Research Note: Simultaneous detection of infectious laryngotracheitis virus, fowlpox virus, and reticuloendotheliosis virus in chicken specimens.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

[Construction of recombinant fowlpox virus coexpressing HA gene from H5N1 avian influenza virus and chicken interleukin-2 gene and assessment of its protective efficacy].

The hemagglutinin (HA) gene from H5N1 avian influenza virus and the chicken interleukin 2 (chiIL-2) gene were inserted into a expressing vector p12LS to construct a recombinant transferring vector p12LSH5AIL2, in which HA gene under the control of the promoter Ps was in inverse tandem connection with the chiIL-2 gene under the control of the promoter PE/L. The p12LSH5AIL2 was then used to transfect the chicken embryo fibroblasts (CEF) pre-infected with a wild-type fowlpox virus 282E4 strain, to generate a recombinant fowlpox virus coexpressing the inserted HA and chiIL2 genes (rFPV-H5AIL2). The rFPV-H5AIL2 was obtained and purified by blue plaque screening on the CEF. The in vitro expression of HA gene by rFPV-H5AIL2 was detected in the recombinant fowlpox virus-infected CEFs with an indirect immunofluorescence assay, and the expression of the chiIL2 gene by rFPV-H5AIL2 was confirmed by detection of the chiIL2 mRNA by RT-PCR and by detection of chiIL2 by the indirect immunofluorescence assay. Experiments on SPF and commercial chickens demonstrated that the titer for HI antibodies induced by the rFPV-H5AIL2 was significantly higher than that by the rFPV-HA. The group immunized with the rFPV-H5AIL2 exhibited the similar ratios of protective efficacy and virus shedding as the group immunized with the rFPV-HA in SPF chicken. However, in commercial chicken, the group immunized with the rFPV-H5AIL2 generated significantly higher protection against H5N1 avian influenza virus challenge and lower virus shedding than the group immunized with the rFPV-HA. This study paved the way for further development of a new AIV recombinant vaccine.

Quantitative assessment of poxvirus promoters in fowlpox and vaccinia virus recombinants.

A comparison was undertaken of poxvirus promoters in vaccinia and fowlpox virus (FPV) recombinants using the level of beta-galactosidase expressed from the LacZ gene as a measure of promoter function. In this study a comparison was made of the vaccinia virus promoters, P 7.5 and P L11, the major late promoter of cowpox virus, P CPX (expressing the abundant inclusion body protein), and the FPV promoters, P E/L and P L. In vaccinia virus recombinants the FPV P E/L promoter expressed one-third to one-half the level of beta-galactosidase expressed by the P L11 promoter. In comparison with the P 7.5 promoter, the FPV P E/L promoter expressed four to five times the level of beta-galactosidase. In FPV recombinants beta-galactosidase activity expressed was equal for the P E/L and P CPX promoters. Levels expressed by P L11 and P L were one-half and one-fifth that level, respectively. The temporal regulation of the promoters was maintained in both vaccinia virus and FPV recombinants. The P E/L promoter of FPV has the TAAATG sequence characteristic of late poxvirus promoters at the transcription initiation site. In an attempt to enhance the utility of this promoter for the expression of foreign genes in FPV and vaccinia virus recombinants, the effect upon promoter function of changing the G of the ATG to A, T, or C was determined using transient expression assays with vaccinia virus. Substitution of A, T, or C for the G abolished promoter function. Because of its early/late function, the level of expression and the presence of the oppositely oriented late P L promoter, the FPV P E/L promoter will be valuable for the expression of foreign genes in poxvirus recombinants.

Transient expression assay for qualitative assessment of gene expression by fowlpox virus.

A transient expression assay for fowlpox virus (FPV) was developed to assess the feasibility of using heterologous promoters in FPV and to qualitatively determine relative promoter strength. A transient expression system for FPV has not been reported, and various methods used for transient expression in vaccinia-virus-infected cells produced negative results when used with FPV. Here a successful method for transient expression of E. coli beta-galactosidase in FPV-infected chick embryo fibroblasts is reported. This transient expression assay has been developed to qualitatively assess promoter recognition and gene expression by FPV. It should also prove useful in the identification of promoters from the FPV genomic library and in testing the accuracy of chimeric promoter-gene constructs.