Kinetics of the appearance of Marek's disease virus DNA and antigens in the feathers of chickens

A comparison was made of the temporal appearance of six isolates of serotype 1 Marek's disease virus (MDV) in the feathers of specific pathogen-free (SPF) infected birds using three assays: agar gel precipitation (AGP), enzyme-linked immunosorbent assay (ELISA) and dot-blot DNA hybridisation. Isolate GA-5 served to standardise the in vivo pathogenicity assay, while the remaining five were recent isolates from Israel. Each isolate was inoculated into susceptible 4-day-old birds housed with an equal number of uninoculated birds. All six caused high mortality (80 to 100%) in the inoculated birds and a wide range of mortality (15 to 80%) in the contact groups. The transmission of infection from the inoculated group to the contact group was demonstrated for all six isolates by AGP and ELISA and for four isolates by dot-blot hybridisation. The other two isolates either showed a concurrent rise in MDV-DNA levels (isolate B) or failed to produce detectable levels of DNA in the inoculated and contact infected groups (isolate Ab). This could be due to the nature of the hybridisation reaction between the probe and the homologous sequence in the genome of isolate Ab. Antigenic activity was detected 11 days post-injection by ELISA, 14 days by AGP in some of the inoculated groups. In the contact infected birds the ELISA and dot-blot assays detected virus about 14 days earlier than did AGP. The time interval between the first detection of virus in the inoculated as compared with the contact infected groups differed for each assay and each isolate, viz; 10 to 14 days by ELISA, 14 to 24 days by AGP and 11 to 18 days by DNA-hybridisation.     

Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein gene

The coat protein gene of isolates of citrus tristeza virus (CTV) from 20 citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR-ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridisation probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridisation assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was compared experimentally. It may be automated to the same extent as any ELISA.     

Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus

A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.     

Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus

A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.     

Trends in dengue diagnosis

The conventional diagnosis of dengue virus infections includes the detection of the virus in serum or tissue samples, both by isolation in culture or through detection of specific viral molecules (genome RNA or dengue antigens) and detection of specific anti-dengue antibodies (serology). Isolation of dengue virus provides the most direct and conclusive approach to diagnosis, despite the demand for high-level equipment, technical skills and manpower. However, it is useless in early diagnosis because several days are required to isolate and classify the virus. Serology, despite being simpler, is not able to afford an accurate early diagnosis in primary infections because 4-5 days are required for the immune system to produce a sufficient amount of antibodies. Moreover, it leads to misleading results in secondary infections owing to cross-reactivity among serotype-specific antibodies and with other flavivirus antibodies. The RT-PCR and other PCR-based techniques are fast, serotype-discriminating, more sensitive and easier to carry out than conventional nucleic-acid hybridisation, but are handicapped by easy sample contamination and high technological demands. Recently, advances in bioelectronics have generated commercial kits and new techniques for detection of dengue antibodies and RNA, based on biosensor technology. Most of them are rapid, easy to operate, reusable, cheap, sensitive and serotype-specific. Nevertheless, their accuracy is still questionable because most still lack validation and standardisation. This review summarises and describes the techniques currently employed and anticipated in the near future for diagnosis of dengue disease.