Infectious Chikungunya Virus in the Saliva of Mice, Monkeys and Humans

Abstract

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.

Fig 1. Hemorrhagic lesions in the olfactory epithelium of CHIKV infected IRF3/7^-/- mice.

(A, C, E) H&E of the head of IRF3/7^-/- mouse day 5 post CHIKV infection and (B, D, F) H&E of the head of a control uninfected IRF3/7^-/- mouse; T—turbinate bones, arrows—red blood cells, NC—nasal cavity. (A) Low magnification image showing extensive red blood cells and mucous exudates (pink staining material) in the nasal cavities. (*—large foci of vacuolization in the olfactory bulb). (B) The same region and magnification as in A for an uninfected mouse. (C) Medium magnification image showing breaches in the olfactory epithelium (dotted white circles). (D) The same region and magnification as in C for an uninfected mouse. (E) High magnification image of a lesion in the olfactory epithelium showing pyknotic nuclei (white arrow heads) and some neutrophils (black arrow head). (F) The same region and magnification as in E for an uninfected mouse.

Fig 2. Virus in oral cavity washings, vaginal washings and urine of CHIKV-infected IRF3/7^-/- mice.

Mice were infected with CHIKV i.p. (n = 9) and when clinical symptoms of hypovolemic shock became apparent (indicating onset of hemorrhagic manifestations) [6], mouth washings (Oral cavity), vaginal washings (Vagina) and urine (where possible), were collected and viral titers determined and expressed as log₁₀CCID₅₀/ml of washing medium on the indicated day (which is indicated in brackets below the titer Figs, respectively). † day when mouse was euthanized. NT–not tested.

Fig 3. CHIKV infection via intranasal and oral routes.

CHIKV was inoculated via pipette into the nose or mouth of restrained IRF3/7^-/- and C57BL/6 mice (n = 3 per group, 12 mice total). Viraemia was monitored as described, with 2 log₁₀CCID₅₀ the limit of detection [6]. †—mouse euthanized.

Fig 4. Presence of CHIKV RNA and infectious virus in the saliva of acute CHIKV infected patients.

All patients were negative for dengue. Symptoms, manifestations and comorbidities at time of saliva collection. ¹ H–Hospital study, C–Cohort study. ² CHIKV detected in saliva by TaqMan RT-PCR. ³ Saliva was incubated with Vero cells, and TaqMan RT-PCR was used to confirm CHIKV in cultures showing cytopathic effect. NT–not tested.