Filoviruses could cause severe viral hemorrhagic fever in human and other primates leading to 50%-90% mortality in patients; however, so far no specific and efficient vaccine has been synthesized , nor have other treatment methods proved to be effective [10, 20, 24]. The pathogen belongs to the order Mononegavirals, family Filoviridae, which further divides into two genera: Ebola virus (EBOV) and Marburg virus (MARV). The first outbreak of Ebola virus was recorded in Zaire, sub-Saharan Africa and four species have been identified: Zaire ebolavirus, Sudan ebolavirus, Ivory Coast ebolavirus and Reston ebolavirus. Its counterpart, the MARV, has only one species, Lake Vicoria marburgvirus isolated in 1967 (http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB). Since EBOV and MARV cause serious diseases with high morbidity and mortality, these viruses are classified as the BSL-4 pathogens and can cause significant threats to public health and affect the economic growth on a global scale due to the increase of international trade and traveling as a consequence of globalization. Consequently the viral pathogen might be able to pass the borders and spread across different countries. Their existence as endemic disease threats and as potential biological warfare weapons suggests that it is of great importance to detect these pathogens specific, rapidly and sensitively. Here, we summary the recent progress in the development of detection and diagnosis methods for EBOV and MARV and mainly focus on virus isolation, electron microscopy, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).
The polymerase chain reaction (PCR) has been used for detecting a wide variety of pathogens across a range of research fields, including virology. In recent years, real-time PCR is widely used in the fields of pathogen detection and diagnosis because of its advantages of speed, simplicity and high sensitivity. It has revolutionized molecular diagnostic technologies. In the past decade, many PCR detection systems have been developed for diagnosis of EBOV and MARV (Table 1).
Table 1. RT-PCR amplification systems for the detection of EBOV and MARV
The use of traditional RT-PCR to detect acute EBO-Z virus disease in humans and animals were developed by Sanchez et al for identifying outbreaks and supporting epidemiologic investigations. RT-PCR assays were proven effective for detecting viral RNA in body fluids and tissues of EBO-Z infected individuals . The first field evaluation of RT-PCR for EBOV disease was reported by Leroy . They used the same primer set as Sanchez et al to detect EBOV viral RNA in peripheral blood mononuclear cells . Twenty-six laboratory confirmed patients of EBOV hemorrhagic fever were studied. The results were compared with ELISA antigen capture, and EBOV specific IgM and IgG antibody. The one-step, real-time RT-PCR method were developed by Drosten et al with the utilization of the DNA-interacting dye SYBR green I using the primer set of Sanchez et al . It demonstrated that RT-PCR was the most sensitive method and able to detect the viruses from early acute disease through early recovery.
In order to address the challenge of diversity of filoviral genomes in PCR-based assays, Zhai et al. developed a consensus PCR method similar to Sanchez et al , which utilized a cocktail of specific primers in a one-step RT-PCR. It yields a long product that could be sequenced for automated phylogenetic analysis. This allowed a more accurate placement of newly identified filovirus positive samples relative to existing species, lineages, and strains.
The introduction of real-time RT-PCR allows detection of filoviruses to be carried out with minimal manipulation and equipment, and can provide results in less than two hours . It may prove to be a useful diagnostic tool for control and management of future outbreaks. Highly sensitive and specific primers and probe sets were reported by Gibb et al [7, 8]for Real-time RT-PCR amplification of MARV and EBOV sequences and targeted genes, respectively. The newly designed primer and probe sets for MARV was equivalent to, or 10 to 100-fold more sensitive than previously designed primer sets. It was able to detect all stains of MARV . Meanwhile, one-tube real-time RT-PCR assay for identification of EBOV-Z and EBOV-S was developed. One common primer set and two differentially labeled fluorescent probes were used to simultaneously detect and differentiate the two subtypes of EBOV. They were unique in their abilities to simultaneously detect and differentiate EBOV-Z and EBOV-S . Rapid detection protocol for filoviruses using TaqMan RT-PCR for detection MARV and EBOV were developed after the series of studies mentioned above . Three primer and probe sets were designated into blocks of nucleoprotein gene conserved sequences of MARV, EBOV-Z and EBOV-S. All three assays were highly sensitive and specific and able to detect and identify filoviruses at an early stage of a suspected filovirus disease.
In the largest outbreak of Ebola hemorrhagic fever to date that occurred in Uganda from August 2000 to January 2001, a nested RT-PCR, combining with two-step real-time RT-PCR and one-step real-time RT-PCR was used in field diagnosis . The greatest value of early case identification in the real-time RT-PCR-based assay was clearly demonstrated by its ability to identify patients. It gave earlier identifi-cation than any other available tests. Recently, in the outbreak of a large hemorrhagic fever which was investigated by the Centers for Disease Control and Prevention in northern Angola, MARV was confirmed as the cause of the outbreak. The primer and probe set was newly designed specifically for the VP40 gene sequences in the outbreak . As well as in the field diagnosis in Uganda mentioned above, real-time RT-PCR assay outperformed all other assays designed to detect acute MARV infection, including the "gold standard" virus isolation assay. It was also sufficiently robust to allow deployment into a field setting in Angola. The extensive virus genomic analysis in this study also confirmed that the VP40 gene target of MARV was an excellent choice for a broadly reactive MARV detection assay. The performance of real-time RT-PCR in such outbreaks suggests that it should be a highly useful assay for detection of potential naturally occurring or terrorist activities outbreaks in the future.
The first industry-standard molecular assay for all filoviruses species was designed by Panning et al . They developed a diagnostic real-time RT-PCR reaction kit for filoviruses based on the strain collections of all participants in the network of European biosafety level 4 laboratories. The kit facilitated reliable detection or exclusion screening of filovirus infections. Meanwhile, they also stated that any kit like this can never be guaranteed that a PCR assay will detect unknown filovirus strains that may emerge in the future.
To conclude, there exist many methods for detection and diagnosis of filoviruses. Among them, PCR methods have shown their values for early detection of the viruses due to their high sensitivity and fast speed. However, due to continuous mutation of the viruses, it is expected that the more traditional methods, such as virus isolation and electron microscopy, would also need developing for better identification of filoviruses during future outbreaks.