In this study, NY99 and Eg101, two phylogenetically closely related lineage 1 WNV strains (95.4 and 99.6 % identical at the nucleotide and amino acid level, respectively) of contrasting pathogenicity, were compared with regard to induction of clinical disease and immune responses in the periphery and brain of mice. We observed that both viral strains were neuroinvasive and induced a robust anti-WNV immune response when inoculated subcutaneously at similar doses. While the production of type 1 interferon and WNV-specific antibodies were higher in WNV NY99 infected mice, the innate and adaptive cellular immune response was more predominant in WNV Eg101 infected mice. Furthermore, by demonstrating that immune cells accumulation was higher in the spleens and brains of mice infected with the non-virulent WNV strain, we suggest that pathogenicity is not directly correlated to the number of immune cells in the spleen and brain. Moreover, our results demonstrating an inverse correlation between the number of immune cells in the brain and the levels of pro-inflammatory mediators indicate that WNV-infected/activated resident brain cells not infiltrating immune cells are the primary source of these inflammatory mediators and their increased levels correlate with high brain viral load and severe WNV disease.
Enhanced WNV-specific antibodies, type 1 interferon and inflammatory response in WNV NY99 infected mice
Despite the high degree of homology between the two strains, WNV Eg101 is non-pathogenic in adult mice after peripheral inoculation. WNV Eg101 infected mice rarely displayed clinical signs and no mortality was observed in these mice [
17]. Similar to our study, it has been demonstrated that peripheral inoculation of the Eg101 strain into MBR/ICR albino Swiss mice rarely resulted in fatal illness in mice older than 4 weeks. However, direct intracranial inoculation of WNV Eg101 in same age-matched mice was lethal [
33], thereby suggesting the role of peripheral immune response in limiting virus replication, neuroinvasion, and severe WNV disease in WNV Eg101-infected mice. Interestingly, initial virus replication was similar in mice after infection with both WNV NY99 and WNV Eg101 strains as demonstrated by high and comparable viremia at day 3 after inoculation in both the groups. However, viremia in WNV Eg101 infected mice was short-lived than WNV NY99 infected mice, suggesting the induction of an early and effective immune response in these mice. It has been demonstrated that type I interferon and WNV-specific immunoglobulins are rapidly produced following WNV infection and are essential for suppressing viremia and virus dissemination [
8,
9]. In this study, we also observed a rapid and robust production of type I interferon and WNV-specific antibodies in both groups. Surprisingly, we observed reduced levels of IFN-α and IFN-β, WNV-specific IgM and IgG antibodies and neutralizing antibodies, in the serum of WNV Eg101 infected mice than WNV NY99 infected mice.
Similar to interferon and WNV-specific immunoglobulins, WNV-induced pro-inflammatory mediators are also known to protect mice from lethal WNV disease [
11,
14,
31]. TNF-α and IL-1β promotes immune cell trafficking into the brain [
14,
31]. CXCL10 promotes trafficking of WNV-specific CD8
+ T cells via binding to its cognate receptor CXCR3 [
11]. Enhanced expression of CCL3, CCL4, and CCL5 by WNV infection leads to CCR5-dependent trafficking of CD4
+ and CD8
+ T cells, NK cells, and macrophages into the brain [
32]. CCL2 is important in the trafficking of inflammatory monocyte into the brain after WNV infection [
33]. Interestingly in this study, we observed an enhanced production of key pro-inflammatory cytokines and chemokines in the serum of WNV NY99 infected mice than WNV Eg101 infected mice despite increased mortality observed in WNV NY99 infected mice. One possible explanation for these differences is increased viral antigen load in WNV NY99 infected mice, leading to enhanced type 1 interferon, antibodies, and inflammatory mediators in these mice.
Increased WNV-specific innate and adaptive immune cell response in WNV Eg101 infected mice
WNV dissemination is also countered by the effector functions of innate and adaptive immune cells. Several studies have demonstrated that immune cells such as monocyte/macrophages and T cells play an indispensable role in the control of WNV infection [
10,
12,
34]. The role of NK cells in regulating tissue tropism to WNV infection or in controlling WNV replication in vivo has not been clearly defined [
35,
36]. However, various in vitro and ex vivo studies have suggested their role in the control of WNV infection by their recognition and elimination of infected cells [
37,
38].
Unlike the interferon and inflammatory response, we observed significantly higher numbers of total leukocytes, T cells, NK cells, and monocytes/macrophages in the spleens of WNV Eg101 infected mice. Moreover, total number and percentage of IFN-γ and TNF-α producing WNV-specific CD8 T cells were also significantly higher in WNV Eg101 infected mice. This is surprising as interferon and various inflammatory mediators have been demonstrated to enhance immune cell responses in WNV infection [
39‐
41]. Another interesting finding is the increased percentage of NK cells and monocytes/macrophages observed in the spleens of WNV Eg101 infected mice suggesting a predominant role of innate immune cells in limiting virus replication in these mice. Collectively, these data demonstrate that induction of virus-specific effector immune cell response limits virus replication and severe WNV disease in Eg101 infected mice.
It could be argued that differences in virulence observed after infection with WNV NY99 and WNV Eg101 strains in this study are virus dose-dependent. For example, a lower dose (10 PFU) of WNV NY99 could give similar results to those seen with 100 PFU of WNV Eg101, or a higher dose of WNV Eg101 (1000 PFU) could give similar results to those seen with 100 PFU of WNV NY99. However, our previously published data demonstrate that a lower dose (10 PFU) of WNV NY99 results in 37 % mortality when compared to 0 % mortality as observed with 100 PFU of WNV Eg101 [
19,
20]. This data clearly demonstrates that a lower dose of WNV NY99 (10 PFU) does not give similar results to those observed with 100 PFU of WNV Eg101. Moreover, our published data demonstrate no significant difference in terms of survival, clinical score, viremia, WNV-specific IgG and IgM antibodies, and neutralizing antibodies in mice infected with different doses (100 or 1000 PFU) of WNV Eg101 [
17].
WNV entry and replication into the CNS represents an important event in the clinical outcome of WNV disease. Following WNV infection, immune cells, especially T cells and macrophages, travel into the brain and participate in viral clearance [
10,
12,
34]. Moreover, the production of IFN-α and –β is essential in suppressing virus replication in the brain [
8]. In this study, we observed significantly higher virus titers in the brains of WNV NY99 infected mice when compared to WNV Eg101 infected mice at both days 8 and 10 after inoculation. Similar to the periphery, the levels of IFN-α and -β were significantly lower in the brains of WNV Eg101 infected mice. Interestingly, similar to the spleen, the total number of immune cells migrating into the brains of WNV Eg101 infected mice was higher than in WNV NY99 infected mice. However, unlike the spleen, we only observed a significantly higher number of T cells in the brains of WNV Eg101 infected mice. No difference was observed between the number of NK cells and monocytes/macrophages in the brains of WNV Eg101 and WNV NY99 infected mice. Similarly, we also observed a high percentage of T cells in the brains of WNV Eg101 infected mice. On the other hand, we observed a low percentage of monocytes/macrophages in the brains of WNV Eg101 infected mice. Collectively, these data are consistent with previous observations that virus-specific T cells are the predominant cell types in clearing WNV from the brain.
Absence of functional CD8
+ or CD4
+ T cells resulted in failure to clear WNV from infected neurons in the CNS [
10,
12]. It has been demonstrated that increased CD8
+ T cells contribute to the immunopathology with high doses of WNV challenge [
42]. It is also known that migration of inflammatory monocytes plays a pathogenic role during WNVE [
33,
43,
44]. Similarly, immune cell-mediated pathology in the brain has been demonstrated in several neurotropic virus infections such as tick-borne encephalitis virus, Murray Valley encephalitis virus, and Theiler's murine encephalomyelitis virus [
45‐
48]. However, in this study, we did not observe any immunopathology associated with increased immune cell migration into the brains of WNV Eg101 infected mice. This may be due to significantly low levels of inflammatory mediators in the brains of WNV Eg101 infected mice.
Negative correlation between migrating immune cells and associated inflammation
One of the most intriguing findings of our study was the difference in the inflammatory responses observed in the brain. The migration of leukocytes into WNV-infected brain is associated with increased expression of several cytokines and chemokines in the brain. Also, increased expression of cytokines and chemokines in the WNV-infected brain is usually associated with enhanced trafficking of leukocytes into the brain [
11,
14,
32]. In contrast, we demonstrate that increased accumulation of leukocytes in the brains of WNV Eg101 (non-virulent strain) infected mice was associated with reduced production of pro-inflammatory cytokines and chemokines. This phenomenon of significantly increased migration of leukocytes in to the brain of WNV Eg101 infected mice despite reduced production of chemokines and cytokines could be due to the higher numbers of WNV-specific primed cells in the periphery of these mice. Lower numbers of infiltrating cells in the brains of WNV NY99 infected mice could be due to different chemokine receptor expression on the leukocytes leading to differential responsiveness to the chemokines emanating from the brain. Differential virus cytopathic effects of WNV NY99 and WNV Eg101 viruses could also result in difference in leukocytes numbers observed in the brain and spleen. Moreover, we observed a dramatic increase in various chemokine and cytokine levels in the brains of WNV NY99 (virulent strain) mice, which correlates with high brain viral load detected in these mice. These data indicate that infiltrating immune cells may play a limited role in the production of these inflammatory mediators and WNV-infected/activated resident brain cells are the primary source(s) of these inflammatory mediators in the brain. However, our data do not rule out the possibility that monocytes/macrophages/microglial may be less activated in Eg101 infected mice brains and may produce less pro-inflammatory cytokines, or the infiltrating immune cell population is suppressing/regulating brain cytokine production.
Resident brain cells such as neurons and astrocytes have been associated with producing inflammatory mediators in several neuroinflammatory scenarios including WNV infection. It has been previously demonstrated by us and others that WNV-infected neurons directly contribute to inflammation by secreting various chemokines and cytokines [
11,
13]. Similarly, we and others have previously demonstrated that WNV-infected and activated astrocytes can produce high levels of cytokines and chemokines [
49,
50]. Recent studies of WNV infection in brain slices cultures demonstrate the ability of the brain cells to produce pro-inflammatory cytokine response and to activate astrocytes and microglial cells in the absence of infiltrating inflammatory cells and systemic immune responses [
51,
52].
Neuronal death is the hallmark of WNVE. Like several encephalitic viruses, WNV induces neuronal death both in a direct or indirect manner, and in the latter case, through inflammation [
13,
53,
54]. Therefore, increased neuronal death and subsequent pathology in WNV NY99 infected mice could be due to (i) virus-mediated neuronal apoptosis and (ii) bystander neuronal damage due to pro-inflammatory molecules released by resident brain cells, such as neurons and activated glial cells. Taken together, these data demonstrate that induction of effective and balanced immune response in the periphery and brain of WNV Eg101 infected mice limits virus replication and severe WNV disease in these mice.