Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Eastern Equine Encephalitis Virus in Mexican Wolf Pups at Zoo, Michigan, USA.

During the 2019 Eastern equine encephalitis virus (EEEV) outbreak in Michigan, two 2-month old Mexican wolf pups experienced neurologic signs, lymphohistiocytic neutrophilic meningoencephalitis with neuronal necrosis and neuronophagia, and acute death. We identified EEEV by reverse transcription real-time PCR and in situ hybridization. Vector mosquitoes were trapped at the zoo.

Rational design of a live-attenuated eastern equine encephalitis virus vaccine through informed mutation of virulence determinants.

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses.

Magnetic resonance imaging, clinicopathologic findings, and clinical progression of a puppy with confirmed Eastern equine encephalitis virus.

A 5-month-old puppy was evaluated for rapidly progressive neurologic signs and pyrexia. Magnetic resonance imaging showed multifocal meningoencephalitis with transtentorial and foramen magnum herniation. A cerebrospinal fluid tap revealed highly cellular fluid, and the puppy was euthanized. Histopathology showed lymphoplasmacytic and neutrophilic meningoencephalitis. Viral polymerase chain reaction testing for Eastern equine encephalitis was positive. Rapid progression of neurologic signs and respiratory arrest necessitated mechanical ventilation. Severe hypernatremia, most consistent with central diabetes insipidus, developed. Key clinical message: Transtentorial and foramen magnum herniation and high cerebrospinal fluid cell counts may be indicators of poor prognosis. Brain death, respiratory arrest, and central diabetes insipidus may also ensue with Eastern equine encephalitis infection.

Imagerie par résonance magnétique, résultats clinicopathologiques et progression clinique d'un cas confirmé d'infection par le virus de l'encéphalite équine de l'Est chez un chiot. Un chiot de 5 mois a été évalué pour des signes neurologiques progressant rapidement et une pyrexie. L'imagerie par résonance magnétique a montré une méningo-encéphalite multifocale avec hernies transtentorielle et au foramen magnum. Un prélèvement de liquide céphalo-rachidien a révélé un liquide hautement cellulaire et le chiot a été euthanasié. L'histopathologie a montré une méningo-encéphalite lymphoplasmocytaire et neutrophilique. Un test de réaction en chaîne par la polymérase pour l'encéphalite équine de l'Est était positif. La progression rapide des signes neurologiques et l'arrêt respiratoire ont nécessité une ventilation mécanique. Une hypernatrémie sévère, plus compatible avec un diabète insipide central, s'est développée.Message clinique clé:Une hernie transtentorielle et au foramen magnum de même qu'un nombre élevé de cellules dans le liquide céphalorachidien peuvent être des indicateurs de mauvais pronostic. La mort cérébrale, l'arrêt respiratoire et le diabète insipide central peuvent également s'ensuivre avec une infection par l'encéphalite équine de l'Est.(Traduit par Dr Serge Messier).

Cases of Eastern equine encephalitis in humans associated with Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes with the virus in New York State from 1971 to 2012 by analysis of aggregated published data.

From 1971 to 2012, in New York State, years with human Eastern equine encephalitis (EEE) were more strongly associated with the presence of Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes infected with the EEE virus (Fisher's exact test, one-sided P = 0.005, 0.03, 0.03) than with Culiseta morsitans, Aedes vexans, Culex pipiens-restuans, Anopheles quadrimaculatus or Anopheles punctipennis (P = 0.05, 0.40, 0.33, 1.00, 1.00). The estimated relative risk of a case in a year in which the virus was detected vs. not detected was 14.67 for Ae. canadensis, 6.38 for Cq. perturbans and 5.50 for Cs. morsitans. In all 5 years with a case, Cs. melanura with the virus was detected. In no year was there a case in the absence of Cs. melanura with the virus. There were 18 years with no case in the presence of Cs. melanura with the virus. Such observations may identify the time of increased risk, and when the methods may be used to prevent or reduce exposure to vector mosquito species in this geographic region.

Five Emerging Neuroinvasive Arboviral Diseases: Cache Valley, Eastern Equine Encephalitis, Jamestown Canyon, Powassan, and Usutu.

There are many arthropod-borne viruses (arboviruses) capable of neuroinvasion, with West Nile virus being one of the most well known. In this review, we highlight five rarer emerging or reemerging arboviruses capable of neuroinvasion: Cache Valley, eastern equine encephalitis, Jamestown Canyon, Powassan, and Usutu viruses. Cache Valley and Jamestown Canyon viruses likely circulate throughout most of North America, while eastern equine encephalitis and Powassan viruses typically circulate in the eastern half. Usutu virus is not currently circulating in North America, but has the potential to be introduced in the future given similar climate, vectors, and host species to Europe (where it has been circulating). Health care providers should contact their state or local health departments with any questions regarding arboviral disease surveillance, diagnosis, treatment, or prevention. To prevent neuroinvasive arboviral diseases, use of insect repellent and other mosquito and tick bite prevention strategies are key.

Characterization and pathogenesis of aerosolized eastern equine encephalitis in the common marmoset (Callithrix jacchus).

BACKGROUND: Licensed antiviral therapeutics and vaccines to protect against eastern equine encephalitis virus (EEEV) in humans currently do not exist. Animal models that faithfully recapitulate the clinical characteristics of human EEEV encephalitic disease, including fever, drowsiness, anorexia, and neurological signs such as seizures, are needed to satisfy requirements of the Food and Drug Administration (FDA) for clinical product licensing under the Animal Rule. METHODS: In an effort to meet this requirement, we estimated the median lethal dose and described the pathogenesis of aerosolized EEEV in the common marmoset (Callithrix jacchus). Five marmosets were exposed to aerosolized EEEV FL93-939 in doses ranging from 2.4 × 101 PFU to 7.95 × 105 PFU. RESULTS: The median lethal dose was estimated to be 2.05 × 102 PFU. Lethality was observed as early as day 4 post-exposure in the highest-dosed marmoset but animals at lower inhaled doses had a protracted disease course where humane study endpoint was not met until as late as day 19 post-exposure. Clinical signs were observed as early as 3 to 4 days post-exposure, including fever, ruffled fur, decreased grooming, and leukocytosis. Clinical signs increased in severity as disease progressed to include decreased body weight, subdued behavior, tremors, and lack of balance. Fever was observed as early as day 2-3 post-exposure in the highest dose groups and hypothermia was observed in several cases as animals became moribund. Infectious virus was found in several key tissues, including brain, liver, kidney, and several lymph nodes. Clinical hematology results included early neutrophilia, lymphopenia, and thrombocytopenia. Key pathological changes included meningoencephalitis and retinitis. Immunohistochemical staining for viral antigen was positive in the brain, retina, and lymph nodes. More intense and widespread IHC labeling occurred with increased aerosol dose. CONCLUSION: We have estimated the medial lethal dose of aerosolized EEEV and described the pathology of clinical disease in the marmoset model. The results demonstrate that the marmoset is an animal model suitable for emulation of human EEEV disease in the development of medical countermeasures.

Characterizing areas of potential human exposure to eastern equine encephalitis virus using serological and clinical data from horses.

Eastern equine encephalitis (EEE) is a rare but severe emerging vector-borne disease affecting human and animal populations in the northeastern United States where it is endemic. Key knowledge gaps remain about the epidemiology of EEE virus (EEEV) in areas where its emergence has more recently been reported. In Eastern Canada, viral activity has been recorded in mosquitoes and horses throughout the 2000s but cases of EEEV in humans have not been reported so far. This study was designed to provide an assessment of possible EEEV human exposure by modelling environmental risk factors for EEEV in horses, identifying high-risk environments and mapping risk in the province of Quebec, Canada. According to logistic models, being located near wooded swamps was a risk factor for seropositivity or disease in horses [odds ratio (OR) 4·15, 95% confidence interval (CI) 1·16-14·8) whereas being located on agricultural lands was identified as protective (OR 0·75, 95% CI 0·62-0·92). A better understanding of the environmental risk of exposure to EEEV in Canada provides veterinary and public health officials with enhanced means to more effectively monitor the emergence of this public health risk and design targeted surveillance and preventive measures.

Eastern equine encephalitis.

We describe a patient who died from a fulminant presentation of encephalitis. After an exhaustive search, we found no treatable cause. Postmortem PCR analysis of brain tissue led to a diagnosis of eastern equine encephalitis. We have identified several clinical pearls that may assist others in making the diagnosis earlier in the disease course.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Antibody and Viral Nucleic Acid Testing of Serum and Cerebrospinal Fluid for Diagnosis of Eastern Equine Encephalitis.

Eastern equine encephalitis diagnostic serum antibody can appear 6 days after the onset of symptoms, and its numbers can increase 4-fold in 4 days, arguing for early and frequent serum testing. In populations where cerebrospinal fluid viral nucleic acid testing sensitivity and specificity remain undetermined, cerebrospinal antibody testing should also be performed.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.

Eastern equine encephalitis: a classical case.

We present the case of a 40-year-old man with rapidly deteriorating acute meningoencephalitis, finally diagnosed as eastern equine encephalitis (EEE). The course and timing in this patient are quite characteristic. EEE is an arthropod borneviral illness in which the mosquito serves as the vector. It is amongst the most severe of the arbovirus encephalitides and has a high mortality and morbidity. In nonfatal cases, residual neurological deficits are often severe and permanent. North American lineage of EEE is mainly found in the northeast especially along the coastal areas. EEE is primarily found in horses and other domestic mammals that remain outdoors nocturnally. In humans, symptoms range from nonspecific constitutional features to catastrophic neurological sequelae including death. Hyponatremia and increased cerebrospinal fluid (CSF) white blood cell count are independent predictors of poor outcomes. Diagnosis is suggestive by demonstrating IgM antibody in blood or CSF samples and can be confirmed by other serologic assay including immunohistochemistry and polymerase chain reaction (PCR). Measures recommended by the Center for Disease Control (CDC) for prevention include using repellents, protective clothing, screens, and eradication of mosquito breeding areas. EEE remains without cure and prevention is the best medicine.

Virology, ecology, epidemiology, pathology, and treatment of eastern equine encephalitis.

Eastern equine encephalitis (EEE) was one of the first-recognized neuroinvasive arboviral diseases in North America, and it remains the most lethal. Although EEE is known to have periodic spikes in infection rates, there is increasing evidence that it may be undergoing a change in its prevalence and its public health burden. Numerous factors shape the scope of EEE in humans, and there are important similarities with other emergent viral diseases that have surfaced or strengthened in recent years. Because environmental and ecological conditions that broadly influence the epidemiology of arboviral diseases also are changing, and the frequency, severity, and scope of outbreaks are expected to worsen, an expanded understanding of EEE will have untold importance in coming years. Here we review the factors shaping EEE transmission cycles and the conditions leading to outbreaks in humans from an updated, multidomain perspective. We also provide special consideration of factors shaping the virology, host-vector-environment relationships, and mechanisms of pathology and treatment as a reference for broadening audiences.

FIRST RECORDS OF AEDES TORMENTOR AND CULEX PANOCOSSA AS A RESULT OF VECTOR SURVEILLANCE ACTIVITIES CONDUCTED DURING THE CHARACTERIZATION OF FOCI OF EASTERN EQUINE ENCEPHALITIS IN TAMAULIPAS, MEXICO.

We report the 1st records of Aedes tormentor and Culex panocossa throughout vector surveillance events carried out in putative foci of eastern equine encephalitis in Tamaulipas, Mexico. Formerly, Ae. tormentor had been reported in, at least, 2 Central American countries and Mexico. In Mexico, reports were from the states of Campeche, Chiapas, Quintana Roo, and Veracruz. Records of Ae. tormentor in these 4 Neotropical states were recently reviewed and eliminated; thus, the southernmost geographic distribution for this species is considered to be the state of Tamaulipas Mexico in its neotropical zone. Further, Cx. panocossa had been collected in Guerrero, Tabasco, and Veracruz. In Tamaulipas, there are 82 species of mosquitoes, being the 4th state accounting for the highest mosquito species diversity of 11 states in which comprehensive studies have been conducted on the subject of mosquito distribution.

Eastern equine encephalitis in children, Massachusetts and New Hampshire,USA, 1970-2010.

We describe the clinical, laboratory, and radiographic characteristics of 15 cases of eastern equine encephalitis in children during 1970-2010. The most common clinical and laboratory features were fever, headache, seizures, peripheral leukocytosis, and cerebrospinal fluid neutrophilic pleocytosis. Radiographic lesions were found in the basal ganglia, thalami, and cerebral cortex. Clinical outcomes included severe neurologic deficits in 5 (33%) patients, death of 4 (27%), full recovery of 4 (27%), and mild neurologic deficits in 2 (13%). We identify an association between a short prodrome and an increased risk for death or for severe disease.

Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

Leukocyte response to eastern equine encephalomyelitis virus in a wild passerine bird.

Leukocyte counts are frequently used to assess the immunologic status of animals; however, few studies have directly looked at the predictive value of leukocyte counts and an animal's ability to respond to an infection with a pathogen. Understanding how an animal's leukocyte profile is altered by an active infection can assist with interpretation of leukocyte profiles in animals for which infection status is not known. In this study we examine the leukocyte counts of gray catbirds (Dumetella carolinensis) infected with eastern equine encephalomyelitis virus (EEEV). Blood smears were collected from infected catbirds on -4, 2, 5, and 14 days postinoculation (dpi) with EEEV, and from a corresponding uninfected control group, to monitor leukocyte counts. Although we found that preinfection leukocyte counts were not a reliable predictive of a catbird's viremia, we did find that infected catbirds exhibited significant hematologic changes in response to EEEV infection. We observed a significant drop in all subpopulations of leukocytes (i.e., lymphocytes, monocytes, and granulocytes) following infection. Lymphocytes and granulocytes still had not recovered to preinfection levels at 14 dpi. Uninfected catbirds also exhibited statistically significant changes in leukocyte counts, but this was due to a slight increase at 14 dpi and was not considered biologically relevant. Studies such as this can provide important information for field ecoimmunologists that use leukocyte counts to assess immunocompetence in free-living animals.