Molecular identification of local field isolated fowl pox virus strain from Giza governorate of Egypt

Aim: Molecular identification of field isolated pox virus from infected chickens in Egyptian farms in 2012 by polymerase chain reaction (PCR). Materials and Methods: Isolation and identification of fowl pox virus (FPV isolate ch-08TK) from 30 day-old chickens manifesting pox lesions. The isolate was propagated successfully on chorioallantioc membrane of specific pathogen free (SPF) embryonated chicken eggs and clear pock lesions were observed. These lesions were homogenated and used to infected 4.5 SPF chickens and pigeons (via wing web route for chickens and via feather follicle route into the thigh of pigeons) using 10 EID /mL; uninoculated birds of each species were used as negative controls for determining the effect of isolated FPV strain. 50 Result: The isolated strain gave pathogenic takes 100% in chickens and 75% in pigeons. Virus identification by PCR was done using dream Tag master mix kit using the primers that targeted thymidinekinase (TK) gene. These primers were designed using Lasergene DNASTAR software Version 10. We used these primers to amplify a 305 bp fragment of the TK gene of FPV. Phylogenetic analysis of sequenced TK amplicone which reflects a new emerging isolate of the field isolated FPV gave very limited similarity (not exceeding 60%) with the published sequences. Thus FPV isolate ch-08 TK gene (with Accession No. KF314718 in Gen Bank) is different than canary, Egypt, 2012, P4b and Elsharqyia-FWPV2 FPV140 (FPV140); TKPV FPV140 (FPV140) and PGPV FPV (FPV140) which have been isolated from cases of avipox virus in 2011 from skin crust of different domesticated birds reared under the Egyptian backyard management system. Our sequencing and phylogenetic analysis of newly isolated virus using DNASTAR software Version 10 revealed that this virus differ from canary, Egypt, 2012, P4b and Elsharqyia-(FWPV2 FPV140 (FPV140); TKPV FPV140 (FPV140) and PGPV FPV (FPV140)) according to published data in Gen Bank. Conclusion: FPV (isolated ch-08 TK gene with Accession number KF314718 in Gen Bank) was isolated from 30 day old laying chickens suffering from pox lesions. We believe that this study is the first molecular identification of FPV strain from laying chickens in Egypt.


Introduction
Fowl pox (FP) is of considerable economic importance as the disease can result in a drop in egg Avipox virus (APV) infection is a highly contagious production, or retarded growth in younger birds.The disease of birds and has been reported in more than 200 chances of mortality increase when the dry form occurs species of birds and affects domesticated and freetogether with the wet form [9].The general signs of the ranging birds around the world [1].APV infection is diseases include weight loss, loss of feathers and scaly caused by a large DNA virus that belongs to the genus skin of the head, neck and back.Secondary bacterial Avipox virus within the subfamily Chordopoxvirinae infections are common with both forms of the disease, in the family Poxviridae [2].Pox viruses are different having the potential to cause pneumonia or other from other DNA viruses in that the viruses replicate and bacterial infections at the site of blistering [5].Intemature in the cytoplasm of the infected cells [3].Wild gration of reticuloendotholiosis virus (REV) sequences birds [4,5] and insects [6] play an important role in the has been observed in the genome of FPV [10,11].spread of pox infection.The DNA of fowl pox virus Virulence is enhanced by the presence of REV provirus (FPV) contains approximately 288 to 300 kilo base in the genome of field strains of FPV.Complete pairs (Kbp) [2,7].The shape of pox viruses resemble sequence of the genome of a vaccine-like strain of FPV around brick [8].It is slow spreading and is charachas been determined [7,12].The virus tends to persist terized by formation of proliferative lesions and scabs in the poultry environment for extended periods of time (dry form) on skin, and diphtheritic lesions (wet form) where other viruses may not survive.In this regard the in the upper part of digestive and respiratory tracts [2].
presence of photolysis gene and A-type inclusion body gene in the virus genome appear to protect the FPV from environmental insults [13,14].Antigenic cross reactivity is observed among APVs and it appears that many genes are conserved.Restriction fragment length Pathogenicity test: Pathogenicity test was carried out polymorphism (RFLP) analysis can be used for as per method described by AAAP [23] for determining comparison of field isolates and vaccine strains of FPV the effect of isolated FPV in different species.25SPF chickens and 20 pigeons (28 days old) were inoculated [15,16].Cloned genomic fragments of FPV can be used standard procedures by using Mega DNA extraction kit Nodular lesions from infected birds were removed with (Biobasic Cat # 00086242) according to the sterile scissors and forceps by cutting deep into the manufacture procedures.epithelial tissue.The materials are ground and the 10% Virus identification by PCR: The CAMs collected at 5 W/V tissue homogenates were prepared in Hank's days post inoculation in SPF eggs were serially diluted balanced salt.Suspension is centrifuged for 10 min at and used in reverse transcriptase PCR (RT-PCR).about 700 Xg. to remove the large tissue particles.
Specific primers were chosen accordingly to the TK Antibiotics (penicillin & streptomycin) were added to gene sequence of an FPV strain [25].Direct PCR was the supernatant to give respective final concentrations done using dream Taq master mix Kit (Fermantase of 1000 IU/ml and 1mg/ml.The suspension was held at O cat#00041067) using the primers that target thymidine room temperature (25 C) for 30 min.About 0.1 ml of kinase (TK) gene These primers were designed using suspension was inoculated onto the chorioallantoic Lasergene DNASTAR software Version 10.The membranes (CAMs) of 10-12 day-old SPF embryoamplicons were electrophoresed on a 1% agarose gel nating chicken eggs obtained from SPF production and visualized under a U.V. transilluminator.A 305 bp farm; Koum Osheim; El-Fayoum, Egypt.This farm is a fragment of TK gene FPV was amplified.The primer part of the Ministry of Agriculture.Inoculated embryos o pair designated and had the following sequences: were incubated at 37 C, observed and candled for 5 ' ' FP-F 5-TAG-AAG-CAT-CCA-TGT-TAT-TACA-3 days.Five non inoculated eggs were kept as negative ' ' FP-R 5-GTT-AAG-CGC-GGC-CAC-AA-AC-3 controls.Five days post inoculation; CAMs were cut, A commercial FPV was used as positive controls in PCR.opened and the white pock lesions and generalized thickening areas were collected, pooled and used for DNA sequencing: The complete nucleotide sequence of further passages on CAMs.
the amplicon was performed in (Macrogen USA).For preparation of the gene for sequencing, the PCR Virus titration: Titration of Fowl Pox isolate was product was separated on 1% low melting agarose.The performed in SPF emberyonated chicken egg (ECE) bands were sliced off and purified with the biospin [21].Ten-fold serial dilutions of isolate were obtained PCR purification kit (Biobasic cat # BSC03S1) as and 5 embryos (10-12 days old) used for dilution were described by the manufacturer.Sequencing reactions inoculated with 0.2 ml in PBS with pH 7.2 via the CAM were performed in a MJ Research PTC-225 Peltier route.All deaths within 24 hours post inoculation were Thermal Cycler using ABI PRISM 3730XL Analyzer not considered.The survived embryos were examined BigDyeTM Terminator Cycle Sequencing Kits with for evidence of infection.Demonstrated pock lesions th AmpliTaq DNA polymerase (FS enzyme Applied or generalized thickening of the CAM, on the 5 day Biosystems), Sequencing Technology (Sanger dideoxy post inoculation ID was calculated as described earlier 50 sequencing). [22].

Phylogenetic analysis: The nucleotide sequence
Pathogenicity test: Inoculation of the isolated virus in analyses and construction of the phylogenetic trees SPF chickens revealed that about 100% showed takes were performed using Lasergene DNASTAR software at fourth day post inoculation at the sites of inoculation.Version 10.
While 15 of the inoculated pigeons showed thickening of the thighs at sites of inoculation in pigeons neither

Results
negative control chickens nor pigeons showed lesions, Egg passage: After Passage in SPF embryonated eggs, symptoms related to avian pox infection.lesions on CAMs were observed in the form of focal Discussion white opaque pocks with a generalized thickening of the CAM (Fig. 2).
Genus Avipox includes FPV or FWPV; Pigeons other species [26].Fowl pox has a world-wide strains: Following proteinase K digestion of the distribution and is caused by a DNA virus of the genus purified virions the extracted FPV DNA from a Avipox viruses of the family poxviridae [2].It is a slow representative strain VI was treated with Eco R1 and spreading virul disease of chickens.All avian species was separated on a 1-2 % agarose gel.The extracted are susceptible to avian pox as indicated by the fact that DNA was identified as the FPV genome and used for natural pox infections have been reported in several detection of FPV strains using PCR.A DNA fragment species; wild birds of different families as well as in with a length of 305 bp was amplified from FPV strain domestic birds [5, 27,28].Most of field strains contain in accordance with the reaction cycle (Fig. 3).
REV provirus.Virulence of virus is enhanced by presence of REV provirus in the genome of field strains Virus specificity and sensitivity: PCR of FPV strain VI of FPV.Restriction fragment length polymorphism was carried out by applying the primer set for testing (RFLP) analysis can be used for comparison of field the specificity of detection of viral DNA.
Uninfected CAMs were used as negative control isolates and vaccine strains of FPV [29].DNA of FPV and total nucleic acids extracted from CAMs can be detected successfully by using PCR [25,30].containing FPV were used as positive control.The Virus isolation: In this study, poxvirus was isolated on results showed that no fragment could be amplified CAMs of SPF chicken egg and further identification with the nucleic acids from any of control cells, was carried out by PCR and sequencing.This however, it could be detected with the nucleic acids technique is useful when there is only extremely small extracted from the FPV strain VI.
amount of viral DNA in the sample [21].Sample To assess the sensitivity of detection viral DNA collection was carried out according to [2].Lesions by PCR of a fragment of size 305bp was simplified in a detected in Egyptian farms in 2012 in our study agree serial 10-fold dilution of infected CAMs with strain VI.
with previously reported study [31] which isolated After electrophoresis, specific bands of expected size FPV from one month old local breed chickens in were visible in the sample.
Grenada with gross lesions in the skin of the head Phylogenic analysis of Avipox viruses TK gene: The region.Virus isolation on CAM was done [21] and we isolated strain from layer chicks is shown in (Fig. 4-6).
observed focal white opaque pocks with a generalized   thickening of the CAM (Fig. 2).Similar results have with previous studies [36][37][38].Based on the definition, been previously reported with variable levels of at least one infectious unit is required for the isolation thickening, ranging from mild to severe, in CAMs of virus, indicating that PCR may also detect infected with avian pox virus isolated from Italy [32].
noninfectious virus particles present in the sample There have been many reports that describe differences preparations.In addition, this primer set appears to be in growth characteristics of the orthopoxviruses and specific for FPV DNA, because it did not amplify the factors that influence poxvirus growth on CAMs which DNA sequences of nucleic acid preparation from include incubation temperature, age of the embryos uninfected tissue.Furthermore, nucleic acid extracted and the source of eggs; so the variability in pock color from unrelated pox virus was not amplified with this has also been ascribed to mutation of specific viral primer set.Thus, PCR seems to be rapid, specific and genes.[33].
highly sensitive and could become a powerful tool for the detection sensitivity of FPV infections [34].Extraction and detection of DNA: Results of extraction and detection of DNA of FPV strains which gave a Pathogenicity test: Inoculation of the isolated virus fragment of 305bp (Fig. 3) agree with that reported by into SPF chickens revealed that about 100% showed other authors where application of the PCR for the takes at fourth day post inoculation in wing web sites of diagnosis of FPV infection gave a positive 578bp inoculation, while 75% of the inoculated pigeons fragment [34] and with others who also used PCR for showed thickening of the thighs (sites of inoculation in identification and characterization of FPV strain pigeons).In addition, neither negative control chickens [11,35].Data obtained by others [30] also adds more nor pigeons showed lesions or symptoms related to support to our results.avian pox infection.These results indicate that the isolated strain is more related to chicken than pigeon.Specificity and sensitivity of FPV: Our results agree PPV); Turkey Pox virus (TKPV); Canary Virus titration: Isolated pox virus has 10 EID / dose.50 Pox virus (CNPV); penguin pox virus (PEPV) and Fowl pox DNA extraction and detection of DNA of FPV

Figure- 1 .
Figure-1.Head of the infected chicken:Notice that infected head showing crusts formation on the comb and on the eyes.

Figure- 2 .
Figure-2.CAM of SPF embryonated chicken eggs inoculated with supernatant fluid of samples collected from infected chicken: Notice that focal white opaque pocks with a generalized thickening of the inoculated CAM with supernatant fluid of samples collected from infected chicken.

Figure- 3 .
Figure-3.PCR of genomic DNA extracted from Pock lesion: PCR amplification: A 305 bp length amplicon was amplified either from the crusts or the propagated virus on CAM.The amplified TK conservative region from either the crusts (Lane 1) or propagated virus on CAM (Lane 2).Note that the amplicons migrate at about 305bp .M a 100 bp DNA ladder.

Figure- 4 .
Figure-4.Phylogenetic analysis of Avipoxviruses TK gene.Notice the sequence analysis of the TK gene of the field isolate of FPV

Figure- 6 .
Figure-6.The phylogenetic tree analysis of the sequenced TK amplicon of the field isolated FPV.Phylogenic analysis of Avipox viruses TK gene: Our chickens in Egypt.results are different from that previously reported on Authors' contributions inoculation of canary, Egypt, 2012, P4b virus in SPF MHHA was responsible for collection of samples from chickens where 7out of 15 chickens (about 46%) rd commercial layer chicken flocks in Giza governorate at showed takes at 3 day post inoculation into the wing end of 2012.YAS did the PCR identification and phyloweb; while none of the inoculated pigeons showed thickening of the thighs at the sites of inoculation in genic analysis.SSEM did the virus isolation, titration, pigeons [39].Therefore, in this study as well as in pathogenicity, drafted and revised the manuscript.All others [25,32,33,40], APVs from the same species of authors read and approved the final manuscript.bird are classified in different sub clades.Conversely, it Nodular lesions were collected pelleted by centrifugation at 5000 Xg for 30 min.The aseptically from a commercial layer chicken flock in pellet was suspended in 1 m M Tris-HCL pH 8.0 and Giza governorate at end of 2012.The flocks have sonicated at 80% amplitude in an ultrasonic nodular lesions on the combs; corners of the mouth and th disintegrator five times for 10 s with intervals of 30 s. around the eyelids.The lesions started from 19 day of The supernatant was collected by low speed age and with 10% mortality rates; these lesions centrifugation and suspended in proteinase K buffer persisted in the flock till 30 day of age.There was no o and digested with proteinase K (1 mg /mL) at 37 C for history of previous pox vaccination in this flock.2h.The viral DNA was extracted according to the Virus isolation using embryonated SPF eggs [19,20]: