MOLECULAR CHARACTERIZATION OF DUCK PLAGUE VIRUS FOR DETERMINATION OF TCID50

Online May, 2021 --------------------

Duck plague is an enveloped DNA virus that belongs to the Anatid Herpes Virus; the Herpesviridae family is an acute and highly infectious duck, geese, and swan disease that causes tremendous economic losses of duck rearing in Bangladesh and other duck rearing countries. Therefore, we decided to isolate duck plague virus from recent fields' outbreaks area and performed molecular detection and phylogenetic analysis to find out the similarities between our findings and other isolates around the world. Visceral organs of 13 suspected ducks from recent outbreaks area were collected by post-mortem examination for inoculum preparation. Several passages were performed to harvest into 9-11 old embryonated eggs Chorioallantoic membrane (CAM) route and duck embryo fibroblast (DEF) primary cell culture. DNA polymerase (446bp) and DNA polymerase (UL, 602bp) genes were used for molecular detection by Polymerase chain reaction (PCR). Pathogenicity was done with duckling and TCID50 on DEF. Molecular characterization was performed from extracted DNA of duckling and 2 Positive PCR products were partially sequenced for phylogenetic analysis of their origin and nucleotide variations. Sequenced data was analyzed to reveal genetic relationships among constructed phylogenetic tree for understanding potential transmission with origin of virus and data was then submitted to gene bank and got accession number for DPV-BR1-MN937272 and DPV-BR-2-MN937273. Among 13 samples, 4(30.77%) were found positive by PCR using DNA polymerase at 446 bp and UL at 602 bp gene. Chorioallantoic membrane (CAM) was observed hemorrhagic after 72 days and duck embryo fibroblast (DEF) become round as showed cytopathic characteristics after 48h of infection .Duckling showed that isolated virus was highly pathogenic as characteristics signs of post-mortem examination. Therefore, this has found that recent isolates have similarity with Bangladesh, India and China isolates. Moreover, TCID50 has confirmed the isolates have accepted titer to be a vaccine strain. In the present study, the DPV isolated from the recent field outbreaks using duck embryo and DEF cell culture systems for biological and molecular characterization of isolated DPV to develop a vaccine from the local strains.

Study of outbreaks area
The research work was conducted the study from June to September 2018 in two unfamiliar areas of theMohanganjUpazila of Netrokona(24°34.5′N90°23.5′E), and TrishalUpazila of Mymensingh districts (24°34.5′N90°23.5′E). Where active affected areas have been evaluated by regular observation and contact with veterinary professionals working in veterinary clinics of the district government.Besides this field we conducted investigations based on information on previous vaccination history and clinical signs where an active outbreak of DPV was reported. During we made sample collection visual inspection to observe typical clinical signs of DPV and it did a detailed physical examination on affected ducks.

Methods of sample collection
After taking the permission from the owners, we collected 13 dead and live ducks from the commercial farms and households. It

Preparation of inoculum and sterility test
Each sample was crushed under sterile conditions prior to the analysis of collected samples according to a 10 % suspension using phosphate buffer solution for preparation (PBS) (Manual, 2012). Then the suspension was centrifuged at 6000 rpm for 10 minutes and it collected supernatant in a sterile falcon tube treated with 100 μg/ml antibiotics (Gentamicin). The sterile suspension was examined for sterility in fresh nutrient and blood agar as described at 37°C for 24 hours. Then stored the prepared sterile solution at -20°C for until use(Ahamed et al., 2015a).

Propagation of DPV in embryonated duck eggs
It propagated the antibiotic-treated suspension containing viruses in the allantoic cavities of 10-day-old embryonated duck eggs through CAM route (Liu et  Inoculated eggs were incubated at 37°C with proper humidified condition and observed twice daily for mortality of the embryo. The embryos that died within 72 hours were discarded as nonspecific deaths (Manual, 2012) . After 6-10 days of inoculation, it chilled embryonated eggs at 4°C for overnight and allantoic fluid (AF) and CAM were collected.

Isolation of Duck Plague Virus in duck Embryo Fibroblast (DEF) cell culture
Duck embryo fibroblasts (DEFs) cells were prepared freshly as described (Shi et (Table 1) and it performed PCR for amplification. For amplification of DNA polymerase gene and UL gene, total 25 µl PCR mixture was prepared containing 12.5 µl master mix (Promega-Madison, WI, USA), Nuclease free water 1.5 µl, Primer Forward 2 µl, Primer Reverse 2 µl and Template (Extracted DNA) 7 µl. Thermal condition used for DNA polymerase gene amplification was: initial denaturation at 94°C for 4 min, 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 7 min. Further, the PCR condition for UL gene amplification was: initial denaturation at 94°C for 5 min; followed by 30 cycles of reaction denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension 72°C for 30 sec, and a final extension at 70°C for 7 min and stored the amplified PCR products at -20°C until confirmed by gel electrophoresis.

Determination of DPV from PCR products by agarose gel electrophoresis
The amplified PCR products were examined by using agarose gel electrophoresis. For UL gene confirmation 1%

Pathogenicity test in adult duckling
A small group of 4-week-old adult ducks was used for these studies. The ducks were inoculated with 1 mL 106.166 TCID50/mL serum-free minimum essential medium diluted virus through the subcutaneous and intramuscular route under the wings at several sites (Liu et al., 2011).Few adult ducks were kept separately for control and each of them inoculated with 1 ml culture medium through the same route. After inoculation, ducks also observed two weeks for clinical signs of disease, and it happened postmortem to re-isolate DPV, which was confirmed by PCR.

Sequencing data analysis and construction of phylogenetic tree
For the further experiment, two positive PCR products of UL gene (602 bp) were sent to Invent Technology Ltd. for partially sequencing. The nucleotide sequence generated in this study has been deposited in the GenBank to obtain accession number. Data analysis and multiple alignments were performed using Codon Code analyzer and MEGA X software. The sequence was compared with the GenBank database using the BLASTn. The evolutionary history was inferred with the Phylogenetic tree using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) and are in the units of the number of base differences per site. This analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 132 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Nei and Kumar, 2000).

Isolation of DPV
Among the 13 DPV suspected cases 4 ducks (30.77%) were found positive for duck plague which propagated and subsequently confirmed by PCR using specific primer. Three ducks of Mymensingh and 1 duck of Netrokona were confirmed as positive for duck plague virus by PCR (Table 3). Overall prevalence rate of duck plague virus was 30.77% where 33.33% was in Netrokona, and 30% in Mymensingh district (Figure 4).

Molecular detection of viral DNA by PCR
The desired band positive at specific 446 bp and 602 bp were observed for DNA polymerase primer (446bp) and UL (602bp) gene primer, respectively which was the indication of duck plague virus.

Propagation of DPV in embryonated duck eggs
The inoculated embryonated eggs death began after 3-5 days of inoculation. The embryos showed dwarfism, subcutaneous hemorrhage in legs, head, neck and abdomen. The CAM was found with hemorrhagic lesions throughout the membrane and irregular congestion resulting in thickening. Besides, positive control of uninfected eggs remains alive without no hemorrhagic lesions on CAM and PCR showed positive results.

Isolation of Duck Plague Virus in duck Embryo Fibroblast (DEF) cell culture
DEF adapted dpv was observed after 72h of infection under inverted microscope that appeared 90℅ of cell died and detached from the cell culture plate surface. On the other hand, positive control plates remain confined to the cell culture plate surface. The cells changed their morphology by clumping and rounding from their normal spindle like shapes. Therefore, it formed large intracellular vacuoles and cytoplasmic granulations. Molecular detection by PCR of adapted DPV showed positive.

Determination of TCID50
After 48 observations of inoculated wells were found the DEF cell was infected with the DPV and showed cytopathic effect under the inverted microscope. The result was evaluated followed by Reed & Muench (1938) ( Table 2) and found the 50% endpoint 0.166 at 10 -5 dilution. Therefore, the titer (TCID50 /mL) of the virus suspension found 10 6.166 /mL.

Pathogenicity test in adult duckling
The infected group ducks revealed typical clinical signs and symptoms after 10 days of post-inoculation, which comprised paralysis of legs, dropping wing, back arched position, greenish diarrhea, soiled vent, pasty eyes, depression, loss of appetite, swelling of the head and neck. During postmortem examination, body cavity was found filled with blood and it observed hemorrhage on the esophagus. The liver was enlarged with blood spots and gray-white areas on the surface. Moreover, hemorrhage in the bursa of Fabricius, annulus trachealis and intestine with annular band was observed. Besides, the positive control remains healthy and found no postmortem lesion. It showed the confirmation of DPV by PCR on targeted gene location.

Study of sequence and phylogenetic analysis
The obtained two partial nucleotide sequence data of UL gene were submitted to GenBank and got accession number MN937272 for DPV-BR1 and MN937273 for DPV-BR-2. After submission of nucleotide sequence data, it confirmed that our isolated viruses are DPV. Analysis of acquired data and phylogenetic tree in comparison with other sequences of

DISCUSSION
DPV is a highly contagious, and fatal disease among the waterfowls that has relatively high mortality and broad host range (Li et al., 2016, Ager-Wick et al., 2018. Due to high mortality, morbidity rates DPV became one of the most widespread and devastating diseases that causes enormous economic losses in commercial waterfowl-based industry throughout the world (Sandhu and Shawky, 2003, El-Samadony et al., 2013, Li et al., 2016. In Bangladesh rural farming has become an integral part but DPV has caused tremendous losses every year due to lack of vaccination and poor immune responses through the several imported vaccines. Thus, in the duck's industry DPV Control became one of the serious challenges, therefore, using quality vaccines can prevent this deadly duck disease. This recent work was carried out to isolate and molecular characterize duck plague (DP) virus by partially sequencing local strain DP virus vaccine seeds from recent field outbreaks in Bangladesh for the production of vaccine seeds.
During this investigation, 13 suspected dead ducks were collected from DPV affected farms of Netrokona, and Mymensingh for virus isolation. Among the DPV suspected cases overall prevalence rate was 30.77%, which was similar to ( Postmortem examination found hemorrhagic intestine and esophagus, body cavities full of blood, enlarged blood spotted liver as found in previous research work (Li et al., 2016, El-Samadony et al., 2013, Huang et al., 2014. Ducklings were also confirmed by PCR. In this research molecular detected viruses with UL gene were partially sequenced and obtained GenBank accession number MN937272 for DPV-BR1 and MN937273 for DPV-BR-2 respectively. After analyzing sequenced data and constructed phylogenetic trees showed that our isolated virus was DPV and which is genetically 100% similar to LC105645 in Vietnam, KX768734 in Bangladesh, KX511893 in India and EF643559 in China and other has 98-99% genetically relationship.