Duck viral enteritis is a disease of waterfowl (duck, goose, and swan) caused by duck enteritis virus (DEV), an alphaherpesvirus. DEV causes considerable economic losses to the commercial duck industry due to mortality and decreased egg production (12). In China, the virulence of DEV has increased in ducklings and geese, and recent reports indicate that the current DEV vaccine lacks long time efficacy. These findings suggest that DEV is constantly mutating (18).
The viral genome of DEV comprises a linear and double stranded DNA genome of approximately 180 kb with a structure similar to that of other alphaherpesviruses. Some glycoprotein genes of DEV have been previously characterized and studies of the gH, UL24, TK, gC, and UL5 genes suggest that DEV should be classified into the subfamily of Alphaherpesvirinae (13, 14, 17), and is more closely related to members of the Mardivirus genus than to other herpesviruses.
Among the herpesviruses, UL4 encodes a non-essential and non-structural protein that enhances virion formation and does not show apparent function in infected cells in culture or in experimental animal systems (2, 7, 10). In other studies, it was reported that UL4 was encoded by a 0.8 kb mRNA (19) and the product of the human herpesvirus 2 (HHV-2) UL4 gene was a very late (γ2) protein that accumulates in the cytoplasm of infected cells observed as punctate structures in the nucleus in late infection (23). It was also found that synthesis of the UL4 protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis (6). However, there are no reports on nucleotide sequence and any function of the DEV UL4 gene. As a result, we could not clone the DEV UL4 gene based on previous studies. Entire genomic comparison revealed that the gene order UL6-UL5-UL4-UL3 is conserved amongst genomes of most alphaherpesviruses, in which UL4 is located between UL5 and UL3 genes (1, 3, 11). This genetic order made it possible for us to design a degenerate primer in a conserved region of the UL3 gene to amplify the DEV UL4 gene.
The objective of the present study was to clone and sequence the UL4 gene of the Chinese DEV vaccine strain by polymerase chain reaction (PCR) using a specific primer and a degenerate oligonucleotide primer. The sequence obtained was compared with those of other herpesviruses. These data are useful for further study of molecular biology and accurate classification of DEV.
A vaccine DEV strain (No. 020318) was provided by Zhejiang Academy of Agricultural Sciences of China and deposited at our laboratory.
The virus was propagated in the allantoic membrane of 11-day-old non-immunized duck embryos by standard methods (22). The preparation of DEV DNA was performed as described by Hansen et al (9). Extracts of normal allantoic fluid were prepared and used as negative controls.
Primers SP1, DP1, P1 and P2 are shown in Table 1. Primer positions are indicated with arrows. (Fig. 1). The conserved sequence of DEV UL3 gene was shown in Table 2. All primers were synthesized by Invitrogen Co., Ltd. (Shanghai) and were suspended in double-distilled water to 50 pmol/μL concentration upon arrival.
Table 1. Primers used in PCR
Figure 1. The PCR amplification of the UL4 gene. The genomic structure scheme presented here is drawn according to human herpesvirus 1. Primer positions and amplification directions are indicated with arrows.
Table 2. The conserved region of herpesvirus UL3 genes that were used to design primers DP1
The primers SP1 and DP1 were used to amplify the sequence of the UL4 gene and partial sequence of the UL3 gene. PCR was performed in 50 μL volumes containing 5.0 μL of 10× PCR buffer (100 mmol/L Tris-HC1 (pH 8.3), 500 mmol/L KC1), 5 μL of 25 mmol/L MgCl2, 8 μL of 2.5 mmol/L dNTP mixture, 1 μL of SP1, 5 μL of DP1 and 0.5 μL of Ex Taq DNA polymerase (TaKaRa). The PCR parameters were 4 min at 95 ℃, and 30 cycles of 50 s at 94 ℃, 1 min at 50℃, 2 min at 72 ℃, and a final extension time of 10 min at 72 ℃. The PCR product was visualized on 1% agarose gel electrophoresis containing 0.8% μg/mL ethidium bromide. The negative control was performed under the same conditions.
The PCR product was purified using a Mini Gel kit (TaKaRa corp). Purified product was cloned into pMD18-T vector and transformed into Escherichia coli (E. coli) DH5α competent cells. The recombinant clones were selected by the Amp/IPTG/X-Gal agar plate. All reagents and vectors were purchased from TaKaRa corp. Sequencing reactions were performed by the Shanghai Invitrogen Co., Ltd. At least two recombinant plasmid sequences were 100% matching, so the nucleotide sequence of target fragment was finally confirmed.
The DNA sequences were analysed using the DNAStar sequence analysis software (version 7.0). One complete open reading frame (ORF) was found, hence P1 and P2 (Table 1) were used to amplify the only complete ORF of the DEV UL4 gene. The PCR product was cloned into pMD18-T as described above.
Comparison and phylogenetic analysis of the nucleotide sequences and deduced amino acids of the DEV UL4 gene with those of other herpesviruses (Table 3) were performed with DNAStar and BLAST. Phylogenetic analysis was performed using the MegAlign program in Lasergene (DNAStar 7.0) with Clustal Ⅴ multiple alignment and weight matrix PAM250 of sixteen reference strains of alphaherpesvirus (Table 3), reliability of the tree was assessed by bootstrap analysis with 1000 replicates.
Table 3. Reference sequences of alphaherpesviruses used for comparison and phylogenetic analyses