2.1. Comparison of lymphocytic immunoreactivity and histopathological scores
Two general approaches can be used to evaluate the severity of cellular inflammatory infiltration in the CNS induced by viruses with different neurovirulence. The conventional semi-quantitative approach relies on histopathological analysis of routinely stained (H&E and/or Nissl) sections and assignment of cellular inflammatory infiltration (CII) scores based on the number of perivascular infiltrates and degree of parenchymal infiltration. The representative H&E-stained sections that contained the entire areas of the CNS Regions of Interest (CNS ROIs) including the basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, and spinal cord (cervical and lumbar regions) were digitalized and are shown in and and Supplementary Fig. 1a and b
. These digitalized whole-tissue sections provide an overall view of the inflammatory cell infiltration in the entire CNS ROIs of a mock-inoculated control or virus-infected monkeys. The new approach is based on automated image analysis (AIA) that measures the immunoreactivity (IR) of the infiltrating T and B cells using specific lymphocytic markers (CD3 and CD20, respectively) and provides quantitative data on the overall level of perivascular and parenchymal lymphocytic infiltration ( and and Supplementary Fig. 1c and d
Fig. 1 Assessment of lymphocytic infiltration in the basal ganglia. (a) Representative section through the basal ganglia of a mock-inoculated monkey (H&E staining). (b–d) Adjacent sections through the basal ganglia of a TBEV/DEN4Δ30-infected (more ...)
Fig. 2 Assessment of lymphocytic infiltration in the thalamus. (a) Representative section through the thalamus of a mock-inoculated monkey (H&E staining). (b–d) Adjacent sections through the thalamus of a TBEV/DEN4Δ30-infected monkey (more ...)
To validate the results of the AIA of lymphocytic IR against the CII scores, we performed Pearson correlation analyses. We compared the CD3-IR, CD20-IR, or pooled CD3/CD20-IR values within the CNS ROIs with the CII scores for each of twelve monkeys (one mock-inoculated control monkey and groups of three or four monkeys infected with TBEV/DEN4Δ30, LGTV, or YF 17D virus) (). This analysis demonstrated that either CD3-IR or CD20-IR alone, or pooled CD3/CD20-IR (reflecting overall lymphocytic IR) correlated strongly and significantly with CII scores in all CNS ROIs (R, 0.719–0.953; P < 0.01). In addition, statistical analysis demonstrated that CD3-IR, CD20-IR, or CD3/CD20-IR values were similar to CII scores in identifying differences between the viruses based on induced inflammatory cell infiltration (as indicated by asterisks in ). These results show that both methods can reliably evaluate the overall inflammatory cell infiltration in the virus-infected CNS.
Fig. 3 Analysis of correlation between CD3-IR, CD20-IR, or pooled CD3/CD20-IR and histopathological scores for cellular inflammatory infiltration (CII) in the CNS. Correlation between CD3-IR (a–c), CD20-IR (d–f), or pooled CD3/CD20-IR (g–i) (more ...)
2.2. Comparison of microglial immunoreactivity and histopathological scores
Two different approaches can also be used to evaluate the magnitude of microglial activation in the virus-infected CNS. A conventional semi-quantitative histopathological analysis generates scores based on the number of microglial nodules and an estimation of the degree of diffuse patterns of microglial activation in the H&E and/or Nissl stained sections. This type of microscopic analysis is complicated and time-consuming since the routine histological stains are not cell type specific and assessment is largely based on the presence of focal and/or diffuse parenchymal “hypercellularity”, which is often topographically associated with degenerating/dying neurons (e.g., neuronophagia). In contrast, the AIA allows direct evaluation of microglial activation by measuring the IR for the specific microglia/macrophage marker CD68 and provides a more accurate assessment of both focal and diffuse microglial activation. The assessment of the CD68-IR in the digitalized whole-tissue sections is illustrated in , .
Fig. 4 Assessment of microglial activation and neuronal degeneration in the basal ganglia and thalamus. Adjacent sections through the basal ganglia (a and b) and thalamus (c and d) of a YF 17D-infected monkey showing immunostaining for CD68 (a and c) or NeuN (more ...)
Fig. 5 Image analysis of microglial activation (CD68-IR) and neurodegeneration (NeuN-IR) in the spinal cord. Adjacent transverse sections of the lumbar spinal cord of a mock-inoculated monkey (a and b) or LGTV-infected monkey (c and d) showing the analysis of (more ...)
Pearson correlation analysis demonstrated that the CD68-IR values correlated strongly and significantly with histopathological scores for microglial activation/neuronal degeneration (MGA/ND Score) within all CNS ROIs (; R
, 0.795–0.877; P
≤ 0.001). However, it should be noted that there were some discrepancies between these two evaluation approaches. First, using the AIA we measured substantial CD68-IR in the zone of virus inoculation (thalamus) and partially in the adjacent areas of basal ganglia [22
]. This microglial response was largely attributed to tissue injury due to inoculation since it was observed in both mock-inoculated and virus-infected monkeys. In contrast, these injection-related lesions were considered non-specific and received a score of zero by histopathological scoring [19
]. Second, CD68-IR may also reflect concomitant recruitment of peripheral macrophages in addition to activated microglia. Third, the histopathological scores included not only microglial activation but also neuronal degeneration. Taken together, the discrepancies in the two evaluation approaches could explain why MGA/ND histopathological scores were often statistically more discriminating (P
< 0.05) for viruses with different neurovirulent phenotypes than CD68-IR values (). Therefore, we sought to address the degree of neurodegeneration in virus-infected CNS quantitatively and independent of microglial activation.
Fig. 6 Analysis of correlation between CD68-IR and NeuN-IR and histopathological scores for microglial activation/neuronal degeneration (MGA/ND) in the CNS. Correlation between CD68-IR (a–c) or NeuN-IR (d–f) and MGA/ND scores in the basal ganglia, (more ...)
In this study, we explored the potential of a high-throughput AIA of neuroinflammation and neurodegeneration for quantitative assessment of virus neurovirulence in monkeys. Using immunohistochemistry to detect cellular markers, digitalized whole-tissue section slides, and AIA algorithms, we quantitatively evaluated major features of the virus-associated CNS histopathology including cellular inflammatory infiltration, microglial activation, and neurodegeneration. Our results provide the first comprehensive comparison of the quantitative data obtained using the AIA and conventional semi-quantitative histopathological scoring.
The histopathological basis for the majority of viral encephalitides is characterized by damage to the CNS parenchyma (neuronal cell death), focal/nodular proliferation of microglia and astroglia, neuronophagia, and focal/nodular infiltration by lymphocytes and macrophages [23
]. The most comprehensive approach to develop a semi-quantitative histopathological grading scale for evaluation of flavivirus neurovirulence in monkeys was undertaken more than 40 years ago by Nathanson et al. [7
]. This grading scale was thereafter adopted or modified to assess the neurovirulence of different viruses [4
]. The published scales usually incorporate most of the histopathological features by grouping the lesions into grades based on their severity/extent and/or qualitative estimate (minimal and/or mild, moderate, severe and/or overwhelming). Some scales described in the literature are more detailed and comprehensive than others. Overall, this type of analysis is prone to subjectivity and bias and is largely influenced by a “dynamic range” of virus-induced CNS histopathology.
It should be emphasized that the original grading scales [7
] were developed to cover the “broadest spectrum” of the CNS histopathology induced by flaviviruses with varying levels of neurovirulence (listed from least to greatest level of neurovirulence): dengue type 4, yellow fever 17D, Langat, West Nile, and Japanese encephalitis viruses. When only attenuated viruses are evaluated using a scale calibrated for the severe neuropathology induced by neurovirulent wild type viruses (usually requiring a high level of biosafety), the use of such a broad grading scale might pose a significant limitation by compressing the usable dynamic range at the lower end of such a scale. In practice, those promising vaccine candidates that demonstrated sufficient attenuation during preclinical in vitro
testing and in vivo
evaluation in small laboratory animals, and then “graduated” to evaluation of neurovirulence in monkeys, would not induce severe CNS histopathology of grade 4 on these scales. Indeed, numerous studies have reported that various attenuated viruses usually induced minimal or mild and rarely moderate CNS histopathology on these scales [13
]. In our recent report [19
] and in this study, we used an expanded grading scale that included separate categorization of cellular inflammatory infiltration and microglial activation/neuronal degeneration and was tailored to the maximal magnitude of the observed virus-associated histopathology. This approach enabled us to better differentiate the degree of neurovirulence associated with a variety of attenuated flaviviruses. We believe that such an approach is favorable and caution against the use of broad-spectrum grading scales as they may not be sufficiently sensitive to detect subtle differences in neurovirulence between attenuated viruses and, if available, appropriate reference viruses. This concern is particularly important for the safety evaluation of live attenuated vaccines against neurotropic viruses and calls for new testing strategies or improvement of existing methodologies.
The need to develop more clearly defined criteria for assessment of virus neurovirulence and vaccine safety has been recognized by regulatory experts and scientists involved in vaccine development [3
]. In addition, there is a concern that expertise in performing the histopathological assessment of virus neurovirulence is waning [3
]. To address these issues, we developed a quantitative method providing an objective assessment of virus neurovirulence by accurate measurement of virus-induced neuroinflammation and neurodegeneration. To this end, the immunoreactivity for the pan-T cell marker CD3 and pan-B cell marker CD20 measured by specific image analysis algorithms was shown to provide an excellent overall quantitative estimate of the lymphocytic infiltration [22
]. In this study, the comparison of lymphocytic immunoreactivity in the CNS measured by AIA and inflammatory cell infiltration scores generated by histopathological analysis demonstrated strong and significant correlation between these two methods. However, the AIA provided a more accurate measure of lymphocytic infiltration since the issue of enumeration of perivascular cuffs which are varying in sizes and “thickness” [10
], as well as possibility of overlooking diffuse parenchymal infiltration, is virtually eliminated as every immunostained lymphocyte in the CNS ROI is identified and analyzed independently of its location.
Our previous and current study demonstrated that AIA of the CD68-IR in virus-infected CNS provides a versatile and valuable tool for quantitative evaluation of the overall microglial activation including both focal and diffuse patterns (generally referred to as microgliosis). It should be emphasized that the diffuse patterns of microglial activation are difficult or impossible to measure using routine histological stains and conventional semi-quantitative scoring analysis. Therefore, the overall microglial activation induced by neurotropic viruses could be largely underestimated, yielding inconsistent and unreliable data. In addition, our observations suggest that the magnitude and extent of CD68-IR may serve as a reliable indirect marker of neurodegeneration because of the strong and significant negative correlation between microglial activation and neurodegeneration.
Finally, we describe the first use of image analysis for direct quantitative assessment of neurodegeneration induced by flaviviruses in the CNS of monkeys. One significant problem associated with the traditional semi-quantitative assessment of neurodegeneration in the virus-infected CNS is that the majority of grading scales include the percentage of neurons that were “changed or lost”. Given the enormous number of neurons in the affected regions of the primate CNS and considering the fact that unbiased stereological methods [25
] were not used to assess the neuronal loss, it seems unlikely that such analysis provides a reliable evaluation of viral neurovirulence, and thus may fail to predict the safety of live virus vaccines. In contrast, our results show that quantitative analysis of immunoreactivity for neuron-specific antigen NeuN, a selective neuronal marker [26
], is a useful and reliable method for the assessment of neurodegeneration in the virus-infected CNS. NeuN-immunostaining is remarkably superior to routine histological stains such as Nissl because it provides selective staining of neuronal nuclei, cytoplasm, and proximal processes with greater intensity and better definition, and has the advantage of clear delineation of neurons from other cell types (personal observation and Refs. [28
]). In this study, AIA revealed significant decrease of NeuN-IR in sites of neurodegeneration attributed to infection with more neurovirulent viruses (i.e., TBEV/DEN4Δ30 or LGTV). The decreased expression of neuronal protein NeuN was indicative of both neurodegeneration and neuronal loss. Although strong and significant correlations between NeuN-IR and relevant semi-quantitative scores were established in this study, we strongly believe that the quantitative analysis of neurodegeneration using neuronal markers provides a more reliable tool for evaluation of virus neurovirulence than the semi-quantitative analysis of routine stains, especially since the predominant cells infected by neurotropic viruses are neurons.
The quantitative approach described here measures the amount of immunoreactivity (number of positive pixels per mm2 of tissue), and it is different from two-dimensional cell profile counting or stereological counting techniques. We believe that neither cell profile counting nor stereology could be reliably applied in the context of complex features of virus-induced neuroinflammation when individual inflammatory cells form densely packed layers or clusters. For assessment of virus neurovirulence, it is not important to know the absolute number of inflammatory cells or exact percentage of neurons that were lost. Rather, the meaningful quantitative comparisons can be made based on the magnitude of the cellular inflammatory responses and degree of virus-induced neurodegeneration measured by the amount of immunoreactivity for the relevant cellular marker using AIA.
In conclusion, we observed strong and significant correlations between quantitative measures of virus-induced neuroinflammation and neurodegeneration obtained by AIA and relevant semi-quantitative scores generated by conventional histopathological analysis suggesting that the outcomes of these two methods can be viewed as functions of one another. However, the presence of a correlation does not imply that these methods possess equal sensitivity and reproducibility. We show that AIA offers a number of significant advantages over traditional semi-quantitative approach. First, neuroinflammation and neurodegeneration are measured using specific cellular markers, thus providing in-depth information regarding the cell types involved in host response to viral infection of the CNS. Second, quantitative analysis of the immunoreactivity for each specific cell type provides an objective and accurate measure of the cellular inflammatory infiltration, microglial activation, and neurodegeneration regardless of their topographical patterns, thus eliminating the need for excessive manual counting and visual impression-based assessment. Third, the use of the AIA algorithms, once established, is relatively simple and time efficient. This is in contrast to semi-quantitative histopathological scoring, which is tedious and time-consuming. AIA algorithms can be applied to entire sections of the CNS regions, which dramatically reduces analysis time. Indeed, the time needed to apply the AIA algorithms within a given CNS region does not depend on the severity of neuroinflammation or neurodegeneration, while the time required for distinguishing, counting, and grading inflammatory lesions using a semi-quantitative approach is dependent on their severity. Fourth, this approach may be generally applicable for rapid and unbiased assessment of a wide variety of CNS pathologies. Because the AIA of neuroinflammation and neurodegeneration is an accurate and reliable method, it may enable robust statistical comparisons of viruses with varying neurovirulence, and may help to establish consistent analytical standards across laboratories and regulatory agencies. Given that an adequate number of cellular markers is analyzed, this method provides objective quantitative criteria for evaluation of virus neurovirulence and will facilitate decision-making regarding the safety of live virus vaccine candidates and their advancement to clinical evaluation in humans.
The feasibility of the AIA for studies of viral neuropathogenesis/neurovirulence and vaccine safety evaluation will rely on the accessibility of digital scanners and image analysis software to the scientific and regulatory communities. Nevertheless, once acquired, the digitalized whole-tissue section slides (virtual slides) can be stored indefinitely without loss of quality, and viewed, shared, annotated, and analyzed by multiple investigators in real time across the world. It is our hope that these outstanding advantages of modern digital pathology will markedly advance many fields of research including but not limited to virology, neuroinflammation, neuroscience, and vaccinology in the near future.