The novel EHV-1 genotype, named H-752, doesn’t fit into either of the two currently recognized strains of EHV-1: D-752, the one that predominantly causes neurologic disease (equine herpesvirus myeloencephalitis, or EHM), and N-752, the one that causes neurologic disease less frequently. Because most diagnostic laboratories in the U.S. and abroad depend on a test that checks for one or the other, novel genotypes like H-752—which causes both respiratory and neurologic signs—can result in negative EHV-1 test results, said Nicola Pusterla, DVM, PhD, Dipl. ACVIM, professor of equine medicine at the University of California, Davis, School of Veterinary Medicine.

“Diagnosticians running molecular diagnostics need to be asking themselves, ‘Are we capturing all the EHV-1 outbreaks, independent of what genotype is causing them, with the assays we’re using right now?’” Pusterla said. “And I would say, for the labs that are using allelic discrimination assays that only screen for N- or D-752, they will miss the new genotype, H-752.”

Laboratories would find EHV-1 variants more successfully by targeting a universal gene for EHV-1 that’s independent of the genotype, he said.

Routine Health Monitoring, Less Limiting qPCR Lead to Early Outbreak Discovery

Veterinarians discovered the H-752 variant at an early phase of a March 2021 outbreak thanks to basic monitoring techniques—mainly taking rectal temperatures of horses that otherwise appeared healthy, said Pusterla.

Two of his co-authors were performing routine care on 31 horses at a show barn in Pennsylvania when they realized 10 of the horses had mild fevers. These horses acted healthy and alert, he said. To understand what was going on, the veterinarians took blood samples and nasal swabs to test for equine influenza virus, equine rhinitis A and B virus, and EHV-1 and -4 using conventional quantitative polymerase chain reaction (qPCR).

Eight of the qPCR tests came back positive for EHV-1, he said. But the results were odd: They only showed positive results with the gB gene target assay (universal gene of EHV-1), whereas the more specific qPCR tests focusing only on the ORF-30 gene—which checks for N-752 or D-752—were negative. And because most laboratories use the ORF-30 test exclusively, they would have seen only negative results for EHV-1 in these horses, he said.

The researchers observed all the horses for the next 35 days. During that time 26 developed fever lasting up to eight days, Pusterla said. Some lost their appetite, had swollen lower legs, and/or developed mild ataxia (incoordination), weakness, or problems with proprioception. By the 35^th day, all the horses had tested positive for EHV-1 with qPCR with the same odd result. They all received the antiviral valacyclovir and, if they had fever, flunixin meglumine and sodium heparin. Viremia dropped from 42% to 0% in a week on this regimen, he said.

Pusterla’s team genotyped the isolated viruses and found they were dealing with a “new” strain—but only in the sense that it had never before been identified in the U.S. “There was a 2018 outbreak in France with the same genotype, so it’s not ‘new’ in that sense,” he said.

Further analyses revealed the mutation occurred at the site of the ORF-30 gene’s Nucleotide 2254—the same spot that discriminates between the classically recognized neuropathogenic (D-752) and non-neuropathogenic (N-752) genotypes.

The researchers traced the U.S. outbreak back to one mare at the barn that had been hospitalized for unrelated reasons at a nearby clinic; other horses at that clinic had later also shown signs of EHM, and two had been euthanized as a result, Pusterla said.

The 2018 French outbreak also included neurologic cases, including one horse that had to be euthanized, he said.

The early use of valacyclovir in the Pennsylvania outbreak might have significantly contributed to a reduction in neurologic problems, said Pusterla.

All the horses had been vaccinated regularly for EHV-1, which probably also helped keep signs relatively mild, he added. While vaccines do not necessarily stop horses from getting or spreading disease, they can help the animals fight the virus better and have less severe clinical signs.

“So the question about H-752 now is, how prevalent is it?” he said. “And nobody really knows. Because until (recently), nobody was really looking at it. So we truly don’t know how many have we missed.”

Take-Home Message

The findings underscore the importance of using diagnostic techniques in the laboratory that are not limited to known variants, he said.

They also highlight the strength of a very simple tool: the thermometer. Elevated rectal temperatures among healthy-appearing show horses clued veterinarians in to the fact that an infection was brewing, well before it caused significant damage.

“These were not horses that had their heads on the ground or were spewing nasal discharge,” Pusterla said. “These were horses that were just looking normal but happened to have elevated rectal temperature.”

Some of those horses would likely have shown clinical signs as time passed, he added. “The barn managers would have been calling the veterinarian a week or 10 days later when they suddenly had three or four horses with neurologic deficits,” he said.

Early intervention and good basic monitoring helped keep the outbreak, and its effects, under control, he said.

“Everybody wants to look for the magic bullet—this or that test,” he said. “But taking rectal temperature is still a very powerful tool.”

Ideally, horses would have thermometers integrated with their identity microchips to allow for quick daily scanning of all horses in barns, he said, something that could soon become a reality.