Assessing rapid viral enumeration/detection systems

By Dr. Ray Nims

In a previous posting, we alluded to the recent availability of rapid methods for identification of viruses. These technologies, together with rapid methods for enumerating viruses, should greatly expedite the quantification and identification of viruses (and bacteriophage) as compared with the existing cell culture-based approaches.

Rapid enumeration technologies are intended to replace the cell-based infectivity endpoints such as plaque assays or tissue-culture infectious dose assays, which typically require 7-10 days for completion. The use of the rapid methods may be appropriate in cases where it is not necessary to determine the infectious titer of a virus stock. An example of this might be for monitoring the amplification of viruses for preparation of live or subunit vaccines. The particle enumeration technologies include those that specifically measure viral particles and those that measure particles in general. As shown in Table 1, the particle enumeration technologies are not specific to any given virus. These are not generally useful, therefore, for viral identification, although the particle detection method associated with the NanoSight system does allow for sizing of the particles. Viral particle size is a key attribute to be aware of when, for instance, attempting to identify an unknown viral contaminant.

Table 1. Characteristics of rapid viral enumeration/identification technologies

The quantitative polymerase chain reaction (Q-PCR) and universal biosensor (Ibis T5000) technologies represent approaches that are capable of providing information both on the relative quantity of a virus in a sample and its identity. The important difference between the two is that in the former case (Q-PCR), the user is typically evaluating the identity and or quantity of a virus which is reactive with the specific primers and probes used in the assay. From an identification standpoint then, the Q-PCR technique has typically been used to confirm whether an unknown virus is related to the virus for which the assay primers and probes was designed. The degree of relatedness required is determined by the specific primers and probes used in the assay, and may be either to the genus level or the species level. Efforts are being made to incorporate primers for more highly conserved sequences to allow for more broad coverage in Q-PCR assays intended for viral screening. In the case of the universal biosensor (Ibis T5000), an unknown virus in a sample may be simultaneously identified and quantified, as long as the virus is or is closely related to one for which mass spectrometry information is present in the software used for assay analysis. Quantification in either case is in genomic units, and as with the particle enumeration methods, the readout of the quantitative nucleic acid methods does not indicate whether the virus detected is infectious. An additional nucleic acid-based method that may prove useful, in cases where relatively rapid identification of an unknown viral contaminant is needed, is deep (massively parallel) sequencing. This method is more labor intensive (and perhaps costly) then the other quantitative nucleic acid methods described above, but has the advantage that it can provide information regarding the completeness (partial vs. full-length) of the viral genomic sequences detected. This approach has displayed utility in identifying a novel picornavirus in harbor seal samples, porcine circovirus in rotavirus vaccines, and a new parvovirus in bovine serum.

Microarray screening is a technology that may be used to rapidly identify (but not enumerate) an unknown virus in a sample, provided that a probe for the virus is part of the microarray chip. Some microarray chips intended for viral identification also contain probes for conserved viral genomic sequences. In this case, the microarray may identify a novel unknown virus, at least to the genus level. As with the other rapid methods that are based on presence of specific genomic material, the assay cannot discriminate between infectious and non-infectious virus.

See Table 1 for some of the important characteristics and limitations of each method. The use of the rapid methods discussed above and in Table 1 should reduce the time needed for viral quantitation from weeks to hours, and for identification of an unknown contaminant in a sample from months (or years) to one or more days. This should greatly facilitate the monitoring of viral proliferation in manufacturing processes and the investigation of viral contamination events.