Photo courtesy of the Connecticut Agricultural Experiment Station. Mosquito Control. For Healthcare Providers. Links with this icon indicate that you are leaving the CDC website.
Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website.
You will be subject to the destination website's privacy policy when you follow the link. Representative photomicrographs showing lesions and virus distribution in selected tissues at 12 hours post-infection PI. A: ISH-positive fibroblasts inset and macrophages in the dermis and connective tissue fascia in the inoculated foot. B: ISH-positive tendon at insertion on bone. Inset shows higher power view of virus-positive cells.
C: IHC-positive synovial cells in metatarsal joint of inoculated foot. D: IHC-positive dendritic macrophages arrow in draining popliteal lymph node. F: Note the ISH-positive osteoblasts lining the primary spongiosa in this sequential section of area shown in D.
The intensity of ISH signal is reduced in the area of necrosis. G: Higher magnification of area shown in E and F , showing the normal morphology and numbers of the virus-positive osteoblasts covering trabeculae. H: IHC staining of sequential section of the same area demonstrating EEE virus antigen in morphologically osteoblasts. In the long bones, although most osteoblasts appeared to be normal, there were rare apoptotic cells lining the bony trabeculae in the metaphyseal regions Figure 1E.
In all four mice, there was intense staining of histologically normal osteoblasts in the lateral metaphysis and periosteum of the femur and tibia Figure 1, F to H. On the contralateral leg, there were also ISH- and IHC-positive clusters of osteoblasts in the metaphyses of long bones of all four mice, and along the metaphyseal periosteum in two of four mice. No viral antigen or nucleic acid was detected in the draining popliteal lymph node of the contralateral leg. Elsewhere, rare virus-positive cells were present multifocally in the bones of the nasal turbinates, hard palate, and skull.
A single cluster of virus-positive neurons located in the somatosensory cortex of one mouse provided the first evidence of CNS invasion. Epineurial and epimysial cells were frequently virus-positive and there were also a few clusters of virus-positive cells in the cutaneous squamous epithelium.
Significantly, the many viral antigen-positive macrophages in the footpad were uniformly negative by ISH. In the femur and tibia, there was widespread necrosis and loss of osteoblasts and other cells in the areas immediately adjacent to the growth plate, with associated viral antigen-positive macrophages and cell debris in affected areas. By ISH, the most intensely stained osteoblasts were located along the diaphysis, with markedly decreased ISH signal in the areas of the epiphysis and metaphysis that contained necrotic osteoblasts.
Viral infection had spread to the osteoblasts and fibroblasts in the connective tissues encircling the growth plate. These included the ring of fibrous tissue and bone comprising the perichondrial ring of LaCroix and the ossification groove of Ranvier Figure 2A. By this time, there was also intense ISH-positive staining of fibroblasts and periosteum at insertion sites of tendons on bone Figure 2E.
However, ISH of serial sections again showed that these sinus macrophages were negative for viral nucleic acids. Representative photomicrographs showing lesions and virus distribution in selected tissues at 1 day post-infection PI. A: ISH of proximal tibia showing intense staining within the periosteum and ring of Ranvier top left inset.
Note the relatively less intense staining of osteoblasts along the growth plate top right inset at this time point. B: IHC of draining popliteal lymph node demonstrates presence of viral antigen in sinus macrophages only. The clumped granular appearance of viral antigen is shown in the inset. At this time point, these macrophages were negatively stained for viral RNA.
E: At this time point, there was intense ISH-positive staining of fibroblasts and periosteum at insertion sites of tendons on bone. Note the focal disruption and necrosis of odontoblasts. In the contralateral leg, there was necrosis and loss of osteoblasts in numerous foci in the metaphysis, with abundant viral antigen detected in the osteoblasts, macrophages, and cell debris in these areas.
The most intense ISH signal was seen in periosteal osteoblasts and in some small clusters of endosteal osteoblasts. Metabolically active chondrocytes and osteoclasts at the growth plate were apparently refractory to infection, as were osteocytes and small osteoblasts in other areas. Significantly, virus-positive subcutaneous fibrocytes in the footpad and synovial cells of the contralateral leg were not detected. Elsewhere, there were scattered necrotic cells in bones and tooth pulp, with virus-positive cells including the osteoblasts lining the marrow cavities of turbinate and skull bones, the odontoblasts and fibroblastic cells in the dental pulp Figure 2F , and even the basal epithelium of a vibrissa in one mouse.
There were also small numbers of weakly positive myocytes in the ventricular myocardium as well as clusters of virus-positive cells in the ovarian stroma and in a few foci in the fallopian tubes and in the myometrium.
Virus-infected neurons were detected in the CNS of three of four mice. The location of the virus-infected neurons in the CNS was highly variable, with one mouse having a few virus-positive neurons at the base of the cerebellum and a single Purkinje cell, but numerous scattered positive granular cell neurons.
There were also virus-infected layer I piriform cortical neurons on one side with minimal extension to layer II neurons, and a few positive neurons within the lateral olfactory tract on one side. In another mouse, we found a cluster of virus-positive neurons in the caudate putamen in addition to a couple of isolated virus-positive neurons in the cerebral cortex. In the third mouse, there were scattered virus-positive neurons in the piriform cortex, and clusters of virus-positive neurons in the secondary motor cortex and caudate putamen.
At the inoculation site, there were numerous antigen-positive but ISH-negative macrophages in the connective tissues. ISH signal in the inoculated footpad was present in fascial fibroblasts, subepithelial fibroblasts, and some myocytes. By day 2 PI, the metaphyseal areas were essentially negative for EEE virus antigen and nucleic acid and abundant necrotic cells and debris were present in the areas of trabecular bone normally lined by osteoblasts.
ISH demonstrated the apparent direct extension of infection from the periosteum to surrounding myocytes. In addition, small numbers of keratinocytes stained positively for both viral antigen and nucleic acid. These individual and small groups of virus-positive keratinocytes usually appeared as a thin band of cells below the stratum corneum. In the draining popliteal lymph node, there was a diffuse moderate lymphoid hyperplasia with a few weakly antigen-positive but ISH-negative macrophages in the subcapsular sinuses.
In other locations, there was multifocal necrosis and loss of odontoblasts, ameloblasts, and tooth pulp in all four mice. In affected areas, there was segmental loss of the odontoblast cell layers and multifocal necrosis and loss of ameloblasts with consequent disruption of normal histological arrangements of the tooth.
There were also some perivascular virus-positive foci within the marrow cavities of the bones of the skull in three of four mice. In one mouse, there was a single focus of virus-positive cells under the olfactory epithelium while in two of the four mice there were small clusters of weakly positive myocardial cells. In one mouse, four virus-positive fibroblast cells were detected in the renal medullary interstitium.
All six remaining mice inoculated with the wild-type virus showed clinical signs of severe neurological disease by day 4 PI and were killed. Histologically, there was widespread necrosis of neuronal cells and some associated rarefaction of adjacent neuropil and white tracts in all six mice. Generally, the inflammatory cell reaction was very mild, consisting mainly of neutrophils and eosinophils. Virus was found chiefly within the perikaryon and dendrites of neurons in the cerebral cortex and midbrain, although some glial cells were also virus-positive.
In the hippocampus, lesions were more prominent in the gyrus dentatus than the pyramidal hippocampus. Scattered Purkinje cells and associated cerebellar granular layer neurons became virus-positive relatively late in disease. Virus-positive neurons were located within the caudate putamen, middle layers of the cerebral cortex, internal nuclei, pyramidal cortex, and multifocally in the frontal, cingulate, and parietal cortex and most brainstem nuclei Figure 3A.
The strongest signal was often detected in the thalamus, caudate putamen, and pons. Notably, the intensity of viral staining was diminished in areas with diffuse neuronal necrosis. Viral infection was essentially absent in the internal and external capsules, pyramidal tract, hippocampal commisure, lateral olfactory tract, cerebellar peduncles, and corpus callosum.
The pyramidal neurons of the ventrolateral CA3 field of hippocampus were usually involved. Except for the polymorph layer, the dentate gyrus of the hippocampus was generally spared. Involvement of the cerebellum was generally less severe than that seen in the cerebrum and brainstem Figure 3B , and generally consisted of variably sized foci of virus-positive Purkinje cells and their associated granular cell neurons.
In terminally ill mice, there were scattered large clusters of virus-infected Purkinje cells Figure 4H. In all six mice, there was necrosis in the olfactory tubercle and multifocally in the piriform cortex, and IHC and ISH staining for virus was most intense in the granular cell layer of the olfactory bulbs, with variable extension to the plexiform and glomerular cell layers.
Representative photomicrographs showing lesions and virus distribution in selected tissues at 4 days post-infection PI. A: ISH of brain shows the diffuse viral infection of cortex, hippocampus, caudate putamen, and thalamus. B: ISH of cerebellum and midbrain of same mouse shows the relative sparing of the cerebellum.
C: IHC of olfactory mucosa. Note the absence of viral antigen within the olfactory neuroepithelium and the virus-positive cells and processes in the submucosa.
D: IHC showing virus-positive keratinocytes in skin. G: Higher magnification of F showing necrotic odontoblasts and ameloblasts in the virus-infected area of tooth.
A: Low magnification photomicrograph of proximal femur of uninfected control mouse. Note the normal thickness of cartilage and bone trabeculae in the primary spongiosa of the metaphysis. B: Low magnification photomicrograph showing the growth plate of an eastern equine encephalitis EEE virus infected mouse on day 4 PI.
Note the increased thickness of the zone of hypertrophic cartilage and the very thin and elongated fibrous trabeculae adjacent to the growth plate. C: Higher magnification photomicrograph of normal growth plate demonstrates that the zone of hypertrophic chondrocytes is only up to four cells thick, and abundant osteoblasts are located at the bone-cartilage interface and along the primary spongiosa. D: This section shows typical changes in growth plate at day 1 PI.
Osteoblasts are generally absent from the growth plate area and the zone of hypertrophic chondrocytes is already up to eight cells thick. E: At day 4 PI, the zone of hypertrophy is up to 15 cells thick. F: High magnification photomicrograph showing the morphology of normal chondrocytes in the hypertrophic zone of uninfected control mouse. G: High magnification showing unusual large eosinophilic cytoplasmic granules within the most basal hypertrophic chondrocytes.
I: IHC demonstrating viral antigen within sustentacular cells in the renal papilla. Significantly, there was minimal involvement of the olfactory neuroepithelium, consisting only of a small amount of virus-specific staining in a few isolated olfactory neurons, and in a few individual cells located beneath the olfactory neuroepithelium Figure 3C and within perisinusoidal nerves in the nasal turbinates surrounding the vomeronasal organs.
In all six mice, virus infection in the eyes typically involved multiple foci in the retinal ganglion cell layer with extensions to the internal and external granular layers. At the inoculation site, there were numerous viral antigen-positive myocytes, fibroblasts, and macrophages, as well as several clusters of intraepithelial cells and rare sebaceous glands in the footpad.
In both legs, there were rare antigen-positive macrophages and fibroblasts in the periosteum and within the ring of Ranvier. Viral nucleic acid was only detected in occasional widely scattered skeletal muscle myocytes. In developing teeth, there was necrosis of odontoblasts and ameloblasts that disrupted the dentin and enamel layers Figure 3, E and G.
In affected areas, there were clusters of virus-positive odontoblasts and ameloblasts and scattered individual positive cells in the dental pulp Figure 3F. By day 4 PI, the lesions in the bones of EEE virus-infected mice were characterized by widespread necrosis and loss of osteoblasts, with accompanying thickening of the epiphyseal growth plate and elongation of the primary spongiosa Figures 4, A and B.
At the epiphyseal growth plate, viral infection induced a marked disruption in the normal process of cartilage maturation and replacement by bone. In uninfected mice, the zone of hypertrophic chondrocytes ranged up to four cells thick Figure 4C. However, as early as day 1 PI, there was multifocal loss of osteoblasts adjacent to the growth plate and the zone of hypertrophy was doubled in thickness Figure 4D.
By day 4 PI, the growth plate was up to four times the normal thickness, with the severe alterations affecting the regions of hypertrophy, mineralization, and ossification Figure 4D. The major alterations included elongation of the zone of cartilage hypertrophy in the areas that had lost osteoblasts, and reduced formation of osteoid in the zone of ossification.
The persistent and altered hypertrophic chondrocytes and changes in the growth plate were present only in specific areas where virus infection had eliminated metaphyseal osteoblasts and resulted abnormal endochondral ossification. Hypertrophic chondrocyte columns were up to four times the normal length and some of the more mature chondrocytes contained large eosinophilic cytoplasmic granules Figure 4, F and G.
The decreased osteoid on primary trabeculae was associated with severely reduced numbers of osteoblasts along the metaphyseal trabeculae. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI.
EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus.
0コメント