Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus

Abstract

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the central nervous system (CNS), were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, VEEV genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates

“…It is anticipated that virus stocks derived from the new human isolate clones could be used as challenge material to satisfy the FDA “Animal Rule” during pre-clinical testing of anti-alphavirus vaccines and therapeutics [ 9 ]. We recently published a detailed examination of the disease phenotypes of the VEEV and EEEV cDNA-derived viruses in the cynomolgus macaque model for these purposes [ 34 , 43 , 44 , 45 ]. Furthermore, in the macaque studies, we sequenced the genome of NHP stock virus generated from the INH9813 clone and the sequences were identical, suggesting that our stock production conditions do not alter genotypes [ 34 ].…”

“…We recently published a detailed examination of the disease phenotypes of the VEEV and EEEV cDNA-derived viruses in the cynomolgus macaque model for these purposes [ 34 , 43 , 44 , 45 ]. Furthermore, in the macaque studies, we sequenced the genome of NHP stock virus generated from the INH9813 clone and the sequences were identical, suggesting that our stock production conditions do not alter genotypes [ 34 ].…”

“…Virus genomic RNA was generated from linearized cDNA templates by in vitro RNA synthesis (mMessage Machine, Invitrogen, Waltham, MA, USA) followed by electroporation of BHK cells and harvesting of supernatants at 24 h post infection (as previously described [ 34 ]). Supernatants were clarified by centrifugation, followed by use to infect Vero cells at an MOI of ~10 in a roller bottle culture and harvesting of supernatants at 24 h.p.i.…”

“…Supernatants were clarified by centrifugation, followed by use to infect Vero cells at an MOI of ~10 in a roller bottle culture and harvesting of supernatants at 24 h.p.i. Vero supernatants were clarified as above and subjected to discontinuous 20%/60% sucrose gradient centrifugation followed by pelleting, both at 24 krpm, and resuspending in OPTIMEM medium for storage and use in infections (as described [ 34 ]). All virus stocks were titered by standard BHK cell plaque assay.…”

Sequelae and Animal Modeling of Encephalitic Alphavirus Infections

“…As neurologic signs of disease in macaques have been unreliable, recent applications of advanced telemetry to monitor intracranial pressure, body temperature, and EEG measurements have contributed greatly to the understanding of neurologic disease [ 59 ]. Combined with daily clinical observations, results of this telemetric study demonstrated biphasic fever as well as increases in intracranial pressure, while changes in EEG measurements indicated reduced sleep during the second bout of fever [ 59 ]. Common neurologic signs included mild tremors, twitching, and photophobia; ataxia and nystagmus were observed, but at infrequent rates [ 59 ].…”