An Early Passage Human Isolate of Kyasanur Forest Disease Virus Shows Acute Neuropathology in Experimentally Infected CD-1 Mice

The Kyasanur Forest disease virus (KFDV) is a tick-borne Flavivirus first isolated in 1957 from the Shimoga region of Karnataka State, a densely forested area in western India and described as the etiologic agent for monkey deaths and a highly morbid human illness with a case fatality of approximately 25% (Work and Trapido 1957). Subsequent studies by researchers of the Virus Research Center, Poona, India, (now National Institute of Virology, Pune) revealed its complex ecology and predominantly sylvatic existence involving a zoonotic cycle of monkeys and Ornithodorus ticks. Human exposure to infected tick bites was the major risk factor for infection (Pavri 1989).

Although the virus has been endemic to Shimoga and surrounding forests for many years, recent studies are suggestive of its possible spread to other parts of southern India because of factors that are not completely described but perhaps significant changes in demography of both humans and susceptible vector species might have played an important role (Yadav et al. 2014). There is an effective vaccine and need for a better one is being currently explored.

The pathogenesis and pathology of KFDV have been a subject of serious investigations. Although the disease was initially described as hemorrhagic, contemporary studies had also suggested a prominent neurological component in KFDV infections (Wadia 1975). More so, observations of laboratory-acquired KFDV-infected cases did not show any hemorrhagic manifestations but lingering neurological sequelae in several cases (Pavri 1989). This has been the major reason for studying the pathology of KFDV infection in detail in both natural and experimental infections. More importantly, such studies including two recent ones have provided important findings toward the understanding of KFDV pathogenesis (Dodd et al. 2014, Sawatsky et al. 2014). In this study, we examined the nature of histopathologic lesions caused by a primary human isolate of KFDV in laboratory mice.

The KFDV isolate NIV 12839 was isolated from an acute phase serum sample of a human case, from Thirthahalli, Shimoga, Karnataka. The serum was also positive for KFDV RNA in real-time RT-PCR (Ct = 22). An aliquot of the serum was inoculated in an infant suckling Swiss albino mouse by the intracranial (i.c.) route as per the standard protocol, and after observing sickness, organs were harvested at fourth postinfection (p.i.). Clarified brain suspension was prepared in normal saline and inoculated in chick embryo cells. After confirming KFD infection by real-time PCR, immunblots, and transmission electron microscopy, the KFD isolate 12893 was adapted in Baby Hamster Kidney (BHK-21) cells for four passages.

Zero- to two-day-old CD-1 infant mice were inoculated through intraperitoneal (i.p.) route with 10^3.0 TCID₅₀ dose of the virus and observed for sickness. Animals were euthanized on 24, 48, and 72 h p.i. as per institutional approved protocols and guidelines for animal experimentations. Visible sickness in the inoculated animal groups was seen as ruffled fur, lethargy, and disorientation by the first day p.i. and all mice died by the fourth day p.i. The lungs, liver, heart, kidneys, spleen, and brain were harvested and processed for routine histopathology. A sensitive immunohistochemical labeling using a murine anti-KFDV antibody developed in house was carried out using a commercially available kit (Novolink Polymer Detection System; Leica Biosystems) as per manufacturers' instructions. Evidence of earliest and most consistent histological damage was detected in the brain within one day after infection. This was seen in the cerebellum and other hind brain areas as neuronal loss, necrosis, moderate degrees of lymphocytic infiltration, frank neuronal necrosis, syncytium formation, and gliosis (Fig. 1a–c). Evidence of KFDV antigens was also detected in the brain by immunohistochemistry (IHC) (Fig. 1d–f). By the third p.i. day, microscopic lesions were evident in liver with vacuolation of hepatocytes and significant parenchymal injury (Fig. 1g). The kidneys showed evidence of cortical necrosis, cytoplasmic hyaline clumps, and variable prominence of glomeruli (Fig. 1h). Histologic lesions in the small intestine were seen as epithelial necrosis and lymphocytic infiltration in the submucosa and muscularis (Fig. 1i). Vascular pathology was also prominent in all these organs. KFDV antigens were detected in brain, liver, and kidney tissues by IHC. Nonspecific changes were detected in the heart by the third p.i. day. The spleen, skin, and lung of the KFDV-infected mice did not show any significant changes (data not shown).

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FIG. 1.

Representative micrographs showing the profile of microscopic lesions seen in KFDV-infected mice. (a) Areas of acute lymphocytic infiltrate shown by arrows. (b) Gliosis and areas of neuronal loss (*) and infiltrating cells shown by arrows. (c) Giant cell and syncytium formation (arrows). (d–f) KFDV antigens in brain by immunohistochemistry (IHC). (g) Acute vascular necrosis and vacuolated hepatocytes stained positive for IHC antigens. (h) Glomerular prominence in the kidney with cortical degeneration and positive labeling for KFDV antigens. (i) Small intestinal histopathology showing epithelial cell necrosis and lesions in submuscularis (red box). Scale bars are built into the micrographs (5 μm). KFDV, Kyasanur Forest disease virus.

The pathology of KFDV remains incompletely understood. Several excellent earlier studies ranging from human autopsies to experimentally inoculated animals have shown interesting findings. In fatal human cases who died by the ninth day of illness, the major pathologic findings showed damage to parenchymal organs, features of hemorrhagic bronchopneumonia, but interestingly no prominent neuropathology (Pavri 1989). Autopsy studies on dead monkeys in the endemic area in 1957 also showed a broad spectrum of changes. Although a hemorrhagic component was observed in majority of the dead animals, the overall profile was that of a nonspecific parenchymal organ injury, showing degenerative changes (Iyer et al. 1959, Pavri 1989).

The first report of pathology in experimentally infected monkeys with KFDV was by Webb and Burston (1966). This study reported nonspecific histopathological changes in the central nervous system that did not have any relevance to clinical correlates of disease. Similar findings were also reported in earlier mice studies (Nayar 1972). It is difficult to carry out such studies now because of the high biosafety and containment level requirement for KFDV as this has been classified as a category 4 agent and requires a BSL4 laboratory for experimental handling. In 2014, two studies reported on the comparative pathology in inbred laboratory mice experimentally infected with KFDV and Alkhurma Hemorrhagic fever virus, a virus genetically related to KFDV (Dodd et al. 2014, Swatsky 2014). Two important points emerged from these studies. First, consistent with the earlier studies, the KFDV pathology in experimentally infected laboratory animals was found to be nonspecific. Second, and more important, the question raised was whether this is a virus isolate-specific phenomenon or not. Interestingly, all the earlier studies including the 2014 work had used laboratory-adapted well-passaged KFDV isolate, namely isolate P 9605.

The importance of our findings lies in the use of a low cell culture-passaged human KFDV isolated in 2012 (NIV12839) for our studies using the i.p. inoculation route. Our findings showed that KFDV replication as detected by IHC and associated microscopic lesions evident through cytopathology were seen primarily in the cerebellum and hind brain structures, whereas organs such as kidney and liver showed delayed parenchymal lesions. Although the spectrum of histopathologic changes overlapped and was consistent with earlier findings, syncytium in cerebellar tissue and evidence of consistent vascular injury in the affected organs were novel observations. These observations bring out two important points. First, is KFDV primarily a neurotrophic virus but after amplification in the host acquires pan-organ trophism? Could endothelial cells be a natural target for KFDV in vivo and contribute to disease pathogenesis? Further studies are needed with more primary virus isolates in vitro to answer these questions of pathophysiology of KFDV and develop a model using early passaged human KFDV isolates instead of laboratory-adapted virus strains for screening of potential therapeutic and antiviral compounds.

Conclusions

This study highlights the potential of a primary human KFDV isolate in causing specific neuropathology. Secondary histopathological changes in other organs could be because of indirect parenchymal injury caused by disease pathogenesis. Therefore, further studies using more primary human KFDV isolates and not laboratory-adapted virus are the need-of-hour issue for developing adequate challenge models that will be relevant in testing potential antivirals and therapeutics in future.