Natural reservoir Rousettus aegyptiacus bat host model of orthonairovirus infection identifies potential zoonotic spillover mechanisms

Abstract

The human-pathogenic Kasokero virus (KASV; genus Orthonairovirus) has been isolated from the sera of Egyptian rousette bats (ERBs; Rousettus aegyptiacus) captured in Uganda and unengorged Ornithodoros (Reticulinasus) faini ticks collected from the rock crevices of ERB colonies in South Africa and Uganda. Although evidence suggests that KASV is maintained in an enzootic transmission cycle between O. (R.) faini ticks and ERBs with potential for incidental virus spillover to humans through the bite of an infected tick, the vertebrate reservoir status of ERBs for KASV has never been experimentally evaluated. Furthermore, the potential for bat-to-bat and bat-to-human transmission of KASV is unknown. Herein, we inoculate two groups of ERBs with KASV; one group of bats is serially sampled to assess viremia, oral, fecal, and urinary shedding and the second group of bats is serially euthanized to assess virus-tissue tropism. Throughout the study, none of the bats exhibit overt signs of clinical disease. Following the detection of high KASV loads of long duration in blood, oral, fecal, and urine specimens collected from ERBs in the serial sampling group, all bats seroconvert to KASV. ERBs from the serial euthanasia group exhibit high KASV loads indicative of virus replication in the skin at the inoculation site, spleen, and inguinal lymph node tissue, and histopathology and in situ hybridization reveal virus replication in the liver and self-limiting, KASV-induced lymphohistiocytic hepatitis. The results of this study suggest that ERBs are competent, natural vertebrate reservoir hosts for KASV that can sustain viremias of appropriate magnitude and duration to support virus maintenance through bat-tick-bat transmission cycles. Viral shedding data suggests that KASV might also be transmitted bat-to-bat and highlights the potential for KASV spillover to humans through contact with infectious oral secretions, feces, or urine.

Pathogenesis of Kasokero virus in experimentally infected Egyptian rousette bats (Rousettus aegyptiacus)

“…Hemorrhage was observed in the liver of one 9 DPI bat at the time of tissue collection. 29 Variable pinpoint hepatic hemorrhages were identified during dissection of 3 additional formalin-fixed carcasses (two 6 DPI bats and one 9 DPI bat). Hepatic hemorrhages were pinpoint and generally totaled less than 10, except for one 6 DPI bat which had numerous pinpoint hemorrhages distributed throughout all liver lobes (Fig.…”

“…The detailed qRT-PCR results for blood, oral, rectal, and urine swabs, and tissues collected at the time of euthanasia were previously reported. 29 Viral loads obtained from the liver are summarized here for correlation with the hepatic morphometrics (Fig. 3).…”

“…At 3 and 6 DPI, the %PP values correlated with the qRT-PCR values previously reported for each bat (Spearman's r = 0.78; P = .032). 29 KASV RNA signal was minimally detectable by ISH at 6 DPI, with all bats having %PP values < 0.03 (2of 3 bats were < 0.001). KASV RNA was undetectable by ISH in 2 of 3 of the 9 DPI bats, and in the 18 DPI bats tested (2 of 2).…”

“…The ERB tissues used in the present study were obtained from an experimental infection study which was performed under biosafety level 4 (BSL-4) conditions in accordance with safety regulations at the Centers for Disease Control and Prevention (CDC, Atlanta, GA). 29 The experimental infections were conducted with approval from the CDC Institutional Animal Care and Use Committee (IACUC), and in accordance with the animal welfare guidelines set forth in the Guide for the Care and Use of Laboratory Animals (Committee for the Update of the Guide for the Care and Use of Laboratory Animals, National Research Council of the National Academies 2011). Husbandry, housing conditions, and diet for the duration of the experiment were previously described.…”

“…ERBs experimentally infected with KASV did not show any overt signs of illness despite developing transient viremias (peak viral loads = 5.1–5.9 log 10 TCID 50 eq/ml) with virus replication in multiple tissues, including liver, spleen, lymph nodes, and skin at the inoculation site, and shedding of virus into the oral secretions, feces, and urine. 29 The present study aimed to build on these findings by using formalin-fixed paraffin-embedded (FFPE) ERB tissues derived from the previous experiment to conduct a comprehensive tissue-based analysis of KASV-ERB interactions within a spatial context. To accomplish this goal, we used histopathology, morphometry, in situ hybridization (ISH) for detection of viral RNA, immunohistochemistry (IHC) for colocalization of KASV RNA with cells of the mononuclear phagocytic system, and quantitative image analysis to investigate virus clearance from the spleen and liver.…”