C-C motif chemokine receptor 2 and 7 synergistically control inflammatory monocyte recruitment but the infecting virus dictates monocyte function in the brain.

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function.

Case Series of Jamestown Canyon Virus Infections with Neurologic Outcomes, Canada, 2011-2016.

Jamestown Canyon virus (JCV) is a mosquitoborne orthobunyavirus in the California serogroup that circulates throughout Canada and the United States. Most JCV exposures result in asymptomatic infection or a mild febrile illness, but JCV can also cause neurologic diseases, such as meningitis and encephalitis. We describe a case series of confirmed JCV-mediated neuroinvasive disease among persons from the provinces of British Columbia, Alberta, Quebec, and Nova Scotia, Canada, during 2011-2016. We highlight the case definitions, epidemiology, unique features and clinical manifestations, disease seasonality, and outcomes for those cases. Two of the patients (from Quebec and Nova Scotia) might have acquired JCV infections during travel to the northeastern region of the United States. This case series collectively demonstrates JCV's wide distribution and indicates the need for increased awareness of JCV as the underlying cause of meningitis/meningoencephalitis during mosquito season.

Current Status of La Crosse Virus in North America and Potential for Future Spread.

La Crosse virus (LACV) is an arthropod-borne RNA virus with substantial potential for future spread in North America. La Crosse virus is responsible for La Crosse encephalitis, a leading cause of arboviral encephalitis in children in the United States. Primarily transmitted by Aedes triseriatus (Eastern treehole) mosquitos and amplified by small mammal hosts, LACV has caused infections throughout the upper Midwest and, more recently, the mid-Atlantic and southeastern United States. Notably, in recent years, infections have also been identified increasingly in the Appalachian region. Anthropogenic and environmental factors have likely contributed to recent LACV spread, including the introduction of invasive vector species (especially Ae. albopictus), biotic interactions between and among vector and host species, land-use change, habitat disturbance, increased human travel and transport, and rising global temperatures. Prevention and control strategies, such as increased surveillance of vector and host populations, increased awareness among populations at risk for infection, and increased awareness among physicians are needed to limit future spread. Continued climate change with increases in global temperatures and erratic weather patterns may result in the expansion of competent mosquito vector species and thus could facilitate the geographic spread of LACV.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

La Crosse Virus Disease in the United States, 2003-2019.

La Crosse virus (LACV) is an arthropod-borne virus that can cause a nonspecific febrile illness, meningitis, or encephalitis. We reviewed U.S. LACV surveillance data for 2003-2019, including human disease cases and nonhuman infections. Overall, 318 counties in 27 states, principally in the Great Lakes, mid-Atlantic, and southeastern regions, reported LACV activity. A total of 1,281 human LACV disease cases were reported, including 1,183 (92%) neuroinvasive disease cases. The median age of cases was 8 years (range: 1 month-95 years); 1,130 (88%) were aged < 18 years, and 754 (59%) were male. The most common clinical syndromes were encephalitis (N = 960; 75%) and meningitis (N = 219, 17%). The case fatality rate was 1% (N = 15). A median of 74 cases (range: 35-130) was reported per year. The average annual national incidence of neuroinvasive disease cases was 0.02 per 100,000 persons. West Virginia, North Carolina, Tennessee, and Ohio had the highest average annual state incidences (0.16-0.61 per 100,000), accounting for 80% (N = 1,030) of cases. No animal LACV infections were reported. Nine states reported LACV-positive mosquito pools, including three states with no reported human disease cases. La Crosse virus is the most common cause of pediatric neuroinvasive arboviral disease in the United States. However, surveillance data likely underestimate LACV disease incidence. Healthcare providers should consider LACV disease in patients, especially children, with febrile illness, meningitis, or encephalitis in areas where the virus circulates and advise their patients on ways to prevent mosquito bites.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

BACKGROUND: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. METHODS: To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. RESULTS: BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. CONCLUSIONS: These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

La Crosse virus spread within the mosquito population in Knox County, TN.

In Appalachia, La Crosse virus (LACV) is a leading pediatric arbovirus and public health concern for children under 16 years. LACV is transmitted via the bite of an infected Aedes mosquito. Thus, it is imperative to understand the dynamics of the local vector population in order to assess risk and transmission. Using entomological data collected from Knox County, Tennessee in 2013, we formulate an environmentally-driven system of ordinary differential equations to model mosquito population dynamics over a single season. Further, we include infected compartments to represent LACV transmission within the mosquito population. Findings suggest that the model, with dependence on degree days and accumulated precipitation, can closely describe field data. This model confirms the need to include these environmental variables when planning control strategies.

Enhancement of infectivity of insect cell-derived La Crosse Virus by human serum.

Given the dual life cycle of arboviruses in insect and animal hosts and the importance of serum factors as a first line antiviral defense, we have examined the outcome of interactions between the arbovirus La Crosse Virus (LACV) and human serum. To mimic the life cycle between species, we used LACV derived from insect (I-LACV) and human keratinocyte (HaCaT) cells. Incubation of I-LACV with normal human serum did not result in neutralization, but instead stabilized I-LACV virions and enhanced the amount of infectious virus. Enhanced infectivity was also seen with heat-inactivated serum devoid of complement activity and with serum from a range of animals including mouse, ferret, and non-human primates. Depletion of antibodies from serum resulted in loss of enhancement of infectivity and sucrose gradient sedimentation assays showed IgG co-sedimenting with I-LACV particles. In agreement with our results with I-LACV, HaCaT-derived LACV was not neutralized by complement or antibodies in normal human serum. However, in contrast to I-LACV, HaCaT-derived LACV infectivity was stable when incubated alone and treatment with serum did not enhance infectivity. Our results indicate that LACV derived from insect cells differs substantially from virus derived from human cells, with I-LACV being dependent on serum factors to enhance infectivity. These findings suggest that understanding differential composition of insect versus animal cell-derived LACV may form the foundation for potential new antiviral approaches.

Seroprevalence of Jamestown Canyon virus in the Japanese general population.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. METHODS: We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. RESULTS: The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (- 0.075) and standard deviation (0.092) values using Japanese donors' sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. CONCLUSIONS: Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.

Novel Ionophores Active against La Crosse Virus Identified through Rapid Antiviral Screening.

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.

Double Crossed: A Case of La Crosse Encephalitis.

CASE REPORT: A 10-year-old male with T1DM and recent travel to North Carolina presented to an ED with 1 day of fever, vomiting, and headaches. He was discharged home with the presumptive diagnosis of viral gastroenteritis but returned nine hours later, agitated, and unable to speak. CSF showed pleocytosis. MRI brain was normal, and EEG showed intermittent seizures. He was started on antiepileptics. Antibiotics were discontinued after negative bacterial work-up. Repeat MRI brain one week later showed enhancement in the left cerebral cortex. IVIG was started due to concern for autoimmune encephalitis. Repeat lumbar puncture was positive for La Crosse virus IgM. DISCUSSION: This is the first case of La Crosse encephalitis (LACe) reported in Rhode Island.1 La Crosse virus (LACv) is a ssRNA Bunyavirus transmitted by the eastern tree-hole mosquito typically between July and September. LACv is endemic to the upper Midwestern US and Appalachia. In 2018, 81 of 86 total cases reported by the CDC were pediatric. Children are more likely to present with vomiting, seizures, and focal cortical inflammation or cerebral edema on brain imaging. IgM may be negative early in the disease course. Treatment is antiepileptics and supportive care.

La Crosse Virus Infection of Human Keratinocytes Leads to Interferon-Dependent Apoptosis of Bystander Non-Infected Cells In Vitro.

Resident cells in the skin serve as the first innate line of defense against insect-borne pathogens, but the role of these cell types in promoting or limiting arbovirus replication is not completely understood. Here, we have examined the outcome of infection of cultured human keratinocyte cells with La Crosse virus (LACV), using a spontaneously transformed cell line, HaCaT. In single cycle infections, keratinocyte HaCaT cells supported rapid and high level LACV replication, resulting in high virus yields and extensive caspase-dependent cell death. By contrast, multi-cycle LACV replication in HaCaT cells was restricted by an antiviral response elicited by the production of both IFN-ß and IFN-λ. During low multiplicity LACV infections, HaCaT cell death was seen in non-infected bystander cells. Media from LACV-infected cells induced caspase-dependent killing of naïve non-infected HaCaT cells, and this bystander cell death was relieved by IFN-ß neutralizing antibodies or by an inhibitor of JAK-STAT signaling. Naïve HaCaT cells showed dose-dependent killing by treatment with exogenous IFN-ß but not IFN-λ. Our data suggest a model whereby keratinocytes produce IFNs which limit virus spread through both antiviral signaling and by induction of bystander cell death of potential new target cells for infection.

Throw out the Map: Neuropathogenesis of the Globally Expanding California Serogroup of Orthobunyaviruses.

The California serogroup (CSG) comprises 18 serologically and genetically related mosquito-borne orthobunyaviruses. Of these viruses, at least seven have been shown to cause neurological disease in humans, including the leading cause of pediatric arboviral encephalitis in the USA, La Crosse virus. Despite the disease burden from these viruses, much is still unknown about the CSG viruses. This review summarizes our current knowledge of the CSG viruses, including human disease and the mechanisms of neuropathogenesis.

Effects of La Crosse virus infection on the host-seeking behavior and levels of two neurotransmitters in Aedes triseriatus.

BACKGROUND: La Crosse virus (LACV) infection has been shown to manipulate the blood-feeding behaviors of its main vector, Aedes triseriatus. Here, we investigated the effects of virus infection on serotonin and dopamine and their potential roles in host-seeking. In mosquitoes, serotonin depletion has been shown to interfere with blood-feeding but not host-seeking. Dopamine depletion does not affect either blood-feeding or host-seeking; elevations of dopamine, however, has been shown to inhibit host-seeking. The purpose of this study was to determine the effects of LACV infection on the host-seeking behavior of and neurotransmitter levels in Ae. triseriatus. METHODS: Host-seeking behavior was evaluated using a uni-port olfactometer and a membrane feeder assay. Levels of serotonin and dopamine in infected and control mosquito heads were measured using HPLC-ED. RESULTS: Infection with LACV significantly inhibited the activation and attraction of Ae. triseriatus females to a host. A higher proportion of uninfected Ae. triseriatus females were activated by the presence of a host compared to infected mosquitoes and more uninfected mosquitoes were full responders (95.7%) compared to infected ones (91.1%). However, infection with LACV did not significantly affect the landing, probing, or blood-feeding rates of female mosquitoes. LACV-infected mosquitoes had lower serotonin levels than controls (104.5 vs 138.3 pg/head) while the dopamine levels were not affected by infection status (282.3 vs 237 pg/head). CONCLUSIONS: Our work suggests that virus-induced reduction of serotonin is related to previously reported blood-feeding alterations in LACV-infected mosquitoes and could lead to enhanced transmission and increased vectorial capacity. In addition, some aspects of host-seeking were inhibited by virus infection.

Differences in Neuropathogenesis of Encephalitic California Serogroup Viruses.

The California serogroup of orthobunyaviruses comprises a group of mosquitoborne viruses, including La Crosse (LACV), snowshoe hare (SSHV), Tahyna (TAHV), Jamestown Canyon (JCV), and Inkoo (INKV) viruses, that cause neurologic disease in humans of differing ages with varying incidences. To determine how the pathogenesis of these viruses differs, we compared their ability to induce disease in mice and replicate and induce cell death in vitro. In mice, LACV, TAHV, and SSHV induced neurologic disease after intraperitoneal and intranasal inoculation, and JCV induced disease only after intranasal inoculation. INKV rarely induced disease, which correlated with less viral antigen in the brain than the other viruses. In vitro, all viruses replicated to high titers; however, LACV, SSHV, and TAHV induced high cell death, whereas JCV and INKV did not. Results demonstrated that CSG viruses differ in neuropathogenesis in vitro and in vivo, which correlates with the differences in pathogenesis reported in humans.

Enhanced Arboviral Surveillance to Increase Detection of Jamestown Canyon Virus Infections, Wisconsin, 2011-2016.

Jamestown Canyon virus (JCV), a mosquito-borne Orthobunyavirus (within the California serogroup), can cause severe neuroinvasive disease. According to national data during 2000-2013, 42% of the 31 documented JCV disease cases in the United States were detected in residents from Wisconsin. The Wisconsin Division of Public Health enhanced JCV surveillance by implementing routine use of JCV-specific immunoglobulin M (IgM) antibody testing followed by confirmatory JCV-specific plaque reduction neutralization testing on all patients with suspected cases of arboviral infection who had tests positive for arboviral immunoglobin at commercial laboratories. During 2011-2016, of the 287 Wisconsin specimens tested on the Arbovirus IgM Antibody Panel, 30 JCV cases were identified (26 confirmed and four probable). Twenty-seven (90%) JCV cases were detected after 2013. Among all cases, 17 (56%) were male and the median age was 54 years (range: 10-84 years). Fifteen patients had neuroinvasive disease, including meningitis (n = 9) and meningoencephalitis (n = 6). Although historically considered rare, the relatively high rate (0.12 cases/100,000 population) of diagnosis of JCV infections among Wisconsin residents during 2013-2016 compared with that in previous years suggests occurrence is widespread throughout Wisconsin and historically may have been under-recognized. This study aims to raise awareness of JCV infection for differential diagnosis among the arboviral diseases. Improved and timely diagnosis of arboviral disease is important in that it will provide more information regarding emerging infections and promote preventive measures to avoid mosquito-borne exposure and infection among residents of and visitors to affected areas.

Serological surveillance for Tahyna virus (California encephalitis orthobunyavirus, Peribunyaviridae) neutralizing antibodies in wild ungulates in Austria, Hungary and Romania.

A serosurvey for Tahyna virus (TAHV), a mosquito-borne California encephalitis orthobunyavirus (Peribunyaviridae) endemic to Europe, was performed to estimate the activity of TAHV on a broad geographic scale. Sera from wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were collected from Austria, Hungary and Romania. Samples were tested for neutralizing antibodies against TAHV using a virus microneutralization assay. The results demonstrate that TAHV transmission to mammals is widespread in Europe, particularly in the wild boar population where the mean rate of seroconversion is 15.2%.

Cutting Edge: CCR2 Is Not Required for Ly6Chi Monocyte Egress from the Bone Marrow but Is Necessary for Migration within the Brain in La Crosse Virus Encephalitis.

Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2-/- mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.