Viral neuronotropism is important in the pathogenesis of Murray Valley encephalitis

Murray Valley encephalitis (MVE) virus, an RNA flavivirus, was first isolated in 1951 during an outbreak of encephalitis in the Murray Valley, Australia [1]. Subsequent outbreaks have been reported in 1956, 1974 and 2011 [2,3]. Similar to other flaviviral encephalitides such as Japanese encephalitis (JE), West Nile (WN) encephalitis and Tick Borne Encephalitis (TBE) [1], MVE is often severe with significant morbidity and mortality [2]. As MVE viral tropism in the central nervous system (CNS) is still unknown, we have examined three suspected (not cultureproven) cases of MVE, and report in one case, neuronal localization of viral antigens and viral RNA using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. These findings strongly suggest that MVE virus is primarily neuronotropic, that is has a predilection for neurons, and that neuronal viral cytolysis plays an important role in neuropathogenesis. During the 1974 MVE outbreak in New South Wales (NSW) [3], Australia, a 21-month-old boy was admitted to the Royal Children’s Hospital (RCH), Melbourne, Australia, with a 7-day history of drowsiness, malaise, convulsions and coma. He was transferred from another hospital in NSW, where he had a temperature of 39.5°C, generalized hypertonia and neck stiffness. His pupils were small, equal and reactive to light, and he maintained response to painful stimuli. The lumbar puncture showed cerebrospinal fluid pressure of 150 mmH2O (normal, 70–180 mmH2O); 18 × 10 lymphocytes/l with no polymorphs; chloride of 106 mmol/l (normal, 118–132 mmol/L); glucose 3.36 mmol/l (normal, 2.2–3.9 mmol/L) and protein <0.1 g/l (normal, <0.4 g/l). On admission to RCH, he was comatose and had opisthotonus and muscle spasms. A repeat lumbar puncture showed 3.7 × 10 polymorphs/l, 3.6 × 10 lymphocytes/l, 0.3 × 10 red cells/l, 0.5 × 10 ‘mononuclear’ cells/l, protein 0.5 g/l and glucose 3.08 mmol/l. Organisms and cultures were negative. He died after 10 days of illness with a clinical diagnosis of viral encephalitis. At autopsy, the congested brain weighed 1040 g (normal 1050 g) and showed no asymmetry, herniation or abnormalities in the meninges or other brain areas. However, on later examination, a few small necrolytic areas were noted in the left thalamus (Figure 1a). These relatively circumscribed lesions of 500–900 μm consisted of neuropil vacuolation/oedema, neuronal loss and a few degenerate/necrotic neurons (Figure 1b). There was mild meningitis (Figure 1c), perivascular cuffing and focal parenchymal infiltration by inflammatory cells mainly in grey matter, including cerebral cortex (temporal lobe and cingulate gyrus) (Figure 1d), thalamus, subthalamic nucleus, corpus striatum, hippocampus, hypothalamus, cerebellum (Figure 1c), midbrain, pons and spinal cord. Viral inclusions were not detected. A pathological diagnosis of MVE was made because of the ongoing MVE outbreak [4], but this was never proven by viral culture. In order to confirm MVE, we retrospectively performed IHC to detect flaviviral antigen and specific ISH to detect MVE viral RNA on formalin-fixed, paraffin-embedded CNS tissues archived in the Department of Anatomical Pathology, Alfred Hospital, Melbourne, Australia. The study was approved by the ethics committee of the Alfred Hospital. In addition, standard IHC was also performed to characterize inflammatory cells. Flaviviral IHC was performed using a standard immunoperoxidase technique [5]. Briefly, dewaxed, rehydrated 4 μm tissue sections were antigen retrieved and blocked with H2O2/methanol and serum. A primary mouse monoclonal antiflavivirus NS1 antibody (4G4, 1:500 dilution; courtesy of Roy Hall, University of Queensland, Australia) was added and incubated overnight at 4°C, followed by HRP-conjugated secondary antibody, 3′3diaminobenzidine (DAB) substrate and haematoxylin counterstaining steps. Similarly, inflammatory cells were detected using specific primary antibodies to human CD3 (1:200 dilution), CD4 (1:100 dilution), CD8 (1:150 dilution), CD20 (1:1000 dilution) and CD68 (1:250 dilution) (Novocastra, Newcastle-upon-Tyne, UK). Negative tissue controls included normal human brain, mouse brain infected with enterovirus 71 (EV71), WN, TBE, dengue [courtesy of Noriyo Nagata, National Institute of Infectious Diseases (NIID), Japan] and JE viruses. MVE virusinfected culture cells (courtesy of Tomohiko Takasaki, NIID, Japan) and mouse brain (courtesy of John Bingham, CSIRO, Australia) were used as positive controls. Duplicate negative control IHC assays were also done by omitting the primary antibody.