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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.

also members of the family Phasianidae. This includes diseases such as avian influenza, Newcastle disease, infectious bursal disease, infectious laryngotracheitis, avian botulism, fowl cholera, mycoplasmosis, salmonellosis and coccidiosis, as summarized recently by Dwight et al. (2022, appendix S1). Additionally, marble spleen disease (MSD) caused by the Turkey siadenovirus A (TAdV-3), a member of the Siadenovirus genus, is of great importance in pheasants, producing a characteristic pathology picture, which includes enlarged, mottled (marbled) spleen and the presence of intranuclear inclusions within mononuclear cells (Rautenschlein et al., 2020). Intranuclear inclusions have been additionally described in hepatitis outbreaks in pheasants in the United States and Italy, with one report associating such pathology with the presence of a parvovirus in the hepatocytes by electron microscopy (Gelmetti et al., 1996;Rosen et al., 1965).
Parvoviruses are small, icosahedral, non-enveloped viruses, which have a linear, single-stranded DNA (ssDNA) genome, and belong to the large and diverse Parvoviridae family (Cotmore et al., 2019). Poultry, especially young birds, are susceptible to infections from parvoviruses within the Parvovirinae subfamily. Thus, chicken and turkey parvoviruses, within the genus Aveparvovirus, are responsible for enteric disease, and are often associated with the runting-stunting syndrome and the poult enteritis complex, respectively, although they might be present in healthy birds as well (Day, 2020). Additionally, strains from the Anseriform dependoparvovirus 1 species, within the genus Dependoparvovirus, are responsible for a highly contagious and fatal disease in young geese and Muscovy ducks, and a less severe, chronic disease in mule and Pekin ducks (Palya, 2020). In 1993, the Parvoviridae family was divided in two subfamilies, based on host association, with the subfamily Parvovirinae consisting of viruses that infect vertebrates, and the subfamily Densovirinae comprising viruses that infect invertebrates (Cotmore et al., 2019). However, since 2012, genomes belonging to a novel lineage of parvoviruses designated 'Chapparvovirus' were detected in different vertebrates. For that reason, the family Parvoviridae was recently reorganized and a new subfamily -Hamaparvovirinae -was created, which accommodates viruses infecting vertebrates and invertebrates (Pénzes et al., 2020).
In the present report, we describe a series of field outbreaks of severe hepatitis in young pheasants from rearing farms in France, caused by a novel Chaphamaparvovirus, from the recently created Hamaparvovirinae subfamily, culminating in high mortality and severe losses.

Case history and post-mortem investigations
Between 2017 and 2021, similar outbreaks were noted in 15 pheasant rearing farms in France, with a clinical picture that consisted of prostration, apathy and increased mortality. Most of the affected flocks were located in the region Pays de la Loire, with two flocks located in the regions of Bretagne and Nouvelle Aquitaine, and comprised between 1000 and 13,000 pheasants. The onset of clinical signs was observed when the birds were 3-6 weeks old, and the cumulative mortality varied between 4% and 41%, with a median value of 12% (Table 1). Culled birds were necropsied according to a standard protocol in the laboratory facilities of Labovet and gross pathologic lesions were recorded.
Samples mostly from liver, but also from spleen, pancreas, intestine and kidney were collected and processed for histopathology, electron microscopy and molecular investigations.

Histopathology
Samples of liver, spleen, intestine and kidney were fixed in 4% neutral buffered formaldehyde solution (SAV LP GmbH, Flintsbach, Germany). Following fixation, samples were dehydrated and embedded in paraffin. Then, the paraffin-embedded samples were cut into 4-µmthick sections in a microtome (Microm HM 360; Microm Laborgerate GmbH, Walldorf, Germany), and the generated tissue sections were mounted on glass slides and stained with haematoxylin and eosin (H&E) for microscopic assessment.

Transmission electron microscopy (TEM)
Previously frozen liver samples, at −20 • C, were used for transmis-

Nucleic acid extraction and construction of a NGS library
Nucleic acids were extracted from formalin-fixed paraffin-embedded

Analysis of NGS-data obtained from NextSeq
The de-multiplexed raw NGS-data were analysed with an in-house established bioinformatics pipeline. In short, the NGS-reads were adaptor trimmed using cutadapt (Marcel Martin, 2011) and assembled with MEGAHIT (default parameters). The resulting metagenomics contigs were divided into 200-bp non-overlapping windows and analysed by the Taxonomic Profiling tool from CLC Genomics Workbench 20 (QIAGEN), against a precompiled database containing all viral and bacterial sequences from NCBI (April 2020

Creating the complete genome sequence
Since the initial assembly with NGS-derived reads produced only a partial genome sequence (1554 bp), the remaining sequence, including gaps and genome ends, was amplified by PCR, cloned into the PCR-Vector and sequenced with the Sanger method. In order to have sufficient DNA for the analyses, liver tissue from sample 20-18963, which was positive for parvovirus by real-time PCR, was used instead of the FFPE sample from case 19-03914. Prior to DNA extraction, the sample was pre-treated (Technical Appendix -Section 1) and DNA was extracted with the DNeasy Blood and Tissue Kit (QIAGEN), according to the manufacturer's instructions. Six PCRs were used to close the gaps whereas specific nested PCRs were applied to amplify the 5′-and 3′-ends of the genome (Technical Appendix -Section 1). Finally, the nearly complete genome was amplified with primers PhChPV-1-compl-F and PhChPV-1-compl-R (Technical Appendix -Section 1) and high fidelity DNA polymerase.
Expected PCR products (Appendix Table A1) were excised and puri-

Genome analysis
The secondary structure of 5′-and 3′-UTR regions of the genome was predicted using the Quikfold application on the UNAfold Web

Phylogenetic analysis
The phylogenetic analysis was based on the complete NS1 protein sequences (deduced amino acids). For that purpose, 51 NS1 protein sequences including at least one species representative of all genera belonging to subfamilies of Hamaparvovirinae and Parvovirinae were downloaded from the NCBI protein database. The amino acid alignment was produced by using the MUSCLE program implemented in the MEGA X software with Neighbour Joining Clustering method (Kumar et al., 2018). The phylogenetic analysis was inferred with maximumlikelihood method and Le and Gascuel model (Le & Gascuel, 2008) using MEGA X software.

Real-time PCR and in situ hybridization
In order to examine the samples for PhChPV-1, DNA was extracted from fresh organs and FFPE samples with the DNeasy Blood and Tissue Kit (QIAGEN) and the QIAamp DNA FFPE Tissue Kit (QIAGEN), respectively, according to the manufacturer's instructions. Primers and probe targeting a NS1 protein sequence (Table 2)  Paraffin-embedded samples were investigated by in situ hybridization (ISH), using a digoxigenin-labelled specific DNA probe based on the NS1 gene of PhChPV-1 (Table 2), and following a protocol earlier described Liebhart et al. (2006).

Post-mortem and histopathology findings
At necropsy, birds from impaired flocks revealed hepatitis characterized by hepatomegaly, with changes in the organ appearance varying from mottled to yellowish-brown, with 20-30% of birds also presenting multifocal to coalescing depressed red foci in the parenchyma (Figure 1a and b). Approximately 80% of birds presented mild to severe haemorrhagic enteritis, nephritis, with 10-20% of birds presenting congested and swollen spleens as well. The histologic investigation showed multifocal to almost diffuse degeneration and necrosis of hepatocytes (most frequently as random hepatocyte necrosis), with generally sparse infiltration by mononuclear inflammatory cells and heterophils, and with degenerating hepatocytes presenting amphophilic to acidophilic prominent intranuclear inclusion bodies, filling the nucle-oplasm (Figure 1c and d). In subacute to chronic forms, which were regularly observed, a moderate to marked hyperplasia of bile ducts was also noticed. In some birds, an inflammation of the intestinal mucosa by mononuclear cells could be observed.

Discovery of a novel Chaphamaparvovirus in pheasants by TEM and NGS
The TEM examination of liver samples revealed the presence of  Table A2). Even though the majority of viral contigs mapped to dsDNA viruses, more precisely to TAdV-3 (haemorrhagic enteri-

Genome structure analysis
In order to complete the genomic sequence, a set of PCRs was developed (Appendix Table A1 (Liu et al., 2020). However, unlike Chaphamaparvoviruses recently identified in North American wild ducks, no coding sequence for VP2 could be identified (Canuti et al., 2021).

Phylogenetic analysis
Phylogenetic analysis based on the NS1 protein revealed that the genome identified in pheasants clusters to the Chaphamaparvovirus genus of the newly recognized subfamily Hamaparvovirinae ( Figure 5).
According to the recently proposed taxonomy, it appears to be a repre-

DISCUSSION
In the present study, we report the occurrence of several outbreaks  (Hess, 2020). FAdVs, which are known to infect various bird species (Fitzgerald, 2020), were once isolated in young pheasants but from the respiratory tract (Cąkała, 1966). In the present report, however, the etiologic role of FAdVs in the hepatitis outbreaks was ruled out by cell culture and PCR investigations on different organ tissues (Technical Appendix -Section 2). Alternatively, parvovirus strains from the Anseriform dependoparvovirus 1 species, within the Dependoparvovirus genus, are capable of causing acute disease in young geese and ducks, with characteristic lesions including hepatitis and the presence of similar intranuclear inclusion bodies (Palya, 2020). Interestingly, early reports described outbreaks of highly infectious viral hepatitis in young pheasants, with an analogous clinical presentation to the present study, which included high mortality, hepatomegaly with congestion and inclusion bodies in the hepatocytes (Rosen et al., 1965;Swarbrick, 1973). However, the etiology of the problem was  investigation provides a conclusive step in establishing parvovirus as the likely etiology agent of acute haemorrhagic hepatitis with inclusion bodies in pheasants reported from several countries.
Advances in molecular diagnostic tools, such as NGS, enabled the discovery of new viruses, and in particular new parvoviruses, with some of them being identified in avian species (Kim et al., 2020;Liu et al., 2020;Pénzes et al., 2020;Vibin et al., 2020;Wang et al., 2019).
Most of these new parvoviruses were classified within the recently established Chaphamaparvovirus genus, within the newly created Hamaparvovirinae subfamily, which accommodates parvoviruses that F I G U R E 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) F I G U R E 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) infect both vertebrate and invertebrate hosts, and have an amino acid sequence identity for the NS1 protein above 30% (Pénzes et al., 2020). Likewise, in the present study a new parvovirus -PhChPV-1belonging to the Chaphamaparvovirus genus was identified by NGS in liver samples from dead pheasants. According to the updated taxon demarcation criteria, parvoviruses can be considered members of different species if their NS1 proteins share less than 85% amino acid sequence identity (Pénzes et al., 2020), thus the PhChPV-1 identified in the present report was categorized in a newly suggested species -Phasianus chaphamaparvovirus 1. Interestingly, the closest phylogenetic sequence to PhChPV-1, with 82.1% amino acid sequence identity, is a partial genome sequence of parvovirus associated with hepatitis in grey partridges, in which eosinophilic inclusion bodies were observed in the liver, thus similar to our observations in pheasants (Grilli et al., 2008). Although partridges were initially categorized in a distinct subfamily from pheasants, more recent molecular phylogeny data revealed that these two bird subfamilies actually constitute only one lineage, with some partridges being more closely affiliated with pheasants (Kimball et al., 1999). Therefore, considering that the outbreak in the partridge-rearing farm was observed in the same geographic region as identical parvovirus hepatitis outbreaks in pheasants reported by Gelmetti et al. (1996), it can be hypothesized that such parvoviruses can cross different host species. Interestingly, early on, chukar partridges were implied in the introduction of the pathogen responsible for outbreaks of highly infectious hepatitis in young pheasant farms (Rosen et al., 1965), which aligns with our hypothesis. Although in our study, parvovirus outbreaks were not observed in nearby farms of grey and red partridges, future epidemiologic studies shall include this species as well, as they might act as asymptomatic carriers.
The clinical significance of most newly discovered Chaphamaparvovirus identified by NGS in avian species remains unknown, as cau-sation between the virus and a disease was not fully established. Thus, in addition to NGS investigations, in the present study we assessed organ tissues by ISH and real-time PCR, using specific primers and probes targeting the PhChPV-1 NS1 gene. As a result, all investigated samples were PCR positive and hepatocytes with intranuclear inclusion bodies became strongly positive by ISH, indicating a direct causation between PhChPV-1 and the observed pathology. Diseased birds also presented mild to severe haemorrhagic enteritis during postmortem investigations, which might be explained by the fact that parvoviruses favour infection of actively dividing cell populations such as the enterocytes in young birds (Cotmore et al., 2019) The affected farms from this study produce pheasants for landscape repopulation that are released into the wild from 20 weeks of age onwards. Even though the disease seems to affect young birds up to 6 weeks of age, the etiologic resolution of the outbreaks here reported stresses the need for further epidemiologic studies. Furthermore, new outbreaks are reported every year during the rearing period, which might be due to the high environmental resistance of parvoviruses (Cotmore et al., 2019). For this reason, new intervention strategies are needed regarding farm biosecurity and hygiene. However, game bird farms present numerous structural challenges hindering the application of such measures, as husbandry and management is not comparable with modern poultry farms. Alternatively, a vaccination strategy might be developed, which implies the culture and isolation of the virus in the laboratory.
In conclusion, in the present study, we report the occurrence of several outbreaks of hepatitis in young pheasants in France, which resulted in severe clinical signs and mortality. Investigations by TEM and NGS revealed a novel parvovirus species, here designated Phasianus chaphamaparvovirus 1, within the recently created Hamaparvovirinae subfamily and Chaphamaparvovirus genus, as the responsible agent for the outbreaks. Analyses of organ tissues from diseased pheasants by in situ hybridization and real-time PCR confirmed the etiology of the outbreaks, as the viral genome was present in the observed lesions. Thus, the present investigation provides a decisive step in establishing the etiology of this pathology in pheasants, more than 50 years after its initial description. Moreover, it highlights the need of more research on this new group of parvoviruses, as it encloses possible important pathogens for different avian species.