NEW YORK (GenomeWeb News) – By combining sequencing, phylogenetic analysis, and epidemiology, researchers from the Novartis Institutes for BioMedical Research have identified a novel virus associated with a type of equine hepatitis whose cause has been unknown for nearly a century.
This type of hepatitis, called Theiler disease, was first identified in 1918, and it has been linked to horses that have been treated with blood products from other horses, but no study had found a chemical or pathogen cause of the disease.
As they reported in the early edition of the Proceedings of the National Academy of Sciences, researchers led by Novartis' Amy Kistler sequenced blood samples obtained from horses during a recent outbreak and homed in on a virus found in their blood and in the serum they were treated with, which they then dubbed "Theiler disease-associated virus," or TDAV. TDAV, the researchers added, appears to belong to the Flaviviridae family, though it is quite divergent from other members of that virus family.
While their subsequent inoculation study showed that horses with the virus developed liver disease, the researchers noted that their findings do not fulfill Koch's postulates "as the TDAV in the inoculum was not first purified by cultivation in vitro (because we have yet to develop suitable culture systems)."
Still, "these studies provided evidence that this previously undescribed virus, here designated 'Theiler disease-associated virus' (TDAV), could be a causative agent of Theiler disease," the researchers wrote.
The outbreak of Theiler disease the researchers investigated arose at a horse farm after 21 horses there had been treated with an equine antiserum to botulinum toxin as a preventive measure; two other horses there had fallen ill with botulism after eating contaminated feed.
Of those 21 horses, four received the same antiserum as the ill horses, while the other 17 were treated with a different antitoxin. Other horses at the farm were not treated. Eight weeks later, two horses treated with the second antitoxin became ill with Theiler disease, and other horses soon also became ill. In all, eight of the treated horses came down with Theiler disease, and all of those had been given the second antitoxin.
To search for a possible viral cause of the outbreak, Kistler and her colleagues developed and sequenced libraries from two horses with Theiler disease as well as from the second antitoxin on Illumina's HiSeq 2000 platform, generating about 22 million 100-nucleotide sequence reads per sample. After complexity and host filtering, about 0.01 percent of reads from the horse samples mapped to the Flaviviridae virus family, as did about 0.06 percent of antitoxin 2 reads. The researchers also found a smaller number of reads that mapped to an adenovirus, an insect virus, a plant virus, and to equid herpesvirus 2, which causes a mild respiratory disease.
Using the paired-end read iterative contig extension, or PRICE, assembly algorithm, the researchers assembled the viral genome into a 10.5 kilobase contig from one horse sample; assemblies developed from the other samples were nearly identical, the researchers noted.
An analysis of the TDAV genome suggested that the virus is closely related to the Pegivirus and Hepacivirus species, though it is still rather divergent from those groups, sharing only about 35.3 percent of its amino acids with its closest cousin. In addition, the TDAV genome appears to encode three putative structural proteins and seven putative non-structural proteins.
The researchers then returned to the horse farm, as well as to two others, to screen 60 horses for the presence of TDAV using a qRT-PCR assay based on a set of TDAV primers that they developed.
At the original farm, the researchers collected samples from 37 horses, 17 of which had received antitoxin 2. Fifteen of those horses had low levels of TDAV in their systems. The other two treated horses were negative for TDAV by the qRT-PCR assay using one set of primers but were positive using another set, suggesting to the authors that the horses were infected, but their viral titers were close to the assay's detection limit. No other horses at that farm, or one other farm, appeared to be infected with TDAV.
The researchers also traced the horses whose serum was used to make antitoxin 2 and tested them for TDAV. One of those three horses was positive for TDAV; when it was tested seven months later, it was negative.
"These results establish a strong association of TDAV infection with antitoxin 2 exposure and additionally suggest the virus is not highly prevalent in untreated horses," Kistler and her colleagues write. "Furthermore, the fact that TDAV was not detected in horses on [the original farm] that did not receive antitoxin 2, despite contact with TDAV-infected horses, suggests that TDAV was not readily spread between horses."
To determine whether TDAV leads to Theiler disease, the researchers inoculated four healthy horses with doses of antitoxin 2 and followed them for 14 weeks. During this time, none of the horses developed clinical signs of the disease, though one horse showed increased liver enzyme levels. The horses also showed variable levels of TDAV.
A year after the outbreak, the researchers re-tested the horses at the original farm. The two index cases were negative for TDAV as were 10 other horses that had been ill. Four horses that did not have clinical symptoms were positive for TDAV, indicating to the researchers that some horses could be asymptotic carriers and that the virus may establish a chronic infection in some animals.
The researchers argued that they have collected several lines of evidence that show that TDAV is the causative agent of Theiler disease in this outbreak. However, since the researchers were unable to purify TDAV, their study does not, as they noted, meet Koch's postulates.
But if further work confirms TDAV as the cause of Theiler disease, the authors wrote, the assay they developed would likely help screen both horses and the blood products they are treated with for the disease.
Five of the authors noted that they are co-inventors on a patent related to this work.