Development and comercial pro - duction of standardised PCR-assays for detection of haemorhagic fever viruses and variola virus and their implementation in the diagnostic service of EU P4 laboratories
The main objective of the VHF/VARIOLA-PCR project was the development and distribution of a set of high-quality, standardised, and evaluated PCR-based detection methods for viral haemorrhagic fever (VHF) viruses and variola virus. The implementation of these assays in the diagnostic service of all existing European P4 laboratories established a laboratory infrastructure that can provide high-quality diagnostic services to all EU Member States.
Furthermore, in order to facilitate wide distribution and safe handling of PCR assays for the most relevant viruses (variola virus, Ebola and Marburg viruses, Lassa virus, and CCHF virus), prototypes of ready-to-use kits were developed by a small or medium-sized enterprise (SME). They will also be made available to experienced laboratories in Member States (e.g. those of the European Network of Imported Viral Diseases (ENIVD)) that do not have direct access to P4 facilities.
A different set of PCR assays was developed for confirmatory testing. These are not produced as a kit and are to be used mainly by the P4 laboratories. The development of these assays required the concerted action of all European laboratories experienced in handling and diagnosing VHF and pox viruses, as well as of a company experienced in developing and producing PCR kits for infectious agents. Significant attention was paid to demonstration activities and interlaboratory quality control measures to ensure that the novel technologies emerging from the project are implemented in high quality in the diagnostic service of all participating laboratories.[+] Read More
Common methods for laboratory diagnosis of smallpox and VHF are isolation of the virus in cell culture or laboratory animals, polymerase chain reaction (PCR), virus antigen detection, electron microscopy, as well as detection of specific antibodies in the serum of the patient.
Virus detection and isolation in cell culture is still the gold standard for establishing a definitive diagnosis. However, it takes days to weeks to isolate a virus. Furthermore, P4 facilities are required in case of VHF and smallpox. PCR and other nucleic acid amplification techniques (NAT) meet the need to rapidly identify VHF or biological warfare agents. These techniques are suitable for identification of viruses, bacteria, parasites and fungi.
In contrast to classical diagnostic methods, which are based on the detection and identification of the intact organism, PCR detects the genetic material of a pathogen and thus reduces the contact with infectious material to a minimum. So, even laboratories of lower security level than P4 can perform PCR diagnostics for VHF and smallpox.
VHF/VARIOLA-PCR targeted the development and distribution of a set of standardised and evaluated PCR-based detection methods for VHF viruses and variola virus. In order to facilitate wide distribution and safe handling of PCR assays for the most relevant viruses (variola virus, Ebola and Marburg viruses, Lassa virus, and CCHF virus) the assays will be manufactured by the small-sized enterprise as ready-to-use kits. They will be made available to experienced laboratories in Member States that do not have direct access to P4 facilities.
Initially, assays available in the consortium were evaluated using an inactivated virus. Based on the quality assessment study and published evaluation data, assays for Lassa virus, Ebola/ Marburg virus, and Orthopox (variola) viruses were selected for further optimisation. Other assays were developed de novo.
In order to design reliable primers and hybridisation probes for real-time PCR, additional sequences of the PCR target regions were generated: 30 sequences for Lassa virus GPC gene PCR; 22 sequences for Lassa virus L gene PCR; 7 sequences for Ebola/Marburg virus L gene PCR; and 14 sequences for variola virus 14-kDa fusion protein gene PCR. Corresponding assays were revised or developed de novo using the new sequence data. Sufficient published sequences were available for designing a New World Arenavirus NP gene real-time PCR. Two reliable orthopox virus real-time PCRs were selected as second-line assays from the literature based on evaluation with a large number of Orthopox virus strains.
Real-time probe detection was developed for the following assays: Ebola/Marburg virus L gene PCR, CCHF virus NP gene PCR, Orthopox viruses 14-kDa fusion protein gene PCR, and New world Arenaviruses NP gene PCR. All RTPCR assays were developed in 1-step format. The protocols were evaluated by the consortium and integrated into the diagnostic service of the BSL-4 laboratories.
Prototypes of the Orthopox PCR kit, Ebola/ Marburg virus L gene PCR kit, and CCHF virus NP gene PCR kit were produced by artus/QIAGEN Hamburg GmbH and successfully evaluated by the consortium.
Use of the established assays in diagnostic service of BSL4 laboratories and public health laboratories in Member States.