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

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.

Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques.

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2-3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.

Applications of minimally invasive multimodal telemetry for continuous monitoring of brain function and intracranial pressure in macaques with acute viral encephalitis.

Alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are arboviruses that can cause severe zoonotic disease in humans. Both VEEV and EEEV are highly infectious when aerosolized and can be used as biological weapons. Vaccines and therapeutics are urgently needed, but efficacy determination requires animal models. The cynomolgus macaque (Macaca fascicularis) provides a relevant model of human disease, but questions remain whether vaccines or therapeutics can mitigate CNS infection or disease in this model. The documentation of alphavirus encephalitis in animals relies on traditional physiological biomarkers and behavioral/neurological observations by veterinary staff; quantitative measurements such as electroencephalography (EEG) and intracranial pressure (ICP) can recapitulate underlying encephalitic processes. We detail a telemetry implantation method suitable for continuous monitoring of both EEG and ICP in awake macaques, as well as methods for collection and analysis of such data. We sought to evaluate whether changes in EEG/ICP suggestive of CNS penetration by virus would be seen after aerosol exposure of naïve macaques to VEEV IC INH9813 or EEEV V105 strains compared to mock-infection in a cohort of twelve adult cynomolgus macaques. Data collection ran continuously from at least four days preceding aerosol exposure and up to 50 days thereafter. EEG signals were processed into frequency spectrum bands (delta: [0.4 - 4Hz); theta: [4 - 8Hz); alpha: [8-12Hz); beta: [12-30] Hz) and assessed for viral encephalitis-associated changes against robust background circadian variation while ICP data was assessed for signal fidelity, circadian variability, and for meaningful differences during encephalitis. Results indicated differences in delta, alpha, and beta band magnitude in infected macaques, disrupted circadian rhythm, and proportional increases in ICP in response to alphavirus infection. This novel enhancement of the cynomolgus macaque model offers utility for timely determination of onset, severity, and resolution of encephalitic disease and for the evaluation of vaccine and therapeutic candidates.

Electrocardiography Abnormalities in Macaques after Infection with Encephalitic Alphaviruses.

Eastern (EEEV) and Venezuelan (VEEV) equine encephalitis viruses (EEVs) are related, (+) ssRNA arboviruses that can cause severe, sometimes fatal, encephalitis in humans. EEVs are highly infectious when aerosolized, raising concerns for potential use as biological weapons. No licensed medical countermeasures exist; given the severity/rarity of natural EEV infections, efficacy studies require animal models. Cynomolgus macaques exposed to EEV aerosols develop fever, encephalitis, and other clinical signs similar to humans. Fever is nonspecific for encephalitis in macaques. Electrocardiography (ECG) metrics may predict onset, severity, or outcome of EEV-attributable disease. Macaques were implanted with thermometry/ECG radiotransmitters and exposed to aerosolized EEV. Data was collected continuously, and repeated-measures ANOVA and frequency-spectrum analyses identified differences between courses of illness and between pre-exposure and post-exposure states. EEEV-infected macaques manifested widened QRS-intervals in severely ill subjects post-exposure. Moreover, QT-intervals and RR-intervals decreased during the febrile period. VEEV-infected macaques suffered decreased QT-intervals and RR-intervals with fever onset. Frequency-spectrum analyses revealed differences in the fundamental frequencies of multiple metrics in the post-exposure and febrile periods compared to baseline and confirmed circadian dysfunction. Heart rate variability (HRV) analyses revealed diminished variability post-exposure. These analyses support using ECG data alongside fever and clinical laboratory findings for evaluating medical countermeasure efficacy.