New Study May Help Improve Quality of Arbovirus Testing in Mosquitoes
Written by NEWVEC Executive Director, Dr. Stephen Rich
Arboviruses are a broad group of disease-causing viruses transmitted by insects and ticks. These include mosquito-borne agents of Eastern Equine Encephalitis virus and West Nile virus. Public health laboratories across the country test batches, also called pools, of mosquitoes for the presence of the viruses that pose a threat to the health of humans and companion animals. Many testing laboratories rely on a technique that detects the genetic material (RNA) of those viruses in mosquitoes. Generally, a tiny fraction of all mosquitoes harbor these viruses and so the tests for them must be highly specific and sensitive.
The Polymerase Chain Reaction (PCR) is a very effective means of conducting that testing. If the PCR test is positive, then there is generally high confidence that the virus is present. Given the sensitive and specific nature of PCR, the likelihood of a false-positive is vanishingly small. However, there are many reasons a PCR test could be negative even though the virus might have been present. In fact, there are two common reasons for a PCR test to yield a false-negative result. First, it might be that the RNA from the virus was damaged or lost somewhere during the process of preparing it for the PCR. RNA is a very delicate molecule and can be degraded if samples are not carefully maintained. Second, there are substances in mosquitoes, including proteins found in mosquito eyes and in remnants of the blood meal, that block the enzyme that makes PCR function. This is called inhibition because it reflects a hindrance of the PCR to detect virus RNA even when it is present. So in short, false positives are very rare, and false negatives might be more common. If false negatives are frequent, it would be possible to underestimate the amount of a virus in mosquito pools.
Connor M. Rich earned a Master of Science in Microbiology from UMass Amherst in spring of 2025. His research project was titled “Calreticulin as an Internal Control Marker to Complement Arbovirus qPCR Detection Assays.” Working under Dr. Guang Xu in the Laboratory of Medical Zoology, Connor aimed to solve this challenge of false negatives in arbovirus testing. The aim of this study was to incorporate into the arbovirus RNA assay an internal control that will simultaneously detect mosquito RNA. The internal control allows researchers to unambiguously determine whether a negative result is reliable or due to an artifact of testing. This will result in more reliable estimates of virus prevalence.
While the project goal was relatively straightforward, the execution was not so simple. To develop a new assay, it is necessary to know something about the genetic content of the mosquito. However, since little information about the common North American arbovirus vectors is available, Connor had to use molecular tools to decipher the genetic content and design his internal control assay. The results of his study will be submitted for publication this summer and, if adopted, could change the way that scientists surveil arboviruses in mosquitoes.
