Japanese encephalitis virus: Biological clones from a clinical isolate quasispecies show differing neurovirulence in vitro and in a mouse model

@#The Japanese encephalitis virus (JEV), a leading cause of encephalitis, exists as quasispecies in clinical isolates. Using a limiting dilution method combined with immunohistochemistry to detect viral antigens, 10 biological clones were isolated and purified from a clinical JEV isolate (CNS138/9) derived from an autopsy brain. These biological clones were tested for neurovirulence in SK-N-MC and NIE-115 neuronal cells, and a 2-week-old, footpad-infected, JE mouse model. Nine clones were found to be neurovirulent; one clone neuroattenuated. Although further studies are needed to determine genotypic differences, if any, in these clones, the limiting dilution purification and neurovirulence testing methods described herein should be useful for phenotypic studies of quasispecies of neurotropic viruses in general, and JEV and other flaviviruses in particular.

The role of heat shock proteins and glucose regulated proteins in cancer

Heat shock proteins (HSPs) are a family of evolutionary conserved proteins that work as molecular chaperones for cellular proteins essential for cell viability and growth as well as having numerous cyto-protective roles. They are sub-categorised based on their molecular weights; amongst which some of the most extensively studied are the HSP90 and HSP70 families. Important members of these two families; Heat shock proteins 70 and heat shock proteins 90 (Hsp70/90), are the glucose regulated proteins (GRP). These stress-inducible chaperones possess distinct roles from that of the other HSPs, residing mostly in the endoplasmic reticulum and mitochondria, but they can also be translocated to other cellular locations. Their ability in adapting to stress conditions in the tumour microenvironment suggests novel functions in cancer. GRPs have been implicated in many crucial steps of carcinogenesis to include stabilization of oncogenic proteins, induction of tumour angiogenesis, inhibition of apoptosis and replicative senescence, and promotion of invasion and metastasis.

Pathological findings in a mouse model for Coxsackievirus A16 infection

Coxsackievirus A16 (CV-A16) is the leading cause of hand-foot-mouth disease (HFMD), which usually presents as mild and self-limiting symptoms in young children. Rarely, CV-A16 has been reported to cause severe and fatal neurological complications but little is known about these complications. In the present study, 1-day and 7-day old mouse models of CV-A16 were developed using a clinical strain via subcutaneous inoculation. All infected mice exhibited clinical signs of infection, including reduced mobility, limb weakness and paralysis between 3 to 6 days post-infection. Pathologically, the main organs involved were the central nervous system (CNS), skeletal muscles and brown fat. In the CNS, viral antigens as demonstrated by immunohistochemistry, were localized mainly to neurons in the brain stem and spinal cord, suggesting that CV-A16 is neurotropic although inflammation is very mild. The skeletal muscles showed necrosis and myositis due to viral infection as evidenced by the dense viral antigens. Focal viral antigens were also detected in the brown fat. These preliminary pathological findings indicate that our mouse models can be further developed to be useful models for pathogenesis studies, and vaccine and anti-viral drug evaluation.

Pathological findings in a mouse model of Japanese encephalitis infected via the footpad

We have developed and characterised a mouse model of Japanese encephalitis virus (JEV) infection via footpad inoculation in order to better mimic viral transmission by mosquito bites. Two-week-old and 5-week-old mice consistently developed signs of infection such as ruffled fur, weight loss, hunchback posture, tremors, mask-like facies and occasionally, hindlimb paralysis at 4 days post infection (dpi) and 11-13 dpi, respectively. Most of the animals died within 24 to 48 hours following the onset of signs of infection, with mortalities of 100% and 33.3% in 2-week-old and 5-week-old mice, respectively. Mild meningitis and variable parenchymal inflammation with formation of microglial nodules, focal necrosis and neuronophagia, and perivascular cuffing by inflammatory cells were observed in the caudate nucleus, putamen, thalamus, cerebral cortex, brainstem, and spinal cord. Viral antigens/RNA were demonstrated by immunohistochemisty and in situ hybridization, respectively, in most of these areas as well as in the hippocampus and cerebellum, albeit more focally. The pathological findings in this mouse model were generally similar to human Japanese encephalitis (JE) and other established JE models but perhaps, compared to other JEV mouse models, it demonstrates lethal encephalitic infection more consistently. We believe that our mouse model should be useful to study the pathogenesis of JE, and for testing anti-viral drugs and vaccines

Pathological findings in a mouse model for Coxsackievirus A16 infection

Coxsackievirus A16 (CV-A16) is the leading cause of hand-foot-mouth disease (HFMD), which usually presents as mild and self-limiting symptoms in young children. Rarely, CV-A16 has been reported to cause severe and fatal neurological complications but little is known about these complications. In the present study, 1-day and 7-day old mouse models of CV-A16 were developed using a clinical strain via subcutaneous inoculation. All infected mice exhibited clinical signs of infection, including reduced mobility, limb weakness and paralysis between 3 to 6 days post-infection. Pathologically, the main organs involved were the central nervous system (CNS), skeletal muscles and brown fat. In the CNS, viral antigens as demonstrated by immunohistochemistry, were localized mainly to neurons in the brain stem and spinal cord, suggesting that CV-A16 is neurotropic although inflammation is very mild. The skeletal muscles showed necrosis and myositis due to viral infection as evidenced by the dense viral antigens. Focal viral antigens were also detected in the brown fat. These preliminary pathological findings indicate that our mouse models can be further developed to be useful models for pathogenesis studies, and vaccine and anti-viral drug evaluation.

Pathological findings in a mouse model of Japanese encephalitis infected via the footpad

We have developed and characterised a mouse model of Japanese encephalitis virus (JEV) infection via footpad inoculation in order to better mimic viral transmission by mosquito bites. Two-week-old and 5-week-old mice consistently developed signs of infection such as ruffled fur, weight loss, hunchback posture, tremors, mask-like facies and occasionally, hindlimb paralysis at 4 days post infection (dpi) and 11-13 dpi, respectively. Most of the animals died within 24 to 48 hours following the onset of signs of infection, with mortalities of 100% and 33.3% in 2-week-old and 5-week-old mice, respectively. Mild meningitis and variable parenchymal inflammation with formation of microglial nodules, focal necrosis and neuronophagia, and perivascular cuffing by inflammatory cells were observed in the caudate nucleus, putamen, thalamus, cerebral cortex, brainstem, and spinal cord. Viral antigens/RNA were demonstrated by immunohistochemisty and in situ hybridization, respectively, in most of these areas as well as in the hippocampus and cerebellum, albeit more focally. The pathological findings in this mouse model were generally similar to human Japanese encephalitis (JE) and other established JE models but perhaps, compared to other JEV mouse models, it demonstrates lethal encephalitic infection more consistently. We believe that our mouse model should be useful to study the pathogenesis of JE, and for testing anti-viral drugs and vaccines