Accepted Manuscript Title: Epidemiology and molecular characterization of duck hepatitis A virus from different duck breeds in Egypt Author: Ahmed M. Erfan Abdullah A. Selim Mohamed K. Morsi Soad A. Nasef E.M. Abdelwhab PII: DOI: Reference:
S0378-1135(15)00124-8 http://dx.doi.org/doi:10.1016/j.vetmic.2015.03.020 VETMIC 6941
To appear in:
VETMIC
Received date: Revised date: Accepted date:
6-12-2014 9-3-2015 16-3-2015
Please cite this article as: Erfan, A.M., Selim, A.A., Morsi, M.K., Nasef, S.A., Abdelwhab, E.M.,Epidemiology and molecular characterization of duck hepatitis A virus from different duck breeds in Egypt, Veterinary Microbiology (2015), http://dx.doi.org/10.1016/j.vetmic.2015.03.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Epidemiology and molecular characterization of duck hepatitis A virus from different duck breeds in
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Egypt
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Ahmed M. Erfan1*, Abdullah A. Selim1, Mohamed K. Morsi1, Soad A. Nasef1 and E.M. Abdelwhab1,2**
National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, P.O. Box 246, Giza 12618, Egypt
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The Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut– Institute of Molecular
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Virology and Cell Biology, Suedufer 10, Greifswald Insel-Riems 17493, Germany
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*Corresponding Author 1: Ahmed M. Erfan
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Tel.: +20233380121
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Fax: +20233370957
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E-Mail: [email protected]
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** Corresponding Author 2: El-Sayed M. Abdelwhab
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Tel.: +49 38351 7 1139
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Fax: +49 38351 7 1188
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E-Mail:
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[email protected]
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Highlights
Little is known about Duck hepatitis A virus (DHAV) in poultry outside Asia
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Here we conducted a surveillance in 46 commercial duck-farms in Egypt in 2012 -2014
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Ducks in 17 farms (37%) in 10 out of 11 Egyptian provinces were positive by RT-PCR
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Genetic analysis of 15 viruses showed differences from the vaccine strain
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The Egyptian strains were genetically similar to the Asian DHAV1 viruses
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Duck hepatitis virus (DHV) is an acute highly contagious disease of ducklings caused by three distinct
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serotypes of duck hepatitis A virus (DHAV), a member of the RNA family Picornaviridae, where serotype 1
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is the most widespread serotype worldwide. To date, little if any is known about the prevalence and genetic
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characterisation of DHAV outside Asia. The current study describes surveillance on DHV in 46 commercial
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duck farms in Egypt with a history of high mortality in young ducklings from 3 to 15 day-old from 2012 to
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2014. Clinical samples were examined by generic RT-PCR assays followed by partial sequence analysis of
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the 5'UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses. The overall positive rate was 37%
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(n=17/46). All duck breeds (Pekin, Muscovy, Mallard and Green Winged) were susceptible to the disease
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with mortality ranged from 15% to 96.7%. Sequence and phylogenetic analyses indicated that the Egyptian
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strains cluster in the DHAV serotype 1 with Asian viruses and distinguishable from the vaccine strains. So
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far, this is the first report on the genetic characterisation of DHAV in Egypt. This study may be useful to
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better understand the epidemiology and evolution of DHAV.
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Keywords
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Duck hepatitis A virus, Picornavirus, Ducklings, Egypt, RT-PCR, Epidemiology
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1.
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Duck virus hepatitis (DVH) is a highly contagious disease of ducks which causes severe morbidity and
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mortality particularly in ducklings less than 4-week-old (Woolcock, 2003). The virus can be transmitted by
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parenteral and oral routes, while vertical transmission has not been yet reported. Vaccination of breeder
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ducks protects the offspring through maternal antibodies (Woolcock, 2003; OIE, 2010) and vaccination of
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ducklings or transfer of hyperimmune sera can be also protective (Woolcock, 2003; Kim et al., 2009). The
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disease is caused by three different viruses. Duck hepatitis A virus (DHAV) type I, genus Avihepatovirus, a
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member of the RNA family Picornaviridae is the most common cause for DHV worldwide. Meanwhile,
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DHAV types II and III, were recently classified as duck astrovirus 1 and 2 (DAstV-I and DAstV-II) and were
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reported in the United Kingdom and the United States of America, respectively. Both viruses belong to the
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family Astroviridae and are antigenically distinct from DHAV type I. The latter has three distinguishable
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serotypes designated serotype 1, 2 and 3. Serotype 1 is the most widespread serotype worldwide, whereas
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serotype 2 was reported in Taiwan and serotype 3 in South Korea and China (Kim et al., 2007; Li et al.,
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2013).
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DHAV is a nonenveloped virus with single-strand positive-sense ~7.8 kb RNA genome that encodes a single
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viral polyprotein (VP) flanked with untranslated region (UTR) at the 5´ and 3´ ends (Pan et al., 2012). The 5´
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UTR possesses probably distinct internal ribosome entry site (IRES) element that is important for translation
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initiation and RNA synthesis of the virus. The VP is a polyprotein of ~2200 amino acids in length that is
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further divided into several structural proteins including VP0 (VP2/VP4), VP1 and VP3 and nine non-
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structural proteins (2A1, 2A2, 2A3, 2B, 2C, 3A, 3B, 3C and 3D). The VP1 protein is the highly variable one
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(Wang et al., 2008; Gao et al., 2012; Wei et al., 2012; Xu et al., 2012) and probably plays a vital role in
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receptor binding, virulence, immunogenicity and protection against DHV (Liu et al., 2008). The 3D protein
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is RNA-dependent RNA-polymerase which is responsible for the synthesis of the viral RNA (Kok and
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McMinn, 2009). The evolution of the virus is driven by accumulation of point mutations and/or
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recombination between different serotypes (Wei et al., 2012). A limited number of sequences of DHAV are
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available which are only from China, South Korea, Taiwan, Vietnam, UK and USA (Tseng et al., 2007).
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Introduction
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Although the virus was described in Egypt in the late 1970s (Shalaby et al., 1978), little is known about the
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current situation of the disease (OIE, 2009). Vaccination of breeder ducks is applied in the commercial sector
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in Egypt (Abd-Elhakim et al., 2009) using attenuated vaccines produced from E52 Rispens strain
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(Anononymous ; Ellakany et al., 2002). In this study, we investigated the prevalence of DHAV in ducklings
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with a history of high mortality using reverse transcription polymerase chain reaction (RT-PCR) and virus
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isolation in embryonated duck eggs (EDE) from several provinces in Egypt. Furthermore, the sequence of
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5´UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses were generated and analysed. To the
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best of our knowledge this is the first published sequence for DVAH in the Middle East and Africa.
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2.
Materials and Methods
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2.1
Samples
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Samples were collected from different breeds of ducks in 46 commercial farms in Egypt; Pekin (n= 29),
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Muscovy (n= 9), Mallard (n= 5) and Green Winged ducks (n= 3). Samples were taken in 2012 (n= 4), 2013
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(n= 12) and 2014 (n= 30). Farms’ capacities ranged from 400 to 10000 birds in 11 different provinces
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(Figure 1). The surveillance targeted commercial farmed ducks with a history of nervous signs and high
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mortality rate during the first two weeks of life (Table 1 and Supplementary Table S1). Samples were
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obtained from the liver, spleen and kidneys of freshly dead or euthanized birds. Organs were homogenised
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using the Tissuelyser (Qiagen) then subjected to three successive freeze–thaw cycles, and the supernatant
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was separated after 12000 rpm centrifugation for 10 min.
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2.2
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RNA extraction from the supernatants of tissue homogenates was performed using the QIAamp Viral RNA
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Mini kit (Qiagen, Germany, GmbH) according to the manufacturer's recommendations. The RT-PCR assays
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using oligonucleotide primers (Metabion) for partial amplification of 5´UTR (Fu et al., 2008), VP1 (Liu et
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al., 2008) and 3D (OIE, 2010) genes were conducted as previously published (Supplementary Table S2). A
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25-µL reaction containing 12.5 µL of Quantitect probe RT-PCR buffer (Qiagen, Germany, GmbH), 0.25 µL
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RT-PCR
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of RT-enzyme, 1 µL of each primer of 20 pmol concentration, 4.25 µL of water and 6 µL of template RNA
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was used. The reactions were performed in a T3 thermal cycler (Biometra). The amplicons were separated by
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electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) along with 100-bp DNA Ladder
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(Qiagen, Germany, GmbH). The gel was photographed by a gel documentation system (Alpha Innotech,
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Biometra) and the data were analysed through computer software (Automatic Image Capture Software,
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ProteinSimple formerly Cell Biosciences, USA).
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2.3
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Virus isolation from RT-PCR-positive samples was attempted by inoculation of 10- to 14-day-old EDE
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through the allantoic cavity according to the standard protocol (OIE, 2010). Eggs were candled daily for up
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to 7 days. The embryos were examined for stunting, oedema and/or haemorrhages as well as for pathological
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changes particularly in the liver, kidneys and spleen (OIE, 2010).
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2.4
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Partial sequences of the 5´UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses were generated
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using forward and reverse primers of the generic PCRs. Amino acid sequences were deduced from the
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generated nucleotides using BioEdit software version 7.1.7 (Hall, 1999). Sequences of query and
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representative DHAVs were retrieved from the GenBank database. All sequences were aligned and identity
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matrices were calculated automatically using BioEdit. Phylogenetic analyses were done using maximum
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likelihood, neighbour joining and maximum parsimony in MEGA6 (Tamura et al., 2013). Consensus
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unrooted trees were generated with 1000 bootstrap replicates and were further edited using Inkscape 0.91
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Software (Free Software Foundation, Inc, USA).
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2.5
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Sequences generated in this study were submitted to the GenBank under accession numbers KP148295 to
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KP148310 for the UTR, KP148279 to KP148294 for the VP1 and KP148263 to KP148278 for the 3D gene
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fragments.
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GenBank accession numbers
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3.
Results
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3.1
Virus detection and isolation
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All RT-PCRs have relatively comparable sensitivity. Respectively 17, 16 and 15 positive samples were
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detected by RT-PCR targeting the 5´UTR, VP1 and 3D genes. Out of the 17 samples tested positive by RT-
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PCR, only 5 samples were positive in virus isolation (Table 1) in terms of stunting, subcutaneous
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haemorrhages of the embryos (5/5 cases), haemorrhagic enlarged liver (4/5), enlarged mottled spleen (4/5)
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and rarely enlarged kidneys (1/5). In 17 positive farms, mortality ranged from 15% (case no. 4) to 96.7%
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(case no. 5) (Table 1). Nevertheless, the virus was not detected from birds with up to 100% mortality
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(Supplementary Table S1). All duck breeds included in this surveillance were susceptible to the disease;
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Pekin (31%; n= 9/29), Muscovy (44.4%; n= 4/9), Mallard (40%; n= 2/5) and Green Winged ducks (66.7%;
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n= 2/3). Positive samples were higher in 2013 (50%; n= 6/12) followed by 2014 (33%; n=10/30) then 2012
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(25%; n= 1/4). As shown in Figure 1, the disease was reported in birds in Lower Egypt (where the majority
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of samples were collected) as well as from birds in Upper Egypt.
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3.2
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About 217 nucleotides were generated corresponding to nucleotides 270 to 486 of the reference strain ATCC
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R85952 (accession number NC_008250). The nucleotide identity for the 5´UTR gene among the Egyptian
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strains ranged from 95.3 to 100% and had homology of 94.4 – 96.3% with the vaccine strain. The Egyptian
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strains had a maximum of 96-98% identity with the 5´UTR of the closest Asian strains (data not shown).
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Three viruses (F10, F243 and F340) isolated from two adjacent governorates showed 100% homology.
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Phylogenetic analysis indicated clustering of the Egyptian strains apart from the vaccine strain. The latter
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clustered with Chinese strains (data not shown).
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VP1 gene
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The VP1 gene of all viruses including the vaccine strain has 714 nucleotides encoding 238 amino acids. The
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identity matrix for the VP1 gene of the Egyptian strains ranged from 96.3 to 100% and 95.9 to 100% for the
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nucleotides and amino acids, respectively (Table 2). The Egyptian strains have nucleotide identity ranged
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from 92.9 to 93.6% and amino acid identity from 95.3 to 96.6% with the vaccine strain (Table 2). Nucleotide
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identity to the closest Asian strains was 96-97% (data not shown). The Egyptian viruses possess V129 and
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S142 (similar to the vaccine strain) linked to attenuation of DHAV1 for ducklings (Liu et al., 2008; Wang et
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al., 2012). In comparison to the vaccine strain, the Egyptian viruses possess D48A/T, Q183R (except strain
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F355), K186N, D187V, R213I/M, Y219H and L232F (except strain F215) in the carboxy terminal region
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(Supplementary Figure 1). Other mutations observed in some attenuated Chinese isolates (S181, H183,
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K184, N193, E205, R217 and N235) (Liu et al., 2008) are L181, R/Q183, G/E184, N/D193, E205,
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R/K217 and L235 in the Egyptian strains, respectively.
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Phylogenetic analysis of VP1 showed that the Egyptian viruses cluster with the Asian DHAV1 viruses and
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further allocated into three main groups: designated group A, B and C. Nine out of 15 strains had 100% VP1
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identity and thus clustered in group A possessing P11S, D48T, S49P, I180T and R213M in comparison to
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the vaccine strain (Figure S1). Those strains are exclusively from Lower Egypt; five strains from Ismailia (in
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2012 and 2014), two from Buhaira (2013 and 2014), one from Sharkia (2014) and one from Giza (2013).
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Group B was reported from three provinces; two in Upper Egypt and one in Lower Egypt (about 700Km
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apart), strains in 2013 in subgroup B1 and one strain from Sharkia in 2014 in subgroup B2. Meanwhile, only
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one virus clustered in group C with the lowest homology to the Egyptian strains from Beni-Suef province
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(between Lower and Upper Egypt; also known as Middle Egypt) in 2014. The vaccine strain clustered with a
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vaccine strain used in Vietnam (accession number JF957697) and many Chinese strains (Liu et al., 2008)
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with >99.9 identity. The topology of VP1 gene was similar in all three analyses and thus only maximum-
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likelihood tree was selected (Figure 2).
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3.4
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Total of 440 nucleotides were generated corresponding to nucleotides 6505 to 6944 of the reference strain
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R85952 which encode 146 amino acids. Nucleotide identity matrix for the 3D gene among the Egyptian
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strain ranged from 98.4 to 100% and had 100% identical amino acid sequences. The Egyptian strains had 95
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3D protein
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to 95.9 % nucleotide homology with the 3D of the vaccine strain and the identity to the closest Asian strains
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was 98% (data not shown). Like other DHAVs, the Egyptian strains contain the GxxCSGxxTxxNS motif
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which is highly conserved region in the 3D of all picornaviruses (Wang et al., 2008).
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4.
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Since decades, DHAV causes severe losses in duck industry. Yet, no information about the prevalence of the
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virus outside Asia, particularly in the Middle East and Africa, is available. Diagnosis of DHAV may be
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confirmed by rapid onset of clinical signs and mortality in inoculated susceptible ducklings, inoculation of
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tissues homogenate into the allantoic sacs of EDE and embryonated chicken eggs or infection of cell culture
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(OIE, 2010). In contrast to the laborious and time-consuming culture techniques, rapid and specific detection
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of the DHAV RNA using different RT-PCR assays were described (Fu et al., 2008; Liu et al., 2008; OIE,
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2010; Wei et al., 2012). In this study, all three PCR assays had a comparable sensitivity, but the isolation rate
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was very low. The high sensitivity of RT-PCR over virus isolation is generally accepted. Nevertheless,
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although the samples were collected from freshly dead/euthanized birds, the death of the virus during the
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transportation to the central laboratory could not be excluded. Furthermore, due to the lack of SPF EDE in
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Egypt, it is possible that the duck eggs used for virus isolation in this study had anti-DHAV antibodies which
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may neutralise the virus and decrease the isolation rate.
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The prevalence of DHAV in ducklings in this study was 37% in 10 out of 11 provinces indicating a serious
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health problem for the duck industry in Egypt. Therefore, nationwide surveillance is recommended. Also,
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other causes than DVH such as Pasteurella, Salmonella or E.coli may be responsible for high mortality
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especially in DHAV-negative flocks which should be further investigated. To date, resistance of certain
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breeds of ducks to DHAV is not well known (Woolcock, 2003). As obtained here, the virus was isolated
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from Pekin, Muscovy, Mallards and Green Winged ducks. Unfortunately, it is not clear why these birds were
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infected because the history of vaccination of the breeder flocks is missing. Whether the current vaccine
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cannot protect birds against the field virus because of antigenic variation or the titer of maternal immunity, if
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any, was low is not known. Thus, antigenic characterisation of the virus and studies to assess protection
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should be conducted. In China several vaccines were updated according to the circulating viruses in the field,
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nevertheless the infection of vaccinated ducklings was reported (Jin et al., 2008; Li et al., 2013). The absence
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of the lesions in EDE and the existence of mutations previously reported in attenuated tissue-adapted strains
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(Wang et al., 2012) may indicate that the Egyptian strains differ in their virulence to the Asian viruses;
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therefore in-vivo pathogenicity testing is highly required.
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Although the number of gene sequences of DHAV is substantially increasing providing useful information
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on the taxonomy, epidemiology and virulence of the virus, however it is limited to few countries only (Kim
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et al., 2006; Ding and Zhang, 2007; Tseng et al., 2007; Tseng and Tsai, 2007). In this study, based on the
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VP1 gene, three different lineages (A, B and C) were detected while the majority of the strains located in the
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geographically-limited lineage A from Lower Egypt, viruses in lineage B was reported from both Lower and
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Upper Egypt which may be due to the rapid and random movement of birds between different regions in
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Egypt. Also, we noticed that one strain in lineage C (isolated from Middle Egypt) was probably resulted
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from recombination of strains from Upper and Lower Egypt. However, due to the limited number of positive
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samples and short gene fragments these data must be interpreted cautiously. Remarkably, the Egyptian
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strains are distinguishable from the vaccine strain where the lowest identity was observed in VP1 followed
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by UTR and 3D genes which is expected because the VP1 gene is the most external surface protein and is
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involved in many vital stages in the viral life-cycle including receptor binding and neutralising epitopes (Liu
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et al., 2008).
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In conclusion, DHAV-1 was detected in 17 out of 46 commercial duck farms of different breeds examined in
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this study. Sequence and phylogenetic analyses indicated that the Egyptian strains are distinguishable from
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the vaccine and similar to the contemporary Asian viruses. So far, this is the first report on the genetic
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characterisation of DHAV in Egypt.
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Acknowledgments
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The authors are grateful to the colleagues and co-workers at the national laboratory for veterinary quality
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control on poultry production, animal health research institute, ministry of agriculture, Egypt for their
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Figure 1. Distribution of duck hepatitis A virus positive samples collected from commercial ducklings in
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Egypt from 2012 to 2014
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* refers to weak positive results after testing by RT-PCR targeting the 5´UTR gene, trials to generate
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sequence from those two viruses failed. Numbers in the map refers to the provinces on the left part of the
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Figure 2. Phylogenetic relatedness of the VP1 gene of the Egyptian DHAV to the vaccine and other
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representative Asian viruses
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Maximum-likelihood unrooted tree generated after 1000 bootstraps indicated clustering of the Egyptian with
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the Asian DHAV1 viruses apart from the vaccine strain. The Egyptian strains allocated into three main
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groups denoted groups A, B (B1 and B2) and C.
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southern China. J. Virol. 86, 10247.
Woolcock, P.R. 2003. Viral infections of waterfowl, In: Saif, Y.M., Barnes, H.J., Glisson, J.R.,
d
304
M
302
Fadly, A.M., McDougald, L.R. (Eds.) Diseases of poultry. Iowa State Press, Ames, Iowa,
306
USA, 1248.
308 309 310 311
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307
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305
Xu, Q., Zhang, R., Chen, L., Yang, L., Li, J., Dou, P., Wang, H., Xie, Z., Wang, Y., Jiang, S., 2012. Complete genome sequence of a duck hepatitis a virus type 3 identified in eastern China. J. Virol. 86, 13848.
15 Page 15 of 20
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Figure 1. Distribution of duck hepatitis A virus positive samples collected from commercial ducklings in
324
Egypt from 2012 to 2014
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326
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327
* refers to weak positive results after testing by RT-PCR targeting the 5´UTR gene, trials to generate
329
sequence from those two viruses failed. Numbers in the map refers to the provinces on the left part of the
330
figure
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Figure 2. Phylogenetic relatedness of the VP1 gene of the Egyptian DHAV to the vaccine and other
332
representative Asian viruses
335 336
te
334
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333
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331
337
Maximum-likelihood unrooted tree generated after 1000 bootstraps indicated clustering of the Egyptian
338
strains with the Asian DHAV1 apart from the vaccine strain. The Egyptian strains allocated into three main
339
groups denoted groups A, B (B1 and B2) and C.
340 341
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Graphical Abstract (for review)
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Table
Table 1 1. Positive flocks for duck hepatitis A virus from 2012 to 2014 in Egypt Sample No.
Province
Code
Year
Birds Breed
Results No.1
Age
Mortality2
Isolation3
RT-PCR UTR
VP1
3D
F340
2012
Ismailia
Pekin
8
4500
1500
+
+A5
+
-
2
F86
2013
Luxor
Pekin
5
1000
400
+
+B1
+
-
3
F163
2013
Luxor
Muscovy
5
2000
600
4
F413
2013
Qena
Muscovy
3
4000
600
5
F729
2013
Qaluibia
Mallard
12
9000
8700
6
F829
2013
Giza
Green Winged
10
2000
7
F953
2013
Buhaira
Pekin
9
2000
8
F112
2014
Ismailia
Pekin
8
9
F147
2014
Ismailia
Pekin
9
10
F215
2014
Beni-Suef
Green Winged
11
F321
2014
Buhaira
Pekin
12
F243
2014
Sharkia
Muscovy
13
F355
2014
Sharkia
Pekin
14
F387
2014
Ismailia
15
F10
2014
16
Gh.3
17
FY.2
ip t
1
an
(day)
+B1
+
-
+
+B1
+
-
+
+B1
+
+
500
+
+A
+
+
1500
+
+A
+
-
us
cr
+
1500
+
+A
+
-
4000
2700
+
+A
+
-
8 & 15
10000
3000
+
+C
+
+
5
2000
900
+
+A
+
-
10
10000
2000
+
+A
+
-
9
2000
600
+
+B2
+
+
Pekin
7
4000
3000
+
+A
+
-
Ismailia
Mallard
9
4000
1000
+
+A
+
+
2014
Gharbiya
Muscovy
8
1000
500
+4
-
-
-
2014
Fayoum
Pekin
10
900
300
+4
+4
-
-
ed
ce pt
Ac
M
2500
1
Total 2 number of birds at the farm
2
Total 3 mortality from one-day old until sampling as told by the owners/farmers
3
Virus 4 isolation was done in embryonated duck eggs, where positive samples showed stunting,
haemorrhages 5 and/or pathological changes of the embryos 4
Weak 6 positive; unable to sequence these samples
5
The 7 genetic group according to the VP1 gene (Figure 1)
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Table
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Table 1 2. Nucleotide (upper right) and amino acid (lower left) identity matrices of VP1 of the vaccine and duck hepatitis A virus isolates from
100 100 100 100 100 100 100 97 97 97 97 96.6
100 100 100 100 100 100 97 97 97 97 96.6
F10 92.9 96.2 100 100 100
F243 92.9 96.2 100 100 100 100
100 100 100 100 100 97 97 97 97 96.6
F112 92.9 96.2 100 100 100 100 100
an
F829 92.9 96.2 100 100
M
100 100 100 100 100 100 100 100 97 97 97 97 96.6
F953 92.9 96.2 100
d
F340 92.9 96.2
ep te
Vaccine F215 93.6 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.7 97.8 95.7 97.8 95.7 97.8 95.7 97.8 96.6 97.4
Ac c
3 Virus Vaccine F215 F340 F953 F829 F10 F243 F112 F147 F321 F387 F86 F163 F413 F729 F355 4
us
commercial 2 ducklings in Egypt
100 100 100 100 97 97 97 97 96.6
100 100 100 97 97 97 97 96.6
F147 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F321 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F387 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F86 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F163 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F413 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F729 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100
F355 93.6 96.9 97.6 97.6 97.6 97.6 97.6 97.6 97.6 97.6 97.6 98.8 98.8 98.8 98.8
98.7 5
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S0378-1135(15)00124-8 http://dx.doi.org/doi:10.1016/j.vetmic.2015.03.020 VETMIC 6941
To appear in:
VETMIC
Received date: Revised date: Accepted date:
6-12-2014 9-3-2015 16-3-2015
Please cite this article as: Erfan, A.M., Selim, A.A., Morsi, M.K., Nasef, S.A., Abdelwhab, E.M.,Epidemiology and molecular characterization of duck hepatitis A virus from different duck breeds in Egypt, Veterinary Microbiology (2015), http://dx.doi.org/10.1016/j.vetmic.2015.03.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Epidemiology and molecular characterization of duck hepatitis A virus from different duck breeds in
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Egypt
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Ahmed M. Erfan1*, Abdullah A. Selim1, Mohamed K. Morsi1, Soad A. Nasef1 and E.M. Abdelwhab1,2**
National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, P.O. Box 246, Giza 12618, Egypt
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The Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut– Institute of Molecular
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Virology and Cell Biology, Suedufer 10, Greifswald Insel-Riems 17493, Germany
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*Corresponding Author 1: Ahmed M. Erfan
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Tel.: +20233380121
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Fax: +20233370957
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E-Mail: [email protected]
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** Corresponding Author 2: El-Sayed M. Abdelwhab
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Tel.: +49 38351 7 1139
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Fax: +49 38351 7 1188
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E-Mail:
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[email protected]
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Highlights
Little is known about Duck hepatitis A virus (DHAV) in poultry outside Asia
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Here we conducted a surveillance in 46 commercial duck-farms in Egypt in 2012 -2014
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Ducks in 17 farms (37%) in 10 out of 11 Egyptian provinces were positive by RT-PCR
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Genetic analysis of 15 viruses showed differences from the vaccine strain
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The Egyptian strains were genetically similar to the Asian DHAV1 viruses
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Duck hepatitis virus (DHV) is an acute highly contagious disease of ducklings caused by three distinct
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serotypes of duck hepatitis A virus (DHAV), a member of the RNA family Picornaviridae, where serotype 1
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is the most widespread serotype worldwide. To date, little if any is known about the prevalence and genetic
32
characterisation of DHAV outside Asia. The current study describes surveillance on DHV in 46 commercial
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duck farms in Egypt with a history of high mortality in young ducklings from 3 to 15 day-old from 2012 to
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2014. Clinical samples were examined by generic RT-PCR assays followed by partial sequence analysis of
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the 5'UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses. The overall positive rate was 37%
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(n=17/46). All duck breeds (Pekin, Muscovy, Mallard and Green Winged) were susceptible to the disease
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with mortality ranged from 15% to 96.7%. Sequence and phylogenetic analyses indicated that the Egyptian
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strains cluster in the DHAV serotype 1 with Asian viruses and distinguishable from the vaccine strains. So
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far, this is the first report on the genetic characterisation of DHAV in Egypt. This study may be useful to
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better understand the epidemiology and evolution of DHAV.
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Keywords
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Duck hepatitis A virus, Picornavirus, Ducklings, Egypt, RT-PCR, Epidemiology
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1.
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Duck virus hepatitis (DVH) is a highly contagious disease of ducks which causes severe morbidity and
51
mortality particularly in ducklings less than 4-week-old (Woolcock, 2003). The virus can be transmitted by
52
parenteral and oral routes, while vertical transmission has not been yet reported. Vaccination of breeder
53
ducks protects the offspring through maternal antibodies (Woolcock, 2003; OIE, 2010) and vaccination of
54
ducklings or transfer of hyperimmune sera can be also protective (Woolcock, 2003; Kim et al., 2009). The
55
disease is caused by three different viruses. Duck hepatitis A virus (DHAV) type I, genus Avihepatovirus, a
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member of the RNA family Picornaviridae is the most common cause for DHV worldwide. Meanwhile,
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DHAV types II and III, were recently classified as duck astrovirus 1 and 2 (DAstV-I and DAstV-II) and were
58
reported in the United Kingdom and the United States of America, respectively. Both viruses belong to the
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family Astroviridae and are antigenically distinct from DHAV type I. The latter has three distinguishable
60
serotypes designated serotype 1, 2 and 3. Serotype 1 is the most widespread serotype worldwide, whereas
61
serotype 2 was reported in Taiwan and serotype 3 in South Korea and China (Kim et al., 2007; Li et al.,
62
2013).
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DHAV is a nonenveloped virus with single-strand positive-sense ~7.8 kb RNA genome that encodes a single
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viral polyprotein (VP) flanked with untranslated region (UTR) at the 5´ and 3´ ends (Pan et al., 2012). The 5´
65
UTR possesses probably distinct internal ribosome entry site (IRES) element that is important for translation
66
initiation and RNA synthesis of the virus. The VP is a polyprotein of ~2200 amino acids in length that is
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further divided into several structural proteins including VP0 (VP2/VP4), VP1 and VP3 and nine non-
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structural proteins (2A1, 2A2, 2A3, 2B, 2C, 3A, 3B, 3C and 3D). The VP1 protein is the highly variable one
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(Wang et al., 2008; Gao et al., 2012; Wei et al., 2012; Xu et al., 2012) and probably plays a vital role in
70
receptor binding, virulence, immunogenicity and protection against DHV (Liu et al., 2008). The 3D protein
71
is RNA-dependent RNA-polymerase which is responsible for the synthesis of the viral RNA (Kok and
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McMinn, 2009). The evolution of the virus is driven by accumulation of point mutations and/or
73
recombination between different serotypes (Wei et al., 2012). A limited number of sequences of DHAV are
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available which are only from China, South Korea, Taiwan, Vietnam, UK and USA (Tseng et al., 2007).
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Although the virus was described in Egypt in the late 1970s (Shalaby et al., 1978), little is known about the
76
current situation of the disease (OIE, 2009). Vaccination of breeder ducks is applied in the commercial sector
77
in Egypt (Abd-Elhakim et al., 2009) using attenuated vaccines produced from E52 Rispens strain
78
(Anononymous ; Ellakany et al., 2002). In this study, we investigated the prevalence of DHAV in ducklings
79
with a history of high mortality using reverse transcription polymerase chain reaction (RT-PCR) and virus
80
isolation in embryonated duck eggs (EDE) from several provinces in Egypt. Furthermore, the sequence of
81
5´UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses were generated and analysed. To the
82
best of our knowledge this is the first published sequence for DVAH in the Middle East and Africa.
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2.
Materials and Methods
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2.1
Samples
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Samples were collected from different breeds of ducks in 46 commercial farms in Egypt; Pekin (n= 29),
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Muscovy (n= 9), Mallard (n= 5) and Green Winged ducks (n= 3). Samples were taken in 2012 (n= 4), 2013
88
(n= 12) and 2014 (n= 30). Farms’ capacities ranged from 400 to 10000 birds in 11 different provinces
89
(Figure 1). The surveillance targeted commercial farmed ducks with a history of nervous signs and high
90
mortality rate during the first two weeks of life (Table 1 and Supplementary Table S1). Samples were
91
obtained from the liver, spleen and kidneys of freshly dead or euthanized birds. Organs were homogenised
92
using the Tissuelyser (Qiagen) then subjected to three successive freeze–thaw cycles, and the supernatant
93
was separated after 12000 rpm centrifugation for 10 min.
94
2.2
95
RNA extraction from the supernatants of tissue homogenates was performed using the QIAamp Viral RNA
96
Mini kit (Qiagen, Germany, GmbH) according to the manufacturer's recommendations. The RT-PCR assays
97
using oligonucleotide primers (Metabion) for partial amplification of 5´UTR (Fu et al., 2008), VP1 (Liu et
98
al., 2008) and 3D (OIE, 2010) genes were conducted as previously published (Supplementary Table S2). A
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25-µL reaction containing 12.5 µL of Quantitect probe RT-PCR buffer (Qiagen, Germany, GmbH), 0.25 µL
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of RT-enzyme, 1 µL of each primer of 20 pmol concentration, 4.25 µL of water and 6 µL of template RNA
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was used. The reactions were performed in a T3 thermal cycler (Biometra). The amplicons were separated by
102
electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) along with 100-bp DNA Ladder
103
(Qiagen, Germany, GmbH). The gel was photographed by a gel documentation system (Alpha Innotech,
104
Biometra) and the data were analysed through computer software (Automatic Image Capture Software,
105
ProteinSimple formerly Cell Biosciences, USA).
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2.3
107
Virus isolation from RT-PCR-positive samples was attempted by inoculation of 10- to 14-day-old EDE
108
through the allantoic cavity according to the standard protocol (OIE, 2010). Eggs were candled daily for up
109
to 7 days. The embryos were examined for stunting, oedema and/or haemorrhages as well as for pathological
110
changes particularly in the liver, kidneys and spleen (OIE, 2010).
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2.4
112
Partial sequences of the 5´UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses were generated
113
using forward and reverse primers of the generic PCRs. Amino acid sequences were deduced from the
114
generated nucleotides using BioEdit software version 7.1.7 (Hall, 1999). Sequences of query and
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representative DHAVs were retrieved from the GenBank database. All sequences were aligned and identity
116
matrices were calculated automatically using BioEdit. Phylogenetic analyses were done using maximum
117
likelihood, neighbour joining and maximum parsimony in MEGA6 (Tamura et al., 2013). Consensus
118
unrooted trees were generated with 1000 bootstrap replicates and were further edited using Inkscape 0.91
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Software (Free Software Foundation, Inc, USA).
120
2.5
121
Sequences generated in this study were submitted to the GenBank under accession numbers KP148295 to
122
KP148310 for the UTR, KP148279 to KP148294 for the VP1 and KP148263 to KP148278 for the 3D gene
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fragments.
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GenBank accession numbers
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3.
Results
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3.1
Virus detection and isolation
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All RT-PCRs have relatively comparable sensitivity. Respectively 17, 16 and 15 positive samples were
128
detected by RT-PCR targeting the 5´UTR, VP1 and 3D genes. Out of the 17 samples tested positive by RT-
129
PCR, only 5 samples were positive in virus isolation (Table 1) in terms of stunting, subcutaneous
130
haemorrhages of the embryos (5/5 cases), haemorrhagic enlarged liver (4/5), enlarged mottled spleen (4/5)
131
and rarely enlarged kidneys (1/5). In 17 positive farms, mortality ranged from 15% (case no. 4) to 96.7%
132
(case no. 5) (Table 1). Nevertheless, the virus was not detected from birds with up to 100% mortality
133
(Supplementary Table S1). All duck breeds included in this surveillance were susceptible to the disease;
134
Pekin (31%; n= 9/29), Muscovy (44.4%; n= 4/9), Mallard (40%; n= 2/5) and Green Winged ducks (66.7%;
135
n= 2/3). Positive samples were higher in 2013 (50%; n= 6/12) followed by 2014 (33%; n=10/30) then 2012
136
(25%; n= 1/4). As shown in Figure 1, the disease was reported in birds in Lower Egypt (where the majority
137
of samples were collected) as well as from birds in Upper Egypt.
138
3.2
139
About 217 nucleotides were generated corresponding to nucleotides 270 to 486 of the reference strain ATCC
140
R85952 (accession number NC_008250). The nucleotide identity for the 5´UTR gene among the Egyptian
141
strains ranged from 95.3 to 100% and had homology of 94.4 – 96.3% with the vaccine strain. The Egyptian
142
strains had a maximum of 96-98% identity with the 5´UTR of the closest Asian strains (data not shown).
143
Three viruses (F10, F243 and F340) isolated from two adjacent governorates showed 100% homology.
144
Phylogenetic analysis indicated clustering of the Egyptian strains apart from the vaccine strain. The latter
145
clustered with Chinese strains (data not shown).
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VP1 gene
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The VP1 gene of all viruses including the vaccine strain has 714 nucleotides encoding 238 amino acids. The
148
identity matrix for the VP1 gene of the Egyptian strains ranged from 96.3 to 100% and 95.9 to 100% for the
149
nucleotides and amino acids, respectively (Table 2). The Egyptian strains have nucleotide identity ranged
150
from 92.9 to 93.6% and amino acid identity from 95.3 to 96.6% with the vaccine strain (Table 2). Nucleotide
151
identity to the closest Asian strains was 96-97% (data not shown). The Egyptian viruses possess V129 and
152
S142 (similar to the vaccine strain) linked to attenuation of DHAV1 for ducklings (Liu et al., 2008; Wang et
153
al., 2012). In comparison to the vaccine strain, the Egyptian viruses possess D48A/T, Q183R (except strain
154
F355), K186N, D187V, R213I/M, Y219H and L232F (except strain F215) in the carboxy terminal region
155
(Supplementary Figure 1). Other mutations observed in some attenuated Chinese isolates (S181, H183,
156
K184, N193, E205, R217 and N235) (Liu et al., 2008) are L181, R/Q183, G/E184, N/D193, E205,
157
R/K217 and L235 in the Egyptian strains, respectively.
158
Phylogenetic analysis of VP1 showed that the Egyptian viruses cluster with the Asian DHAV1 viruses and
159
further allocated into three main groups: designated group A, B and C. Nine out of 15 strains had 100% VP1
160
identity and thus clustered in group A possessing P11S, D48T, S49P, I180T and R213M in comparison to
161
the vaccine strain (Figure S1). Those strains are exclusively from Lower Egypt; five strains from Ismailia (in
162
2012 and 2014), two from Buhaira (2013 and 2014), one from Sharkia (2014) and one from Giza (2013).
163
Group B was reported from three provinces; two in Upper Egypt and one in Lower Egypt (about 700Km
164
apart), strains in 2013 in subgroup B1 and one strain from Sharkia in 2014 in subgroup B2. Meanwhile, only
165
one virus clustered in group C with the lowest homology to the Egyptian strains from Beni-Suef province
166
(between Lower and Upper Egypt; also known as Middle Egypt) in 2014. The vaccine strain clustered with a
167
vaccine strain used in Vietnam (accession number JF957697) and many Chinese strains (Liu et al., 2008)
168
with >99.9 identity. The topology of VP1 gene was similar in all three analyses and thus only maximum-
169
likelihood tree was selected (Figure 2).
170
3.4
171
Total of 440 nucleotides were generated corresponding to nucleotides 6505 to 6944 of the reference strain
172
R85952 which encode 146 amino acids. Nucleotide identity matrix for the 3D gene among the Egyptian
173
strain ranged from 98.4 to 100% and had 100% identical amino acid sequences. The Egyptian strains had 95
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3D protein
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174
to 95.9 % nucleotide homology with the 3D of the vaccine strain and the identity to the closest Asian strains
175
was 98% (data not shown). Like other DHAVs, the Egyptian strains contain the GxxCSGxxTxxNS motif
176
which is highly conserved region in the 3D of all picornaviruses (Wang et al., 2008).
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4.
179
Since decades, DHAV causes severe losses in duck industry. Yet, no information about the prevalence of the
180
virus outside Asia, particularly in the Middle East and Africa, is available. Diagnosis of DHAV may be
181
confirmed by rapid onset of clinical signs and mortality in inoculated susceptible ducklings, inoculation of
182
tissues homogenate into the allantoic sacs of EDE and embryonated chicken eggs or infection of cell culture
183
(OIE, 2010). In contrast to the laborious and time-consuming culture techniques, rapid and specific detection
184
of the DHAV RNA using different RT-PCR assays were described (Fu et al., 2008; Liu et al., 2008; OIE,
185
2010; Wei et al., 2012). In this study, all three PCR assays had a comparable sensitivity, but the isolation rate
186
was very low. The high sensitivity of RT-PCR over virus isolation is generally accepted. Nevertheless,
187
although the samples were collected from freshly dead/euthanized birds, the death of the virus during the
188
transportation to the central laboratory could not be excluded. Furthermore, due to the lack of SPF EDE in
189
Egypt, it is possible that the duck eggs used for virus isolation in this study had anti-DHAV antibodies which
190
may neutralise the virus and decrease the isolation rate.
191
The prevalence of DHAV in ducklings in this study was 37% in 10 out of 11 provinces indicating a serious
192
health problem for the duck industry in Egypt. Therefore, nationwide surveillance is recommended. Also,
193
other causes than DVH such as Pasteurella, Salmonella or E.coli may be responsible for high mortality
194
especially in DHAV-negative flocks which should be further investigated. To date, resistance of certain
195
breeds of ducks to DHAV is not well known (Woolcock, 2003). As obtained here, the virus was isolated
196
from Pekin, Muscovy, Mallards and Green Winged ducks. Unfortunately, it is not clear why these birds were
197
infected because the history of vaccination of the breeder flocks is missing. Whether the current vaccine
198
cannot protect birds against the field virus because of antigenic variation or the titer of maternal immunity, if
199
any, was low is not known. Thus, antigenic characterisation of the virus and studies to assess protection
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should be conducted. In China several vaccines were updated according to the circulating viruses in the field,
201
nevertheless the infection of vaccinated ducklings was reported (Jin et al., 2008; Li et al., 2013). The absence
202
of the lesions in EDE and the existence of mutations previously reported in attenuated tissue-adapted strains
203
(Wang et al., 2012) may indicate that the Egyptian strains differ in their virulence to the Asian viruses;
204
therefore in-vivo pathogenicity testing is highly required.
205
Although the number of gene sequences of DHAV is substantially increasing providing useful information
206
on the taxonomy, epidemiology and virulence of the virus, however it is limited to few countries only (Kim
207
et al., 2006; Ding and Zhang, 2007; Tseng et al., 2007; Tseng and Tsai, 2007). In this study, based on the
208
VP1 gene, three different lineages (A, B and C) were detected while the majority of the strains located in the
209
geographically-limited lineage A from Lower Egypt, viruses in lineage B was reported from both Lower and
210
Upper Egypt which may be due to the rapid and random movement of birds between different regions in
211
Egypt. Also, we noticed that one strain in lineage C (isolated from Middle Egypt) was probably resulted
212
from recombination of strains from Upper and Lower Egypt. However, due to the limited number of positive
213
samples and short gene fragments these data must be interpreted cautiously. Remarkably, the Egyptian
214
strains are distinguishable from the vaccine strain where the lowest identity was observed in VP1 followed
215
by UTR and 3D genes which is expected because the VP1 gene is the most external surface protein and is
216
involved in many vital stages in the viral life-cycle including receptor binding and neutralising epitopes (Liu
217
et al., 2008).
218
In conclusion, DHAV-1 was detected in 17 out of 46 commercial duck farms of different breeds examined in
219
this study. Sequence and phylogenetic analyses indicated that the Egyptian strains are distinguishable from
220
the vaccine and similar to the contemporary Asian viruses. So far, this is the first report on the genetic
221
characterisation of DHAV in Egypt.
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Acknowledgments
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The authors are grateful to the colleagues and co-workers at the national laboratory for veterinary quality
225
control on poultry production, animal health research institute, ministry of agriculture, Egypt for their
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support
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Figure 1. Distribution of duck hepatitis A virus positive samples collected from commercial ducklings in
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Egypt from 2012 to 2014
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* refers to weak positive results after testing by RT-PCR targeting the 5´UTR gene, trials to generate
231
sequence from those two viruses failed. Numbers in the map refers to the provinces on the left part of the
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figure
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Figure 2. Phylogenetic relatedness of the VP1 gene of the Egyptian DHAV to the vaccine and other
236
representative Asian viruses
an
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Maximum-likelihood unrooted tree generated after 1000 bootstraps indicated clustering of the Egyptian with
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the Asian DHAV1 viruses apart from the vaccine strain. The Egyptian strains allocated into three main
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groups denoted groups A, B (B1 and B2) and C.
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Pan, M., Yang, X., Zhou, L., Ge, X., Guo, X., Liu, J., Zhang, D., Yang, H., 2012. Duck Hepatitis A virus possesses a distinct type IV internal ribosome entry site element of picornavirus. J. Virol. 86, 1129-1144. Shalaby, M.A., Ayoub, M.N.K., Reda, I.M., 1978. A study on a new isolate of duck hepatitis virus
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Woolcock, P.R. 2003. Viral infections of waterfowl, In: Saif, Y.M., Barnes, H.J., Glisson, J.R.,
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Xu, Q., Zhang, R., Chen, L., Yang, L., Li, J., Dou, P., Wang, H., Xie, Z., Wang, Y., Jiang, S., 2012. Complete genome sequence of a duck hepatitis a virus type 3 identified in eastern China. J. Virol. 86, 13848.
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Figure 1. Distribution of duck hepatitis A virus positive samples collected from commercial ducklings in
324
Egypt from 2012 to 2014
M
an
us
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325
326
d
327
* refers to weak positive results after testing by RT-PCR targeting the 5´UTR gene, trials to generate
329
sequence from those two viruses failed. Numbers in the map refers to the provinces on the left part of the
330
figure
Ac ce p
331
te
328
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Figure 2. Phylogenetic relatedness of the VP1 gene of the Egyptian DHAV to the vaccine and other
332
representative Asian viruses
335 336
te
334
Ac ce p
333
d
M
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us
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331
337
Maximum-likelihood unrooted tree generated after 1000 bootstraps indicated clustering of the Egyptian
338
strains with the Asian DHAV1 apart from the vaccine strain. The Egyptian strains allocated into three main
339
groups denoted groups A, B (B1 and B2) and C.
340 341
20 Page 17 of 20
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Graphical Abstract (for review)
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Table
Table 1 1. Positive flocks for duck hepatitis A virus from 2012 to 2014 in Egypt Sample No.
Province
Code
Year
Birds Breed
Results No.1
Age
Mortality2
Isolation3
RT-PCR UTR
VP1
3D
F340
2012
Ismailia
Pekin
8
4500
1500
+
+A5
+
-
2
F86
2013
Luxor
Pekin
5
1000
400
+
+B1
+
-
3
F163
2013
Luxor
Muscovy
5
2000
600
4
F413
2013
Qena
Muscovy
3
4000
600
5
F729
2013
Qaluibia
Mallard
12
9000
8700
6
F829
2013
Giza
Green Winged
10
2000
7
F953
2013
Buhaira
Pekin
9
2000
8
F112
2014
Ismailia
Pekin
8
9
F147
2014
Ismailia
Pekin
9
10
F215
2014
Beni-Suef
Green Winged
11
F321
2014
Buhaira
Pekin
12
F243
2014
Sharkia
Muscovy
13
F355
2014
Sharkia
Pekin
14
F387
2014
Ismailia
15
F10
2014
16
Gh.3
17
FY.2
ip t
1
an
(day)
+B1
+
-
+
+B1
+
-
+
+B1
+
+
500
+
+A
+
+
1500
+
+A
+
-
us
cr
+
1500
+
+A
+
-
4000
2700
+
+A
+
-
8 & 15
10000
3000
+
+C
+
+
5
2000
900
+
+A
+
-
10
10000
2000
+
+A
+
-
9
2000
600
+
+B2
+
+
Pekin
7
4000
3000
+
+A
+
-
Ismailia
Mallard
9
4000
1000
+
+A
+
+
2014
Gharbiya
Muscovy
8
1000
500
+4
-
-
-
2014
Fayoum
Pekin
10
900
300
+4
+4
-
-
ed
ce pt
Ac
M
2500
1
Total 2 number of birds at the farm
2
Total 3 mortality from one-day old until sampling as told by the owners/farmers
3
Virus 4 isolation was done in embryonated duck eggs, where positive samples showed stunting,
haemorrhages 5 and/or pathological changes of the embryos 4
Weak 6 positive; unable to sequence these samples
5
The 7 genetic group according to the VP1 gene (Figure 1)
Page 19 of 20
ip t
Table
cr
Table 1 2. Nucleotide (upper right) and amino acid (lower left) identity matrices of VP1 of the vaccine and duck hepatitis A virus isolates from
100 100 100 100 100 100 100 97 97 97 97 96.6
100 100 100 100 100 100 97 97 97 97 96.6
F10 92.9 96.2 100 100 100
F243 92.9 96.2 100 100 100 100
100 100 100 100 100 97 97 97 97 96.6
F112 92.9 96.2 100 100 100 100 100
an
F829 92.9 96.2 100 100
M
100 100 100 100 100 100 100 100 97 97 97 97 96.6
F953 92.9 96.2 100
d
F340 92.9 96.2
ep te
Vaccine F215 93.6 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.3 95.7 95.7 97.8 95.7 97.8 95.7 97.8 95.7 97.8 96.6 97.4
Ac c
3 Virus Vaccine F215 F340 F953 F829 F10 F243 F112 F147 F321 F387 F86 F163 F413 F729 F355 4
us
commercial 2 ducklings in Egypt
100 100 100 100 97 97 97 97 96.6
100 100 100 97 97 97 97 96.6
F147 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F321 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F387 92.9 96.2 100 100 100 100 100 100 100 100 97 97 97 97 96.6
F86 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F163 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F413 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100 98.7
F729 93.1 96.6 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 97.3 100 100 100
F355 93.6 96.9 97.6 97.6 97.6 97.6 97.6 97.6 97.6 97.6 97.6 98.8 98.8 98.8 98.8
98.7 5
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