Detection of chicken chapparvovirus 2 in chickens with hemorrhagic hepatitis in Japan

Abstract

Chicken chaphamaparvovirus causes diarrheal symptoms and can be detected in fecal samples. This study reports the detection of chicken chapparvovirus 2 in debilitated chickens with hemorrhagic hepatitis at a broiler farm in Japan. After euthanasia and necropsy, liver hemorrhage was observed. Nuclear inclusion bodies in the hepatocytes were identified using histological analysis. High-throughput sequencing analysis using RNA from livers of three affected chickens revealed infection by chicken chapparvovirus 2 and chicken anemia virus. Polymerase chain reaction analysis showed that all three chickens were positive for chicken chapparvovirus 2, and only one was positive for both chicken chapparvovirus 2 and chicken anemia virus. In conclusion, chicken chapparvovirus 2 causes infection in chickens in Japan and might be involved in hemorrhagic hepatitis.

Parvoviruses (family Parvoviridae) are non-enveloped viruses with single-stranded DNA genomes enclosed in icosahedral capsids [4]. Conventionally, family Parvoviridae is classified into two subfamilies, Parvovirinae and Densovirinae, which consist of viruses that infect vertebrates and arthropods, respectively [4]. However, the discovery of new viruses has challenged these traditional classifications, leading to the establishment of a new subfamily, Hamaparvovirinae [15]. Viruses belonging to the genus Chaphamaparvovirus, a novel genus classified under Hamaparvovirinae, have been detected in various vertebrates, including rodents [17, 21], pigs [14], dogs [7], cats [6], turkeys [16], pheasants [11], and chickens [5, 9, 10, 18]. The virus can be identified in fecal samples, and diarrhea is a common symptom among the infected pigs, dogs, cats, and chickens [5,6,7, 14].

Chicken chaphamaparvovirus (CkChpV) was first reported in chicken fecal samples collected in Brazil in 2019 [10]. In 2023, an epidemiological study conducted in China aimed to detect several viruses, including CkChpV, in the feces of both healthy chickens and those exhibiting diarrhea [5]. CkChpV was detected in 5.7% of healthy chickens and 32.5% of those with diarrhea, indicating a statistically significant correlation between viral infection and diarrheal symptoms [5]. In addition, galliform chaphamaparvovirus 4 was detected in bile samples from chickens diagnosed with spotty liver disease at a free-range poultry farm in Australia [18]. In avian species, several young pheasants in France that succumbed to necrotizing hepatitis between 2017 and 2021 were infected with Phasianus chaphamaparvovirus 1 [11]. In these cases, nuclear inclusion bodies were observed in the hepatocytes of the deceased pheasants, and the existence of parvovirus-like virions was confirmed using electron microscopy [11]. The virulence of chaphamaparvoviruses in chickens may vary depending on the specific viral strains involved. However, this variability is not yet fully understood. Thus, further studies on the distribution of chaphamaparvoviruses are necessary to enhance our understanding of their virulence in chickens.

In February 2023, three debilitated chickens were found at a poultry farm in Kagoshima Prefecture. The chickens raised on the farm were Ross 308 broiler chickens, and the chicken coop was an open structure with floor rearing, accommodating 8,500 birds per house (mixed genders). The administered vaccines included a live Marek’s disease vaccine (in ovo), a live avian infectious bronchitis vaccine (at hatch, via spray), a combined live vaccine for Newcastle disease and avian infectious bronchitis (administered when chickens were 2 weeks old, through drinking water), and a live infectious bursal disease virus vaccine (administered when chickens were 3 weeks old, through drinking water). Following euthanasia, necropsy was performed, revealing liver hemorrhage and gizzard hyperemia. Histology revealed nuclear inclusion bodies in the hepatocytes. Avian influenza virus, fowl adenovirus, and infectious bronchitis virus infections were ruled out using routine examinations, including polymerase chain reaction (PCR), reverse-transcription PCR, immunochromatography, and virus isolation. Further, no clinically significant bacteria were detected. Details of the examinations are noted in the Supplementary File. On April 22, 2023, 23 chickens were found dead on the same farm. Necropsies on eight of these chickens revealed liver hemorrhages similar to those observed in the February cases. Two days later, three more chickens were identified in a weakened state, their necropsies also indicated liver hemorrhage (Fig. 1A), and the livers were obtained for histopathological examination. The chickens were 39 days old, comprising two females and one male; however, individual identification was not possible at the time of receiving the liver samples. Furthermore, no histological examinations were conducted on organs other than the liver. Liver sections were fixed in formalin, embedded with paraffin, and then stained with hematoxylin and eosin. Histopathological examination revealed multifocal hemorrhage and lymphocytes infiltration, and apoptosis of hepatocytes around hemorrhage areas in the liver of the three chickens. Interestingly, many eosinophilic intranuclear inclusion bodies formed in the hepatocytes around apoptotic areas (Fig. 1B). These results suggest that the disease in chickens was caused by viral infections other than those listed above.

Fig. 1.

Gross pathological and histopathological findings in the chickens. (A) Numerous ecchymotic lesions are observed on the surface of the liver. (B) Hematoxylin and eosin-stained image of the liver. Large inclusion bodies are observed in the nuclei of the hepatocytes. Eosinophilic intranuclear inclusion bodies are observed in the hepatocytes (arrowheads).

To identify the possible etiological agent of this disease, high-throughput sequencing was conducted. RNA was extracted from the livers of the three chickens in a weakened state using a FastGene™ RNA Premium Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan). The RNA extracts were pooled and subjected to library preparation after rRNA depletion. Sequencing was performed using the Illumina NovaSeq 6000 platform, yielding 71,151,968 pairs of 150-bp reads. The reads were preprocessed by fastp [2] and mapped to the chicken genome using HISAT2 [8], and the unmapped reads were subjected to de-novo assembly using rnaviralSPAdes [12]. The resulting contigs were analyzed using a comprehensive sequence similarity search by MMseqs2 [19]. The detailed methods are available in the Supplementary File. Several contigs similar to chicken chapparvovirus 2 were detected, among which the longest contig (2,026 bp) showed 96.95% nucleotide identity to chicken chapparvovirus 2 strain RS/BR/15/2S (NC_076004.1) found in Brazil. Further, a contig (533 bp) showed 99.44% nucleotide identity to chicken anemia virus strain Oita-3/20 (MT975520) identified in Japan in 2020. Other top-hit contigs from the BLAST results are shown in Supplementary Table 1 and the Supplementary File. We then mapped the original reads to chicken chapparvovirus 2 strain RS/BR/15/2S and chicken anemia virus strain Oita-3/20 genomes (Fig. 2). The mapping pattern showed that splicing may have resulted in the expression of genes homologous to the NS2 gene of chaphamaparvovirus galliform 3 (QRK03699.1) and the hypothetical protein of Phasianus chaphamaparvovirus (UQT67959.1).

Fig. 2.

Read mapping of chicken chapparvovirus 2 and chicken anemia virus. Integrative Genomics Viewer genome browser view showing the depth of the RNA-seq reads mapped to chicken chapparvovirus 2 and chicken anemia virus.

As the high-throughput sequencing was performed using pooled samples, PCR was performed with specific primers for chicken chapparvovirus 2 (5′-CTGCTTTCAACAATTGCACGTA-3′ and 5′-TTTTCCAGCTCGCAATTCACC-3′) [5] and chicken anemia virus (5′-GACTGTAAGATGGCAAGACGAGCTC-3′ and 5′-GGCTGAAGGATCCCTCATTC-3′) [20] using the DNA extracted from each individual sample. DNA extracted from chicken liver purchased at a supermarket and from Leghorn male hepatoma (LMH) cells, cells from a chicken hepatocellular carcinoma cell line, were used as negative controls. Primers targeting β-actin (5′-CTCTTTTCGGGGTTCTTTCC-3′ and 5′-GCTTCAAAAGACGCTTCCAC-3′) were used as loading controls. The detailed methods are available in the Supplementary File. The results showed the amplification of bands of the expected size using the chicken chapparvovirus 2-specific primers in all three chickens and that using chicken anemia virus-specific primers in one chicken (Fig. 3). Sequencing confirmed the identity of the amplified bands as chicken chapparvovirus 2 (LC790296) and chicken anemia virus. To detect the viral gene within the liver tissue, in-situ hybridization (ISH) targeting chicken chapparvovirus 2 was conducted using a probe with the same sequence as that used in PCR (see Supplementary File). The positive signal was observed at the same site as the nuclear inclusions in the hepatocytes (Fig. 4).

Fig. 3.

Detection of chicken chapparvovirus 2 and chicken anemia virus using polymerase chain reaction (PCR). PCR was performed using specific primers for chicken chapparvovirus 2, chicken anemia virus, and β-actin. For #1–3, DNA extracted from the livers of the three chickens were used as a template; ctrl, DNA extracted from commercial chicken liver; LMH, DNA extracted from LMH cells; DW, distilled water.

Fig. 4.

Detection of the chicken chapparvovirus 2 gene by in-situ hybridization (ISH). ISH was conducted using the region targeted in PCR as a probe for chicken chapparvovirus 2. The negative control represents staining results obtained in the absence of probes. Arrowheads indicate nuclear inclusion bodies.

In this study, we showed that chicken chapparvovirus 2 infection might be lethal, which is different from diarrhea symptoms caused by chicken chapparvovirus 2 infection in the past. Chapparvovirus was detected in chickens during the search for viruses associated with runting and stunting syndrome; however, no association between the virus and symptoms was reported [9, 10]. Other reports also detected CkChpV in feces, and infected chickens exhibited diarrhea. In an epidemiological study conducted in China, CkChpV was detected in 5.7 and 32.5% of healthy chickens and chickens with diarrhea, respectively [5]. In the present study, chicken chapparvovirus 2 was detected in the livers of three chickens with hemorrhagic hepatitis. In addition, one of the three birds were co-infected with chicken anemia virus. Chicken anemia virus causes immunosuppression [1], and co-infection with fowl adenovirus results in inclusion body hepatitis [3, 13].

As vaccination against chicken anemia virus was not performed on the farm where the present case occurred, immunosuppression caused by chicken anemia virus infection may have enhanced the virulence of chicken chapparvovirus 2. For the remaining two chickens with hemorrhagic hepatitis, but not chicken anemia virus, we cannot rule out the possibility that chicken anemia virus had already been eliminated or was below the detection limit of our PCR system. In contrast, the possibility that a single infection with chicken chapparvovirus 2 could cause chicken hemorrhagic hepatitis is also worth considering. In fact, only diarrhea was reported as a symptom of chicken chapparvovirus 2 infection in chickens [5]; however, other pathologies have been reported in other animals. In pheasants, hemorrhagic hepatitis caused by a novel virus, Phasianus chaphamaparvovirus 1, has been reported [11]. Mouse kidney parvovirus may be associated with chronic kidney infection in immunosuppressed mice [17]. Therefore, it is possible that the hemorrhagic hepatitis observed in this study was caused by infection with chicken chapparvovirus 2 alone.

No reports have documented CkChpV isolation using major chicken cell culture systems [5]. If isolation succeeds in future investigations, it could facilitate studies related to pathogenicity confirmation through single infection.

To the best of our knowledge, this is the first report on CkChpV infection in Japan. Previous reports have documented the detection of chaphamaparvovirus from chickens in Australia, Brazil, China, and Switzerland [5, 9, 10, 18]. Parvoviruses are highly stable in the environment, and they may spread with no notable (or only minor) symptoms, such as diarrhea, even after infection [5, 9, 10]. Thus, CkChpV may have already been spreading nationwide in Japan, and its transmission in Japan should be investigated using fecal samples and PCR. For epidemiological studies and diagnosis, there is also a need to establish detection systems for viral antigens and antibodies, in addition to PCR, so that CkChpV can be detected by various methods.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.