Research Article

Molecular Diagnosis of Avihepatovirus a in Naturally Infected Duck Flocks in Egypt

A. El-Shemy1*, Hoda M. Mekky2, M. A. Bosila2, A. M. Allam1, Kh. M. Elbayoumi2, M. M. Amer3

1Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, P.O. 12622, Giza, Egypt; 2Poultry Diseases Department, Veterinary Research Institute, National Research Centre, P.O. 12622, Giza, Egypt; 3Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, P.O. 12211, Giza, Egypt.

Received | December 19, 2021; Accepted | January 26, 2022; Published | July 14, 2022

*Correspondence | A El-Shemy, Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, P.O. 12622, Giza, Egypt; Email: dr.ahmedelshemy@gmail.com

Citation | El-Shemy A, Mekky HM, Bosila MA, Allam AM, M. Elbayoumi K, Amer MM (2022). Molecular diagnosis of avihepatovirus a in naturally infected duck flocks in egypt. Adv. Anim. Vet. Sci. 10(8): 1752-1760.

DOI | http://dx.doi.org/10.17582/journal.aavs/2022/10.8.1752.1760

ISSN (Online) | 2307-8316

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Duck virus hepatitis (DVH) is an acute viral disease that commonly affects flock of ducklings that are up to 3 weeks old. It is characterized by a sudden onset causing severe losses because of its high mortality, ranging from 90% to 100% within 3–4 days. The disease is caused by three distinct serotypes of duck hepatitis virus (DHV-1–3), which is a member of the genus Avihepatovirus of the Picornaviridae family (Fu et al., 2008, ICTV, 2017). Serotype 1 (DHV-1) is renamed as duck hepatitis A virus-1 (DHAV-1) and represents the most virulent serotype with an important effect on the worldwide poultry industry (Ding and Zhang, 2007; Tseng et al., 2007). The disease was first reported in 1945 inthe USA (Levine and Fabricant, 1950). After that, the spread of the disease was recorded in several countries (Guerin et al., 2007; Sandhu et al., 1992; Woolcock, 2008). The clinical manifestations are characterized by systemic convulsions, locomotor disorders, feet spasm, and opisthotonos. In addition, the liver appears enlarged with petechial and ecchymotic hemorrhages on the surface (Liu et al., 2008; Tsai, 2020). Therefore, both prevention and rapid diagnosis are the logical and most effective strategies for controlling the disease (Woolcock, 2003).

The DHAV-1 viral genome is formed from a nonenveloped, positive-sense, single-stranded RNA of approximately 7.8 kb. It comprises the open reading frame encoding leader protein (L), structural proteins (VP0, VP1, and VP3), and nonstructural proteins (2A1, 2A2, 2A3, 2B, 2C, 3A, 3B, 3C, and 3D) (Wei et al., 2012). The 3D gene is located in the P3 segment of the picornavirus genome. VP1 capsid protein is considered the main antigenic determinant, as in other picornaviruses. Therefore, it shared its viral pathogenicity and virulence with DHAV (Gao et al., 2012). In addition, the 3D gene encodes a protein that implicates a conserved domain known as RNA-dependent-RNA polymerase (RdRp) that contributes toviral RNA synthesis throughout virus replication (Kim et al., 2006; Kerkvliet et al., 2010).

Conventional diagnoses are based on clinical signs, such as gross lesions confirmed using virus isolation and serological tests, which are laborious, time-consuming, and exhibit low sensitivity. To overcome these drawbacks, various bioassays and techniques of polymerase chain reactionhave been developedfor the rapid, simple, and potent diagnosis and detection of the persistent evolution of DHAV-1. Moreover, the application of the PCR assay offered a great understanding of the pathogenesis and diagnosis of DHAV-1. The successful detection of DHAV-1 in cell culture and tissues from infected ducks via PCR using species-specific primer sets against viral DNA polymerase enzymes has been reported (Kim et al., 2007; Liu et al., 2007; Anchun et al., 2009; Wen et al., 2014; El-Samadony et al., 2018).

In Egypt, DVH was discovered in the1970s. Since the prevalence of duck farms in Egypt, DHAV-1 has posed destructive damages and constitutes a major menace for commercial duck farms (Ellakany et al., 2002; Erfan et al., 2015; Hisham et al., 2020). For influential domination on the DHAV-1, massive immunization was performed in Egypt (Ellakany et al., 2002; Abd-Elhakim et al., 2009). However, despite the attenuated Rispens strain E52 vaccine being commercially used to immunize breeder ducks against DHAV-1, severe mortalities were recorded in both vaccinated and non-vaccinated ducks and DHAV-1 still poses a continual significant threat in the duck sector in Egypt (Abd-Elhakim et al., 2009). Lately, research has been applied for the surveillance and molecular characterization of DHAV-1 in various studies (Erfan et al., 2015; Yehia et al., 2021; Rohaim et al., 2021).

This study aimed to perform a molecular diagnosis of the DHAV-1 circulating in naturally infected flock of ducks using reverse transcription PCR (RT-PCR) followed by obtaining a genetic sequence of the 3D gene to track the evolutional variations at the molecular level.

MATERIAL AND METHODS

Duck flocks

Three mixed-sexflock of ducks (two Muscovy and one Pekin) aged 2–4 weeks with flock capacity range of 4500–5000 birds were used in the study. These flocks showed signs and lesions of DVH, with a mortality rate of 8.9% on the firstday that increased till 75% in all flocks within 4 days. Flocks of duck were reared in commercial farms in Monofia and Al-Gharbia governorates, Egypt. As per the breed management manuals, the flocks were fed on a balanced ration (NRC, 1984).

Samples

The liver samples were aseptically collected from freshly dead ducklings or ducklings with clinical signs and lesions between August and October 2018. Each sample was classified into two groups. One group was sent for histopathological examination, and the other group was sent for RT-PCR application. First, each sample for RT-PCR was homogenized and suspended 20% W/V in sterile antibiotics saline and then subjected to three successive freeze–thaw cycles, and the suspension fluid was separated via centrifugation at 3000 rpm for 15 min. Finally, the clear supernatant was separated and kept at −20°C till further use in RT-PCR (Woolcok, 1998).

Histopathological examination

The liver specimens of the affected ducklings were fixed in 10% buffered neutral formalin solution, processed in paraffin sections of4–6 μm, stained with hematoxylin and eosin (H&E), and then microscopically examined (Suvarna et al., 2012).

 

Table 1: Published sequences of 3D of DHV used for multiple alignment analysis.

NO.	Accession No.	DHAV-1 strain name	Governorate	Breed	Collection date
1	MT157210	DHV/Duck/Egypt/Monofia-Shemy-1/2018*	Monofia	Muscovy	2018
2	MT157211	DHV/Duck/Egypt/Al-Gharbia-Shemy-3/2018*	Al-Gharbia	Pekin	2018
3	MT157212	DHV/Duck/Egypt/Monofia-Shemy-2/2018*	Monofia	Muscovy	2018
4	KP148263.1	DVH A virus strain vaccine
5	KP148264.1	F340	Ismailia	Pekin	2012
6	KP148265.1	F953	Behara	Pekin	2013
7	KP148266.1	F86	Luxor	Pekin	2013
8	KP148267.1	F163	Luxor	Muscovy	2013
9	KP148268.1	F413	Qena	Muscovy	2013
10	KP148269.1	F729	Qaluibia	Mallard	2013
11	KP148270.1	F829	Giza	Green Winged	2013
12	KP148271.1	F10	Ismailia	Mallard	2014
13	KP148272.1	F243	Sharkia	Muscovy	2014
14	KP148273.1	F355	Sharkia	Pekin	2014
15	KP148274.1	F112	Ismailia	Pekin	2014
16	KP148275.1	F147	Ismailia	Pekin	2014
17	KP148276.1	F321	Behara	Pekin	2014
18	KP148277.1	F215	Beni-Suef	Green Winged	2014
19	KP148278.1	F387	Ismailia	Pekin	2014
20	KX639146.1	DHV-EGYPT/16160F-2016	Sharkia	Muscovy	2016
21	KX639141.1	DHV-EGYPT/141122F-2014	Behara	Pekin	2014
22	KX639142.1	DHV-EGYPT/151283F(3)-2015	Sharkia	Pekin	2015
23	KX639143.1	DHV-EGYPT/15298F-2015	Fayoum	Mallard	2015
24	KX639144.1	DHV-EGYPT/15P18-2015	Menoufiya	Muscovy	2015
25	KX639139.1	DHV-EGYPT/14921F-2014	Qaluibia	Pekin	2014
26	KX639145.1	DHV-EGYPT/151283F(4)-2015	Beni-Suef	Mallard	2015
27	KX639140.1	DHV-EGYPT/14923F-2014	Giza	Muscovy	2014
28	KX639147.1	DHV-EGYPT/151283F(2)-2015	Gharbia	Muscovy	2015
29	KX639148.1	DHV-EGYPT/151283F(1)-2015	Ismailia	Muscovy	2015
30	KP202874.1	DHV/Duck/Egypt/Al-Gharbia/2014	Al-Gharbia	Pekin	2014

(*) indicates DHV field isolates of the current study

Detection of RNA of DHAV-1 by one-step RT-PCR

RNA extraction methods

Viral RNA extraction from the liver tissue was performed using the Patho Gene-spin™ DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea), following the manufacturer’s instructions. RNA concentrations were measured using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE). The samples were storedat −20°C for one-step RT-PCR (OIE, 2018).

Oligonucleotide primers design

The one-step RT-PCR was performed using the specific primers, DHAV-1 Com F (5’-AAG-AAG-GAG-AAA-ATY-[C or T]- AAG-GAA-GG-3’) and DHAV-1 Com R (5’-TTG-ATG-TCA-TAG CCC-AAS- [C or G]-ACA-GC-3’). The primers were flanked by an expected 467 bp DNA sequence in the 3D gene (OIE, 2018).’

One-Step RT-PCR: The one-step RT-PCR was conducted using 20μL of reaction mixtures DHV-1 ComF and DHV-1 ComR, a T-gradient thermal cycler (Biometra, Gottingen, Germany) was used to perform one-step RT-PCR. First, reverse transcription was performed at 45°C for 30 min, after which the enzyme was inactivated at 94°C for 5 min.

PCR amplification was conducted using the following steps:an initial denaturation for 20 s at 94°C, followed by 40 cycles of annealing for 30 s at 52°C, extension for 30 s at 72°C, denaturation for 20 s at 94°C, final extension for 5 min at 72°C, and reactions were stored at 4°C (OIE, 2018).

DNA Molecular weight marker

The amplified products were read using a 100 bp ladder (NEW ENGLAND BioLabs).

Agarose gel electrophoreses

Electrophoresis for amplicon separation was applied on 1.5% agarose gel prepared according to Sambrook et al. (1989).

The gel was visualized and captured using a gel documentation system, and the data was analyzed using acomputer software.

Sequencing and phylogenetic analyses of the amplified part of the 3D gene

Sequencing of the PCR amplified products was conducted using the GATC company ABI 3730xl DNA sequencer and forward/reverse primers. The traditional Sanger sequencing technique was combined with the new 454 technology for sequencing 467 bp PCR product of the DHAV-1 3D gene. To establish the sequence identity for GenBank accessions, a BLAST® analysis (Basic Local Alignment Search Tool) (Altschul et al., 1990) was initially performed. The published data of 26 Egyptian viruses and one reference vaccine strain from the GenBank were available from the National Center for Biotechnology Information (NCBI) Duck hepatitis A virus resource (https://www.ncbi.nlm.nih.gov/nuccore/?term=duck+hepatitis+a+virus+3D+protein) (Table 1). The alignment of the viruses in this study was performedusing the MegAlign module of Lasergene DNAStar (Thompson et al., 1994). Then, the identity percent and divergence amongall viruses were determined. Finally, phylogenetic analysis was conducted using the MEGA v6.0 software (Tamura et al., 2013). The three reported partial sequences of the 3D gene were submitted to GenBank under the following accession numbers: MT157210, MT157211,and MT157212.

RESULTS AND DISCUSSION

DVH is one of the most significant viral diseases in young ducklings, causing high potential mortality and great economic losses. Shalaby et al. (1978) reported the first occurrence of this disease in Egypt in the 1970s. Because of the immature immune system in young ducklings, which is incapable of protecting them from virus infection and propagation, the seriousness of infection with DHAV-1 is chiefly age-dependent (Song et al., 2014). Earlier research recorded that acute infections caused by DHAV-1 was correlated with Pekin, Mallard, and hybrid breeds, whereas Muscovy ducklings appeared to be the stable carriers of DHAV-1 infections. However, pancreatitis and encephalitis have been reported to be correlated to Muscovy ducklings’ infection (Zhu et al., 2019). Conventional methods used for diagnoses are based on clinical signs,such as gross lesions in ducklings confirmed usingvirus isolation and serological tests. Lately, a number ofquick and specific RT-PCR techniques have been designed to identify the presence of DHAV-1 RNA (Kim et al., 2007; Liu et al., 2007; Anchun et al., 2009; Wen et al., 2014; OIE, 2018).

In the current investigation, the liver samples were collected from three duck farms representing Muscovy and Pekin breeds aged 2–4 weeks, and located in two Egyptian governorates (Monofia and Al- Gharbia) between August and October 2018. The commonly recorded clinical signs among examined ducklings were nervousrelated,such as ataxia, imbalance, lateral recumbency, keel over, kick spasmodically, and opisthotonos followed by death (Figure 1). The mortality rate reported in affected flocks reached 75% within 3–4 days. The results were consistent with those previously recorded in DHV infection studies (OIE, 2010; Erfan et al., 2015).

The liver of the examined ducklings was enlarged with hemorrhages. In addition, enlarged spleen and kidneys with congested renal blood vessels were also observed (Figure 2). Similarly, variable lesions were recorded in the liver and kidneys (OIE, 2010; Hisham et al., 2020).

Histopathological examination of the liver sections of the affected ducklings with clinical signs showed portal vein and bile duct congestion accompanied byhepatic cord disorganization. Focal areas of coagulative necrosis characterized by inflammatory cells infiltration and vacuolar degeneration in the cytoplasm of the hepatocyte were also

observed. Further lesions, including narrowing of the bile duct due to epithelial lining hypertrophy and the existence of large eosinophilic intranuclear inclusion bodies surrounded by a clear halo in the hepatocytes, were markedly seen (Figures 3 and 4). These results were analogous to those reported by Fitzgerald et al. (1969), Sheng et al. (2014), Hassaan et al. (2018), and Mansour et al. (2019).

In this study, implementation of one-step RT-PCR for processed livers using specific primers aiming the 3D gene of DHAV-1 showed the output of the amplified band in all examined specimens at acertain supposed size of the 3D encoding gene (467 bp). These results were analogous to those obtained by El-Samadony et al. (2016) and Zanaty et al. (2017).

In our study, the successfully amplified 3D gene of DHAV-1 isolates named DHV/Duck/Egypt/Monofia-Shemy-1/2018, DHV/Duck/Egypt/Al-Gharbia-Shemy-3/2018, and DHV/Duck/Egypt/Monofia-Shemy-2/2018 was subjected to sequencing and then submitted to the NCBI GenBank; the accession numbers were MT157210, MT157211,and MT157212, respectively.

In Egypt, live attenuated vaccine prepared from the E52 Rispens strain is used for vaccination of breeder duck intramuscularly, which is administered 2–3 weeks before laying to yield high titers of passively transferred maternal antibody in newly hatched ducklings, thereby maintaining them for the first two weeks of their lives (Ellakany et al., 2002; Abd-Elhakim et al., 2009). Occasionally, there is an infection in non-vaccinated ducklings with low immunity because of vaccinal shedding from vaccinated ducklings. Further, the occurrence of the DHAV-1 disease in immunized ducklings was recorded (Mahdy, 2005; Jin et al., 2008; Li et al., 2013). However, the actual reason for the appearance of DHAV-1 infection in immunized flock of ducks in Egypt wasnot identified. This may bebecause of the antigenic variation that occurred in the field virus. Thus, the accustomed vaccine cannot provide enough protection (Zanaty et al., 2017).

The alignment of the sequenced amplified 3D gene of three examined field isolates with the published 3D sequence of some Egyptian DHV isolates and vaccine strain was performed (Figure 5). Sequence analysis of nucleotide alignment for the 3D gene revealed 92.5%–98.6% homology between our sequenced isolates.

Our isolate, DHV/Duck/Egypt/Monofia-Shemy-1/2018, showed 79.9%–97.3% nucleotide identity with the previous isolates from Egyptian governorates. Further, the field isolate, DHV/Duck/Egypt/Al-Gharbia-Shemy-3/2018,showed 77.4%–93.1% similarity with the previous field isolates fromEgyptian governorates. Our isolate, DHV/Duck/Egypt/Monofia-Shemy-2/2018, showed a nucleotide identity of 80.2%–98.8% withtheprevious isolates from Egyptian governorates. The three examined isolates (DHV/Duck/Egypt/Monofia-Shemy-1/2018, DHV/Duck/Egypt/Al-Gharbia-Shemy-3/2018, and DHV/Duck/Egypt/Monofia-Shemy-2/2018) showed nucleotide similarities of 77.4%–80.2% with the most frequently used DHAV-1 vaccine in Egypt. The phylogenetic relationship using the partial 3D protein revealed that the isolated Egyptian strains used in this study were distant from the vaccine strain used in Egypt (Figure 6). The deduced amino acid substitutions of the 3D gene of our investigated strains was studied (Figure 7) to discover the DHAV-1 genome evolution. The area of analysis constituted 115 amino acid residues. Substitution mutations for DHV/Duck/Egypt/Monofia-Shemy-3/2018 strain were noted at several amino acid residues in comparison to amino acid

of other examined Egyptian strains during this study or those previously isolated from Egyptian governorates. Deduced amino acid substitutions analysis of the investigated strains, DHV/Duck/Egypt/Monofia-Shemy-1/2018 and DHV/Duck/Egypt/Monofia-Shemy-2/2018, revealed the same amino acids (except at position I4T and Y5I). Both strains showedmany amino acids changes compared with some previously isolated Egyptian strains (F340, F953, F86, F163, F413, F729, F829, F10, F243, F355, F112, F147, F321, F215, and F387), and it also showed low amino acids changesin relation to other previously isolated Egyptian strains (DHV-EGYPT/16160F-2016, DHV-EGYPT/141122F-2014, DHV-EGYPT/151283F(3)-2015, DHV-EGYPT/15298F-2015, DHVEGYPT/15P18-2015, DHV-EGYPT/14921F-2014, DHV-EGYPT/151283F(4)-2015, DHV-EGYPT/14923F-2014, and DHV-EGYPT/151283F(2)-2015). All isolated Egyptian strains in this study were also completely unrelated to the Egyptian DHAV-1-based vaccine and was further confirmed by the low amino acid similarities to this vaccine.

Phylogenetic analysis in addition to nucleotide and amino acid similarities between our isolated strains with other Egyptian DHAV-1 strains and vaccinal strain corresponded to those obtained by Zanaty et al. (2017), who stated that Egyptian strains are different from the vaccine.

CONCLUSION AND RECOMMENDATIONS

Three isolates of DHAV-1 were identified from the examined flocks of duck. A phylogenetic analysis of these isolates revealed differentiation between our field DHAV-1 isolates and the vaccine strain.

For correct controlling of the DHAV-1 infection in flocksof duck, avaccination program for breeder duck flocks against DHAV-1 is important. Thus, further studies are neededto detect the efficacy of the used vaccine against the current field strains of DHAV-1.

ETHICAL APPROVAL

All work procedures were reviewed and approved by Medical Research Ethics Committee of the National Research Centre, Egypt (Approval no. 7417082021).

ACKNOWLEDGMENTS

The authors thank the laboratory of Veterinary Vaccines Technology (VVT), Central Labs, National Research Centre, Giza, Egypt, for all kinds of support.

CONFLICT OF INTEREST

The authors have no conflict of interests to declare regarding the publication of this paper. Also, the authors declare that the work was self-funded.

AUTHORS’ CONTRIBUTIONS

M.M. Amer and Kh. M. Elbayoumi designed and planned this study. A. El-Shemy, Hoda M. Mekky and A. Allam collected samples, and performed all laboratory work. M. Bosila carried out pathological examination. All authors shared sample collection, performing the tests, collecting research data for writing, manuscript writing, drafted, revised the manuscript, and approved the final manuscript.

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