under certain conditions, one can be labeled with a donor fluorophore that will emit light of a certain wavelength. If the two proteins are farther apart than 10 nm, only the donor color will be observed. However, if the two proteins are in close contact, then fluorescence of the second protein, which is linked to an acceptor fluorophore, will take place.
Another commonly used fluorescent microscopy technique in virology is fluorescence recovery after photo-bleaching (FRAP), a method for determining the kinetics of diffusion in cells. A viral or cellular protein is labeled with a fluorescent molecule, a portion of the cell is photobleached to eliminate fluorescence, and then recovery of fluorescence is observed over time. Fluorescence in the bleached area recovers as bleached fluorophore-linked proteins are replaced with unbleached molecules from a different part of the cell.
Detection of Viral Nucleic Acids
The detection of viruses in cell cultures is being increasingly supplanted by molecular methods such as the polymerase chain reaction and high-throughput sequencing, especially for discovery of new viruses associated with human diseases. These methods can be used to identify viruses that cannot be propagated in cell culture, offering new ways to fulfill Koch’s postulates (Box 1.4).
Polymerase chain reaction. In this technique, specific oligonucleotides are used to amplify viral DNA sequences from infected cells or clinical specimens. Amplification is done in cycles, using a thermostable DNA polymerase (Fig. 2.16). Each cycle consists of thermal denaturation, primer annealing, and extension, carried out by automated cycler machines. The result is exponential amplification (a 2n-fold increase after n cycles of amplification) of the target sequence that is located between the two DNA primers.
Figure 2.15 Using fluorescent proteins to study virus particles and virus-infected cells. (A) Submandibular ganglia after infection of the salivary gland with three recombinant pseudorabies viruses, each expressing a different color fluorescent protein. Courtesy of Lynn Enquist, Princeton University. (B) Single-virus-particle imaging with green fluorescent protein illustrates microtubule-dependent movement of human immunodeficiency virus type 1 particles in cells. The cells were infected with virus particles that contain a fusion of green fluorescent protein with a viral protein. Rhodamine-tubulin was injected into cells to label microtubules (red). Virus particles can be seen as green dots (white arrow). Bar, 5 μm. Courtesy of David McDonald, University of Illinois.
Figure 2.16 Polymerase chain reaction. The DNA to be amplified is mixed with nucleotides, thermostable DNA polymerase, and a large excess of DNA primers. DNA polymerase initiates synthesis at the primers bound to both strands of denatured DNA, which are then copied. The product DNA strands are then separated by heating. Primer annealing, DNA synthesis steps, and DNA duplex denaturation steps are repeated multiple times, leading to geometric amplification of a specific DNA.
Clinical laboratories employ PCR assays to detect evidence for infection by a single type of virus (singleplex PCR), while screening for the presence of hundreds of different viruses can be accomplished with multiplex PCR. In contrast to conventional PCR, real-time PCR can be used to quantitate the amount of DNA or RNA in a sample. In this procedure, also called quantitative PCR, the amplified DNA is detected as the reaction progresses, not after it is completed as in conventional PCR. The product is detected either by incorporation of a dsDNA specific dye or by release of a fluorescence resonance energy transfer probe via the 5′-to-3′ exonuclease activity of DNA polymerase. The number of cycles needed to detect fluorescence above background can then be compared between standard and experimental samples. Quantitative PCR is widely used in research and clinical applications for genotyping, gene expression analysis, copy number variation assays, and pathogen detection. While PCR is often used to detect viral genomes in clinical specimens or during experimental research, it is important to recognize that the nucleic acid detected does not necessarily correspond to infectious virus (Box 2.8).
EXPERIMENTS
Viral RNA is not infectious virus
A study of sexual transmission of Zika virus among mice demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus particles.
Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid, by PCR to detect viral RNA and by plaque assay to detect infectious virus particles. The results (see figure) show that Zika virus RNA persisted in semen for up to 60 days, far longer than did infectious virus, which could not be detected after about three weeks.
There is a lower limit of detection of virus via the plaque assay of approximately 10 plaque forming units/ml. Whether this low concentration of infectious particles would be sufficient to transmit the virus is not known. However, it seems unlikely that these mice are able to transmit virus after a few weeks, despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection.
Recently many papers have been published demonstrating that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm by both by scientists and science writers. However, in most cases detection was by PCR, not by plaque assay, and therefore, we do not know if infectious virus particles were present. Viral RNA would not constitute a threat to transmission, while infectious virus would.
Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood—it detects nucleic acids, not infectious virus.
The lesson from this study is very clear: in novel experimental or epidemiological studies it is important to prove that any viral nucleic acid detected by PCR represents infectious virus. Failing to do so clouds the conclusions of the study.
Detection of Zika virus RNA and infectious virus in seminal fluid. Male mice were infected with Zika virus. At different times after infection, viral RNA and infectious virus particles were measured in seminal fluid by PCR (blue line) and by plaque assay (red line).
Duggal NK, Ritter JM, Pestorius SE, Zaki SR, Davis BS, Chang GJ, Bowen RA, Brault AC. 2017. Frequent Zika virus sexual transmission and prolonged viral RNA shedding in an immunodeficient mouse model. Cell Rep 18:1751–1760.
High-throughput sequencing. The development of DNA sequencing methods in the 1970s revolutionized biology by allowing the decoding of viral genes and entire viral genomes. While powerful, these methods were laborious: in 1980 it took one year for a single person to determine the nucleotide
