Foot-and-Mouth Disease in Red Deer - Experimental Infection and Test Methods Performance.

The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Evaluation of two commercial, rapid, ELISA kits testing for scrapie in retro-pharyngeal lymph nodes in sheep.

AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes. METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID). RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3-75.3)% for ELISA-BR and 91.6 (95% CI=88.2-94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID. CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity. CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.

Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules.

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.