (H & E; bar = 100 μm). A high magnification shows (d) an eosinophilic intranuclear inclusion body in detail (→), surrounded by degenerating hepatocytes and inflammatory cells (H & E; bar = 20 μm)

FIGURE 2

TEM micrograph of a liver sample revealing small, icosahedral viral particles of 21–24 nm. The insert represents a higher magnification of the particles. Scale bars = 250 nm and 100 nm (insert)

FIGURE 3

(a) Metagenomic profiling of sample from case 19–03914 showing the presence of multiple viral and bacterial species. The arrow points to the tiny fraction of the viral content that was classified as parvovirus. (b) Alignment of 200‐bp non‐overlapping windows derived from metagenomic contigs to the representative strain of the Muscovy duck parvovirus (strain FM) highlighting the low coverage of the preliminary assembly derived from NGS data

FIGURE 4

PhChPV‐1 genome characteristics. (a) Schematic representation of PhChPV‐1 genomic features with in silico predicted ORFs. (b) The predicted secondary structure of the terminal repeats of PhChPV‐1

FIGURE 5

Phylogenetic analysis. The evolutionary history was inferred by using the maximum‐likelihood method and Le_Gascuel_2008 model (Le & Gascuel, 2008). The tree with the highest log likelihood (−61123.54) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour‐Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 1.6388]). The rate variation model allowed some sites to be evolutionarily invariable ([+I], 0.31% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 52 amino acid sequences. Accession numbers for NS1 are given in parenthesis next to each sequence except the newly derived PhChPV‐1. There were 1242 positions in total in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018)

FIGURE 6

Demonstration of PhChPV‐1 DNA in liver by ISH. Generalized and strong positive signals (arrow) in histologic section of a liver of a dead pheasant from case 20–18963. The arrowhead labels a non‐stained nucleus (ISH bar = 100 μm)