Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

As Frequent Travelers Go, Viruses Are Among the Least Welcome Arrivals

Increased global travel, climate change, and changes in lifestyle have contributed to the evolution of viruses endemic to tropical and sub-tropical global areas into world-traveling pathogens. And experts say the relatively recent emergence of Ebola virus, Nipah virus, Sin Nombre Hantavirus, SARS, Influenza viruses (H1N1, H7N9), and MERS viruses “clearly signify” the onset of many others in the near future.

The World Health Organization (WHO) prophetically cautioned in 2007, prior to Dengue and Zika outbreaks, that, “It would be extremely naïve and complacent to assume that there will be no other disease like AIDS, Ebola, or SARS, sooner or later,” (World Health Annual Report 2007).  Emergent and re-emergent pathogens have required the development of unique technologies to provide rapid identification of virus to identify and follow nascent outbreaks as well as guide treatment decisions.

The relatively new field of metagenomics whereby investigators can inventory the complex microbial communities found in humans, domesticated animals, wildlife, plants, and various environments, has also enabled the wider application of these technologies.

But as known and unknown pathogens create profound threats to public health, platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Existing detection technologies are based on nucleic acid amplification of sequences from one or a small set of organisms. While they are able to rapidly identify selected pathogens at the species or strain level, they cannot be multiplexed to the degree required to detect hundreds to thousands of different organisms. Microarray-based technology may hold a potential solution to these challenges by offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing.

Reporting in the Journal of Virological Methods in 2015, Glushakova  et al., described development of a novel assay that may allow routine identification of multiple viruses in a “single tube” setting . Noting that with increased multiplexing, the detection of viral RNAs has been complicated by noise, false positives and negatives, the investigators combined two new kinds of nucleic acids emerging from the field of synthetic biology.

The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables, the authors said, clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads.

When Luminex “liquid microarrays” are exploited for downstream detection, the combination of SAMRS and AEGIS supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely related viruses, such as Dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with Dengue viruses and infected mosquitoes; as few as 6–10 Dengue virions can be detected in a single mosquito.

In July 2014, researchers from Lawrence Livermore National Laboratory (LLNL) described a microbe detection array technology that could provide a new rapid method for public health authorities to conduct surveillance for emerging viral diseases.

“The disease symptoms for emerging viruses are often similar to those of other more common viruses, posing a diagnostic challenge to clinicians unfamiliar with the novel organism. “In the case of emerging viruses, it is crucial for patient treatment and for containment of a potential epidemic to quickly identify the correct virus,” the investigators noted.

The multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated using minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition, the performance of the array was tested when hybridization time was reduced from 17 h to 1 h.

The current version of the array, with 180,000 probes, can identify 4,377 viruses and 5,457 bacteria, as well as a combined total of more than 775 protozoa, fungi, and archaea species.

The LLMDA could detect unamplified vaccinia virus DNA at a concentration of 14 femtomolar (fM), or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material.

When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. While detection proved accurate, a fourfold drop in sensitivity for a 1h compared to 17h hybridization was observed. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain-level resolution.

And strain-level resolution matters.  In the case of Dengue virus (DENV), now believed to be the most common arthropod-borne disease in the world, infection with one infecting serotype (DENV 1-4 ) induces long-life protection against the original infecting strain, but confers only short-lived cross protective immunity against the other types.  While the first infection may cause minor disease, self-limited Dengue fever, secondary infections can cause fatal Dengue Hemorrhagic Fever or Dengue Shock Syndrome .

Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies, experts have said.

Writing in the journal BMC Infectious Diseases in 2015, Erik Alm and colleagues reported development and clinical validation of four DENV serotype-specific real-time RT-PCR assays for situations requiring serotyping. Like many RNA viruses, the authors explain, DENV displays considerable genetic diversity. Consequently, these serotype-specific RTPCR assays were set up in singleplex format, allowing easy modification of the individual assays when new genetic variants emerge.

But, as Amy L. Altman, Ph.D., VP, Luminex Biodefense and Protein Diagnostics, told Clinical OMICs, “The need for multiplexed assays remains. There are mosquito-borne diseases similar to Zika (ZIKV), such as Dengue, and Chikungunya, that are all transmitted by the Aedes aegypti mosquito and that are prevalent in the same areas.”

ZIKV infection in humans produces an illness clinically similar to Dengue and many other tropical infectious diseases, and as investigators have noted may be greatly misdiagnosed and underreported.  “Physicians make assumptions based on symptoms, making a clinical difference in how your you might be treated or followed. “Depending on what you really are infected with, monitoring could be very different, as in becoming pregnant while you have Zika.”

Dr.  Altman also believes it is important to distinguish between the two extant Zika strains, one originating in Asia and the other in Africa.  The outbreak in Brazil, scientists have determined, seems derived from the Asian strain, which they say, may have evolved to be better at invading nerve cells or evading the immune system.

From an epidemiological standpoint, understanding the viral lineage may help scientists map clinical manifestations to genetic changes seen in the virus.

In February, 2016, Luminex announced that it was collaborating with the Laboratory of Molecular Evolution & Bioinformatics, Biomedical Sciences Institute, University of São Paulo, Brazil to validate a multi-analyte Zika virus assay developed by GenArraytion. The MultiFLEX™ Mosquito-borne Panel, currently available as a research use only (RUO) assay,  is a multiplex panel designed to detect multiple disease agents, including the Zika virus.

Luminex recently submitted the multiplexed viral  Zika assay to the FDA for approval for emergency use. “We think it’s a benefit to do higher level multiplex testing because we can detect all the strains out there,” Dr. Altman said.

This is a digitally colorized transmission electron micrograph (TEM) of Zika virus, which is a member of the family Flaviviridae. Virus particles, here colored red, are 40 nm in diameter and have an outer envelope and an inner dense core. [CDC/Cynthia Goldsmith]

This article was originally published in the June 2016 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to

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