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. 2024 Jun 12;103(9):103919. doi: 10.1016/j.psj.2024.103919

Pathogenicity studies and molecular characterization of DPV infection in ducklings

Wentao Tang *,†,‡,1, Mengdi Yuan ⁎,†,‡,1, Mingtian Mao *,†,‡, Yitong Cui *,†,‡, Qiong Wu *,†,‡, Bingrong Wu *,†,‡, Dalin He *,†,‡, Feng Wei *,†,‡, Yudong Zhu *,†,‡, Youxiang Diao ⁎,†,‡, Jingdong Hu ⁎,†,‡, Yi Tang ⁎,†,‡,2
PMCID: PMC11264171  PMID: 38970847

Abstract

In the spring of 2023, 10 to 21-day-old chicks in a broiler duck farm in Shandong Province, China, developed swelling of the head and neck, moist eyes with mucous discharge, difficulty in walking, shrinking of the neck, and loose and disorganized coat. Anatomical observation revealed hemorrhages in the esophageal mucosa, myocardium, and liver, and severe hemorrhages in the trachea with copious inflammatory secretions. Soon after, similar symptoms appeared in a large number of ducks in the flock, which eventually led to the elimination of all the 20,000-odd newly introduced ducklings on the farm, resulting in huge economic losses. We detected duck plague virus in the tissues of liver, spleen and lungs of diseased and dead ducks, and successfully isolated the pathogenic strain, named SD423, by inoculating duck embryos and inoculating duck embryo fibroblasts. We successfully conducted animal regression experiments with the isolated strain, and the experimental animals in the 1 d of age group showed symptoms of swollen eyes and tearing, shrinking of the neck, crouching, and hemorrhage in organs such as the liver and intestines successively from the 3rd d. We sequenced the whole genome of the isolated duck plague strain, and by comparing the homology with the published duck plague virus whole sequences in Genbank, the virus strain obtained in this study had the highest homology with the Chinese virulent strain SD (MN518864.1), with nucleotide (nt) homology of about 99.90% and amino acid (aa) homology of about 99.75%, which indicated that the isolate is a virulent strain. Previously, it was reported that the natural infection of duck plague virus mainly occurs above 30 d of age, but the duck plague virus found in this study can naturally infect ducklings up to 20 d of age, and the mortality rate is as high as 100%. In this study, the pathogenicity test and whole genome sequence analysis of this isolate provided data support and theoretical basis for further research on pathogenicity and virulence-related gene analysis of duck plague virus.

Key words: duck plague virus, duckling, whole genome sequencing, pathogenicity

INTRODUCTION

Duck plague, also known as duck viral enteritis (DVE), is an acute, febrile, septic, virulent infectious disease of ducks, geese and other geese-like birds caused by duck plague virus (DPV) (Fauquet et al., 2005). The disease was first reported in the Netherlands in 1923 and was recognized as a new viral disease different from fowl plague (Baudet et al., 1923). The presence of the disease has since been reported in various countries in Europe and North America. Huang Yinxian first discovered the disease in Guangdong, China in 1957, followed by Wuhan, Shanghai, Zhejiang, Guangxi, Jiangsu, Hunan and Fujian (Huang, 1959). After infection with duck plague virus, the body temperature rises rapidly to more than 43 ℃, the spirit is depressed, the appetite decreases, both eyes tear, eyelid edema and adhesion (Aravind et al., 2015). Autopsy of sick and dead ducks showed hemorrhagic spots in the liver, a gray-yellow necrotic or hemorrhagic band at the junction of the dilated esophagus and the glandular stomach, congestion, hemorrhage, necrosis and detachment of the intestinal mucosa, and pathological changes such as the appearance of button ulcer foci in part of the intestinal mucosa (El-Tholoth et al., 2019). Histopathological observations also showed severe lesions in lymphoid and digestive organs, including decreased numbers of lymphocytes in central immune organs, swelling and detachment of epithelial cells in the intestinal mucosa, and hyperplasia of connective tissue in the intestinal lamina propria (Aravind et al., 2015). Duck plague virus is generally commonly cultivated in duck embryos or duck embryo fibroblasts (DEF) (Kocan, 1976). When inoculated with duck embryos, the main manifestations are death of duck embryos and hemorrhage of embryos body; when inoculated with duck embryo fibroblasts, the transparency will generally become lower after 2 to 3 d the particles will increase, the cytoplasm will be concentrated, and the cells will be gradually smooth (Liu et al., 2020). The virus spreads rapidly and has a high lethality rate, causing significant losses to the waterfowl industry globally (Kong et al., 2023). The infection of ducklings up to 20 d of age caused by the current outbreak differed from that reported in the data (Liang et al., 2022). Therefore, in this study, the pathogenicity of this isolate was investigated by virus isolation and identification and animal regression experiments, which provided basic data for the epidemiological investigation of duck plague virus in waterfowl.

Currently, the main published whole genome sequences of duck plague virus include strains CHv (162175 bp), SD (160945 bp), VAC (158091 bp) and 2085 (160649 bp) (Wu et al., 2012). The structure of duck plague virus is envelope, outer membrane, vesicle and core from outside to inside, and the vesicle membrane is icosahedral, the genome is linear double-stranded DNA (Spieker et al., 1996), which consists of covalently bound long unique region (UL), short unique region (US), internal reverse repeat sequence (IRS) and terminal reverse sequence (TRS), and is arranged in the manner of UL-IRS-US-TRS (Chang et al., 2011). There were a total of 78 open reading frames (ORFs), of which 65 ORFs were located in the UL region, 11 ORFs were located in the US region, and the remaining two ORFs were located in the IRS region and the TRS region (Akter et al., 1970). In this study, we sequenced the whole genome of duck plague virus isolated from this outbreak and analyzed virulence genes including UL12, UL41, UL47, LORF11, and UL2, which provided a theoretical basis for further investigation of the down-regulation of the age of naturally infected hosts by duck plague virus.

MATERIALS AND METHODS

Clinical Examination and Pathogen Detection

At a farm suspected to be infected with duck plague virus in Shandong Province, we collected 10 samples of diseased ducks for autopsy study. After clinical and autopsy dissect observations, a preliminary diagnosis of duck plague was made. Separate samples were collected from liver, brain, lung, esophagus, spleen, intestine and heart. Total DNA and RNA were extracted from the supernatant of the tissue milling solution, and the tissues were subjected to routine viral testing, while the rest of the samples were stored in an ultra-low-temperature refrigerator at -80 ℃.

Virus Isolation

We selected the livers, spleens, and lungs of 10 collected samples, respectively, and added 5 times the volume of saline for grinding, and then put them into a -80℃ ultra-low-temperature refrigerator to freeze-thaw them repeatedly for 3 times. The thawed tissue milling solution was centrifuged at 8,000 r/min for 10 min, and the supernatant was filtered through a 0.45 μm bacterial filter to remove the bacteria, and then stored in an ultra-low temperature refrigerator at -80 ℃. Twenty 9 d of age duck embryos were taken out from the incubator, of which 15 were inoculated with the preserved virus solution and 5 were injected with saline as control, and the inoculation volume of each duck embryo was 200 μL. Then they were put into the incubator to continue incubation, and the mortality rate and lesions were observed and recorded every day. The allantoic membrane and allantoic fluid of duck embryos that died after 24 h were collected, ground with DMEM medium and then repeatedly frozen-thawed for 3 times. The supernatant was extracted by centrifugation, filtered to remove bacteria, and 200 μL of the filtrate was inoculated with duck embryo fibroblasts.

Determination of TCID50

One duck embryo of 9 to 11 d old was taken from the incubator, thoroughly sterilized in an ultra-clean bench, and the embryo head, limbs, and internal organs were discarded. The remaining tissue was rinsed with PBS and minced with scissors, and double the volume of trypsin was added, mixed with a pipette, and digested in a 37 ℃ water bath for 10 min. Add 5mL of medium to terminate digestion, centrifuge at 800 to 1,000 r/min for 5 min, discard the supernatant, add 30 to 40 mL DMEM to resuspend the cells, and slowly filter through 4 layers of gauze. The filtrate was added to a 96-well cell culture plate with 100 μL per well, and then incubated in a cell culture incubator for 12 h. Take the 2nd generation DPV virus liquid isolated, use serum-free DMEM medium in sterile EP tubes for 10-fold gradient (10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, 10−11) dilution, according to the different dilutions inoculated into the prepared 96-well cell culture plate, each dilution to do 8 replicate wells, closed and put into 37℃, 5% CO2 incubator for 1 h. Remove the 96-well cell culture plate, carefully aspirate the virus solution with a pipette gun, and add 200 μL of DMEM cell maintenance medium containing 2% fetal bovine serum to each well. The cell status was observed and recorded for 5 consecutive days and the cellular half-infection level (TCID50) was calculated according to the Reed-Muench method. The TCID50 of the isolated virus strain was calculated to be 10−5.8/0.1 mL according to the formula, TCID50 = logarithm of the critical dilution of 50% infection rate + distance ratio × logarithm of the dilution factor, where: distance ratio = (critical infection rate higher than 50–50%)/(critical infection rate higher than 50% - critical infection rate lower than 50%).

Animal Infection Assay

In order to verify whether the isolated viral fluids have the same pathogenicity to ducklings of different ages, 20 healthy ducks each of 1 d of age and 35 d of age were selected for animal infection experiments in this study. Ducklings of different ages were artificially infected with DPV, in which 15 ducklings in the 1 d of age experimental group were inoculated with 200 μL of viral solution, 15 ducklings in the 35 d of age experimental group were inoculated with 400 μL of viral solution, and 5 ducklings at each age were inoculated with saline as the control group. The deaths and clinical symptoms of experimental ducks in each group were observed and recorded every day. On the 15th d, all ducks were culled for autopsy, and photos were taken to record and observe the pathological lesions of each visceral tissue, while liver tissues of ducklings in each group were collected for the isolation and identification of duck plague virus.

Whole Genome Sequencing

We extracted viral DNA using the Genomic DNA Extraction Kit and used the standard Illumina TruSeq Nano DNA LT Illumina TruSeq DNA Sample Preparation Guide to construct the required genomic input libraries. Analysis and De novo assembly of all NGS raw data were carried out by different modules of CLC Genomics Workbench software (QIAGEN, Boston, MA). Based on the known fragment sizes of the DPV genome, all large gene fragments were extracted for further submission to BLAST, the NCBI module of the software.Based on the results of the BLASTN search, all DPV-related genes were selected for the construction of the full-length genome of DPV. The Map reads to reference module was utilized to remap all NGS raw reads to DPV-related genes, further increasing the length and sequencing coverage of each assembled fragment. Finally, the consensus sequence was obtained from the remapped reads and considered as the final assembly result of the DPV whole genome. Viral open reading frames (ORFs) prediction, amino acid (aa) translation, sequence alignment, and genetic evolution analysis were performed using the EditSeq and MegAlign modules of DNASTAR Lasergene 12 Core Suite (DNASTAR, Inc. Madison, WI). The highest degree of similarity between the studied DPV genomic fragments and published sequences was determined by the BLASTN online search program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS

Clinical and Histopathological Features

We carried out clinical diagnosis and pathologic observation of diseased and dead ducks collected from the morbid farms. The main clinical manifestations of diseased ducks are depression, loss of appetite, lying down, difficulty in walking, swelling of the head and neck, and swelling of the eyes with viscous secretions. Autopsy revealed hemorrhages in the esophageal mucosa, myocardium, and liver, and hemorrhages in the tracheal rings with a large amount of inflammatory secretion (Figure 1). The samples collected were tested for common duck pathogens such as Duck Plague virus, Avian Influenza virus, Newcastle Disease virus, Duck Hepatitis virus, Tambusu virus, Novel Goose Parvovirus, Avian Adenovirus, Avian Reovirus, Duck Astrovirus, and Duck Circovirus, etc., and the results were positive for Duck Plague virus, while the rest of the pathogens tested were negative.

Figure 1.

Figure 1

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Clinical symptoms and pathologic changes in 17-day-old ducklings infected with DPV on a farm. (A) sick ducks with swollen eyes, shrunken necks, and recumbent; (B) pitting hemorrhage in the esophagus; (C) severe hemorrhage in the tracheal ring with a large amount of viscous secretion; (D) Massive hemorrhage on the surface of the myocardium; (E) hemorrhage and enlargement of the liver.

Virus Isolation and Characterization

We selected the visceral tissues with obvious lesions in the collected samples, processed them and inoculated them with duck embryos, and then continued the culture. The changes of duck embryos were observed every day, and the dead duck embryos within 24 h were rejected. Obvious lesions began to appear on the 2nd day, duck embryos began to die in large numbers, and the dead duck embryos had thickened cystic membranes and extensive hemorrhage throughout the embryo body (Figure 2) . Carefully aspirate the dead duck embryo allantoic fluid into a 50 mL centrifuge tube, and at the same time take the chorionic allantoic membrane, add 20 mL DMEM medium milled and repeatedly freeze-thawed, centrifugation and filtration of the viral liquid into the -80 ℃ ultra-low-temperature refrigerator for storage. 200 μL was taken to inoculate duck embryo fibroblasts, and the cell status was observed and photographed daily. Cytopathic lesions began to appear 24 h after inoculation, and after 48 h the transparency of the cells decreased, the cytoplasmic granules increased and condensed, and the cells became rounded and finally detached (Figure 3). The cellular viral solution was collected after 72 h, frozen-thawed three times repeatedly, and the supernatant was centrifuged and identified by PCR, which showed only duck plague virus infection.

Figure 2.

Figure 2

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Lesions appearing in DPV-infected duck embryos. (A) duck embryo infected with DPV with severe hemorrhage of the embryo; (B) normal duck embryo without lesions.

Figure 3.

Figure 3

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Lesions appearing in DPV-infected duck embryo fibroblasts. (A) negative control; (B) DPV-infected DEF showed cell crumpling and rounding, and finally shedding.

DPV Infection in Ducklings of Different Days of Age

In the artificially infected 1 d of age group, clinical symptoms began to appear on the 2nd d, with depressed spirit, loss of appetite, coarse and disheveled coat, and viscous secretion from the eyes; on the 3rd d, the experimental ducks had swollen heads, curved necks, difficulty in walking, and swollen and adherent eyes; deaths began to appear on the 5th d; and all of the ducks were dead by the 10th d. The dissection observed a flushed pancreas, punctate hemorrhages in the thymus, esophagus, myocardium, bursa, and spleen, massive hemorrhages in the liver, necrotic detachment of the intestinal mucosa with massive hemorrhages, and severe congestion, hemorrhages, and edema in the cloaca (Figure 4). In the artificially infected 35 days of age group, slight swollen eyes appeared at d 4 post-infection and returned to normal at d 6, and some ducks excreted greenish thin feces at d 13. Dissection at day 15 post-infection revealed punctate hemorrhage in the thymus, slight hemorrhage in the duodenum, splenomegaly, and hemorrhage in the lungs (Figure 5). Clinical signs and histopathological changes were milder in the 35 d of age infected group compared with the 1 d of age infected group, and there were no deaths.

Figure 4.

Figure 4

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Clinical symptoms and pathological changes exhibited by artificially infected DPV 1-day-old group. (A) sick ducks with swollen eyes, shrunken necks, and recumbency; (B) severe hemorrhages in the duodenum and rectum, and detachment of intestinal mucosa; (C) edema and hemorrhages in the cloaca; (D) hemorrhages in the thymus gland; (E) punctate hemorrhages in the bursa of the falciparum; (F) punctate hemorrhages in the heart muscle; (G) hemorrhages and enlargement of the liver; (H) hemorrhages in the lungs; (I) flushing in the pancreas; (J): punctate hemorrhages in the esophagus; (K) Bleeding spots in the spleen.

Figure 5.

Figure 5

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clinical signs and pathologic changes exhibited by the artificially infected DPV 35-day-old group. (A) normal clinical performance; (B) slight hemorrhage in duodenum; (C) normal cloaca; (D) hemorrhagic spot in thymus; (E) normal bursa of Fasciola; (F) green feces; (G) normal heart; (H) normal liver; (I) hemorrhage in lungs; (J) normal pancreas; (K) normal esophagus; (L) enlargement and hemorrhage in spleen.

Whole Genome Sequence Analysis

The complete genome sequence of strain SD423 was 162,048 nucleotides (nt) with a G and C content of (22.21 + 22.69) 44.9%, and a total of 78 open reading frames (ORFs) were predicted to encode potential functional proteins. The final sequence has been deposited in the Genbank database under accession number: . The results showed that the virulence genes UL2, UL12, UL41, UL47, and LORF11 were successfully sequenced. Compared with European strains, Indian strains and strains reported from China, strain SD423 was more similar to the 2020 Chinese isolate of the virulent strain SD (MN518864.1), with 99.90% nucleotide homology and 99.75% amino acid homology, and least similar to the DEV attenuated vaccine strain C-KCE (KF263690), with 99.89% nucleotide homology and 99.62% amino acid homology (Table 1). The results indicated that the outbreak on the farm was caused by virulent strain of duck plague.

Table 1.

Comparison of whole genome sequence homology between SD423 and other representative strains of duck plague virus.

Strain Complete sequence
nt aa
CHv (JQ647509.1) 99.84 99.59
CV (JQ673560.1) 99.89 99.63
SD (MN518864.1) 99.90 99.75
2085 (JF999965.1) 99.90 99.10
DP-AS-Km-19 (MZ574076.1) 99.90 99.63
DEV-K-p63 (KF477736.1) 99.90 99.63
C-KCE (KF263690.1) 99.89 99.62
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nt: nucleotide sequence.aa:amino sequence.

SD423 unique long open reading frame UL2 length of 1002 bp, encodes 334aa, and shares up to 100% nucleotide homology with the Chinese virulent strains CV (JQ673560.1), CHv (JQ647509.1), SD (MN518864.1), LH2011 (KC480262.1), the European virulent strain 2085 (JF999965.1), and the Indian virulent strain DP-AS-Km-19 (MZ574076.1) has up to 100% nucleotide homology. However, compared to the DEV attenuated vaccine strain C-KCE (KF263690) and the attenuated duck enteritis virus DEV K p63 (KF487736.1), the SD423 strain had an insertion of 529 bp after 281 bp (Figure 6). This finding was present between all virulent and attenuated strains of duck plague and may be one of the reasons for the reduced virulence of the attenuated vaccine strains.

Figure 6.

Figure 6

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Gene structure types of UL2. (A) a deletion of 529 bp was found in strains such as K-p63 and C-KCE; (B) strains such as SD and 2085 are highly homologous to strain SD423; (C) strain SD423.

The SD423 LORF11 reading frame was 4341 bp long, with a non-coding region of 861 bp in the middle, which was divided into the LORF11-A coding region of 708 bp long and the LORF11-B coding region of 2769 bp long. The structure of LORF11 was similar to that of LH2011 (KC480262.1) and SD (MN518864.1), the virulent strains isolated from China. Virulent strain 2085 (JF999965.1) and DP-AS-Km-19 (MZ574076.1) strains had 493 bp at the 5′ end that was highly homologous to SD423 LORF11-A, 2678 bp at the 3′ end that was highly homologous to SD423 LORF11-B, and a deletion of 1,171 bp after 493 bp; The attenuated strains C-KCE (KF263690) and DEV K p63 (KF487736.1) were found to be highly homologous to SD423 LORF11-A at the 5′ end for 647 bp, and to SD423 LORF11-B for 178 bp at the 3′ end, with a deletion of 3516 bp following 647 bp (Figure 7).

Figure 7.

Figure 7

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Gene structure types of LORF11. (A) Insertion of 1171 bp was found in strains 2085 and DP-AS-Km-19; (B) Deletion of 3516 bp was found in strains K-p63 and C-KCE; (C) Strains SD and LH2011 were highly homologous to SD423 strain; (D) SD423 strain.

The SD423 UL12 reading frame is 1,689 bp long and encodes 563aa. Compared to the duck plague viruses listed in the text, the nucleotide homology is 99.9% and the amino acid homology is 99.8%, with only a single base mutation at 1,102 bp (G mutated to T) resulting in a mutation of amino acid at 368 from S to A (Table 2). The SD423 UL41 reading frame was 1,494 bp long and encoded 498aa. It showed the highest similarity to the Chinese isolate of the virulent strain, with 99.9% nucleotide homology and 99.8% amino acid homology. Compared with the Chinese virulent and attenuated strains DEV K p63 (KF487736.1), SD423 has a deletion of amino acid D at 126. Nucleotide homology with the virulent European strain was 99.8% and amino acid homology was 99.6%, with the amino acid at 303 mutated from C to Y. Compared to all strains listed herein, the amino acid at 172 was mutated from M to I. The amino acid at 172 was mutated from M to I. The amino acid at 303 was mutated from C to Y. The amino acid at 303 was mutated from C to Y (Table 2).

Table 2.

Comparison of amino acid mutations in UL12, UL41 and UL47 of SD423 with other representative strains.

ORF Amino acid position(s) Amino acid
K p63
(KF477736.1)		 CHv
(JQ647509.1)		 CV
(JQ673560.1)		 SD
(MN518864.1)		 LH2011
(MZ574076.1)		 2085
(JF999965.1)		 DP-AS-Km-19
(MZ574076.1)
UL12 1102 S→A S→A S→A S→A S→A S→A S→A
UL41 126 *D *D *D *D *D - -
166 - #C - - - - -
172 M→I M→I M→I M→I M→I M→I M→I
303 - - - - - C→Y C→Y
UL47 36 V→A - - - - - -
113 R→H R→H R→H - - - -
149 *D *D *D - - - -
234 P→S P→S P→S P→S - P→S P→S
420 #S - - - - - -
599 T→A T→A T→A - - - -
727 - - - F→L - - -
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* represents deletion, # represents addition.

SD423 reading frame UL47 is 828 bp long and encodes 276aa. Highly similar to the European strain 2085 (JF999965.1) and the Indian strain DP-AS-Km-19 (MZ574076.1), with up to 99.9% amino acid homology and 99.7% nucleotide homology, with only a single amino acid substitution at 234 (P mutated to S), which is also present in other strains. It differed most from the attenuated strain DEV K p63 (KF487736.1), with only 99.4% amino acid homology. Sequence comparison revealed a deletion of amino acid D at 149, mutation of amino acid V to A at 36, mutation of amino acid R to H at 113, and mutation of amino acid T to A at 599, for a total of four mutation sites. Compared to the SD (MN518864.1) strain, there is an insertion of amino acid S at 420 and a mutation from F to L at amino acid 727 (Table 2), which also shares a high degree of homology with SD423 nucleotides and amino acids.

In summary, all five virulence-related genes of strain SD423 had the highest homology with the virulent strain, among which the UL47 gene was highly homologous to the European and Indian strains, and each of the virulence-related genes was highly similar to that of the Chinese SD virulent strain, which can further indicate that the isolate belongs to the virulent strain. At present, the functions of the mutation sites of all virulence-related genes are not known to determine whether these alterations are related to the down-regulation of the day of age of naturally infected hosts of duck plague virus, and further in-depth studies are needed.

Genetic Evolutionary Analysis of the LORF11 and UL47 Genes

In this study, we drew the genetic evolution tree by matching the LORF11 gene of SD423 strain with the reference strain. The results showed that the LORF11 gene categorized duck plague virus into three branches, namely: the European American branch, the attenuated strain branch, and the Chinese branch, and strain SD423 belonged to the Chinese branch (Figure 8). It was previously reported that the Indian isolate DP-AS-Km-19 (MZ574076.1) belonged to the European invasive strain, which was consistent with the results of the genetic evolution analysis. However, the SD423 UL47 gene belongs to the same branch as the European strains, suggesting that the gene may be mutated towards the European strains.

Figure 8.

Figure 8

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Genetic evolutionary analysis of the LORF11 and UL47 genes.

DISCUSSION

This outbreak of infectious disease in ducklings was initially judged to be caused by duck plague virus infection from clinical symptoms and pathological changes. The collected diseased material was analyzed by PCR identification, pathogen isolation and culture, and whole genome sequencing, and the results determined that this outbreak was caused by duck plague virus infection. Since duck plague virus could not directly proliferate in chicken embryos for transmission, duck embryos were selected for virus isolation (Sarmah et al., 2023). The initial isolation was performed by inoculation of 9- to 12-day-old duck embryo chorionic allantoic membrane, and the collected allantoic fluid was used to inoculate duck embryo fibroblasts, and the pure duck plague virus was successfully isolated and named SD423 strain.

In this study, generalized hemorrhage of the embryo occurred after inoculation of the duck embryo with tissue grinding solution, and the allantoic fluid was collected and inoculated with duck embryo fibroblasts, which appeared to be vacuolated and crumpled and dislodged after 48 h. The cells were then inoculated with the tissue grinding solution. Different clinical signs and pathological changes were observed in ducklings infected with DPV test at different days of age. The 1 d of age group showed symptoms such as swollen eyes and tears and intestinal hemorrhage, which were similar to previously reported symptoms after DPV infection of ducks (Pathak et al., 2019). However, the 35 d of age infected group showed only abnormal defecation and slight hemorrhage in the intestines and other tissues, which differed considerably from the symptoms of DPV infection in the older day-old ducks in the data. The results showed that the infectious host of duck plague virus is no longer limited to large day-old ducks, but causes infection in ducks of all ages, and the virus isolated this time is more pathogenic to smaller day-old chicks, and these findings provide a scientific basis for the diagnosis of duck plague virus infection and epidemiological investigations.

DPV genomes from different countries are highly homologous to each other, but can be categorized into virulent and attenuated strains based on a number of virulence-related genes (Aasdev et al., 2021). It has been reported that the UL2 gene predicted to encode uracil DNA glycosylasewhich play an important role in viral replication, and is significantly different between virulent and attenuated strains (Xie et al., 2017). The UL2 gene of SD423 was analyzed and was identical to the Chinese, European and Indian strains; There is a large difference with the attenuated strain DEV K p63 (Zou et al., 2014), SD423 has a 529 bp insertion after 281 bp. The results suggest that deletion of the UL2 gene may be an important factor in the reduced virulence of attenuated DPV.

The structure of the LORF11 gene of the SD423 strain was similar to that of the Chinese SD and LH2011 strains, with an 861 bp non-coding region in the middle dividing LORF11 into two coding regions (Apinda et al., 2022), LORF11-A and LORF11-B. The LORF11 gene of European strong strain 2085 and Indian strong strain DP-AS-Km-19 had a deletion of 1,171 bp after 493 bp, resulting in a portion of the gene at both the 5′ and 3′ ends that was highly homologous to SD423 LORF11-A and SD423 LORF11-B, respectively; The LORF11 gene of attenuated duck enteritis virus K p63 has a 647 bp homologous portion at the 5′ end, followed by a deletion of the 3516 bp gene, and then a 178 bp homologous portion.

Recent studies have found that pUL47, the protein encoded by the UL47 gene of DPV (He et al., 2020), can promote the entry of the transcriptional activator pUL48 into the nucleus through its nuclear localization signal, thus enhancing the transcriptional activation function of pUL48, promoting the transcription of viral early genes, and creating an optimal environment for viral replication (Deng et al., 2022). It is evident that the UL47 gene encodes a protein that plays an important role during herpesvirus infection (Luo et al., 2010). In this study, the analysis of UL12, UL41 and UL47 genes of SD423 showed that the SD423 UL47 gene was highly homologous to the Chinese SD, European 2085 and Indian DP-AS-Km-19 virulent strains, but there were mutations in individual bases. The effect of these mutations on virulence is not clear from this study, but it is basically certain that the SD423 UL47 gene is shifting in the direction of European strains, which may lead to failure of immunization with existing vaccines in China. The UL2 gene is highly homologous to the virulent strain, but differs significantly from the attenuated strain, and further verification is needed to determine whether this gene determines the strength of DPV virulence. The LORF11 gene structures of Chinese, European and attenuated strains have their own characteristics, and this finding helps to distinguish strains of different origins. The combined results suggest that it is difficult to attribute the strength of virulence to changes in specific genes, and the reasons that led to this down-regulation in the day-old age of naturally infected hosts of DPV need to be further analyzed. The content of this study will contribute to our understanding of genes related to DPV virulence and evolution, and provide theoretical support for vaccine studies of DPV and the establishment of methods to distinguish strains of different origins.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by China Agriculture Research System (CARS-42-19).

Author Contributions: Conceived and designed the experiments: DH YD YT WT. Performed the experiments: WT MY. Analyzed the data: WT MY MM. Analyzed the data: WT YC QW. Provided reagents/materials/analysis tools: WT BW DH YZ FW. Wrote the paper: WT.

Ethics Statement: This study was approved by the Animal Care and Use Committee of Shandong Agricultural University (permit number: 20190322) and performed in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China). All broilers in this study were bred and cared for in accordance with humane procedures.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.103919.

Appendix. Supplementary materials

mmc1.docx (11.5KB, docx)

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