Clinically relevant involvement of the central nervous system (CNS) by viruses is an uncommon event, considering the overwhelming number of individuals affected by the different human viral infections. Most commonly, clinically relevant viral encephalitis affects children, young adults, or elderly patients, but the spectrum of involvement depends on the specific viral agent, host immune status, and genetic and environmental factors.[1, 2]
The term “acute viral encephalitis” (from Greek enkephalos + -itis, meaning brain inflammation) is used to describe restricted CNS involvement (ie, involvement of the brain, sparing the meninges); however, most CNS viral infections involve the meninges to a greater or lesser extent, leading to aseptic meningitis or causing mild meningoencephalitis rather than pure encephalitis.
In addition to acute viral encephalitis, other less established and more unusual manifestations of viral infections include progressive neurologic disorders, such as postinfectious encephalomyelitis (such as may occur after measles or Nipah virus encephalitis) and conditions such as postpoliomyelitis syndrome, which has been considered by some to be as a persistent manifestation of poliovirus infection.
More recently, provocative studies have found high antibody seroprevalence to viruses such as Ebola, Marburg, and Lyssa viruses in multiple African countries, indicating the presence of a high number of undiagnosed cases every year, including high neutralizing titers of antibodies to rabies virus in 11% of a small cohort of asymptomatic Peruvians living in the Amazon with prior exposure to bats. These studies raise the possibility that in some populations, those conditions may be more common than previously recognized.[3] The emergence of new types of viral infections, such as the Toscana virus (in the Western European countries located on the northern border of the Mediterranean sea, Cyprus and Turkey) where seropositivity in the population is not matched by clinical symptoms (indicating that most infections are mild) also highlight the fact that we need to be alert about the possible threats from unknown pathogens, even in areas that are not necessarily tropical or surrounded by rain forests.
An unusual CNS involvement leading to microcephaly due to infection of pregnant women by Zika virus has also been recently reported and highlights the constant need to look for new types of neurological manifestations of viral infections in humans.[4]
This article is a general overview of the most common viral encephalitides and provides details about general workup and treatment for these important conditions. More detailed descriptions of each viral family are provided elsewhere.
The initial event in the replicative cycle of a virus is its interaction with receptors present on the surface of a cell. Knowledge of this interaction is important in understanding viral spread, tropism, and pathogenesis. The following cellular receptors have been described for these viruses (see Table 1, below):
Measles virus - CD46
Poliovirus - CD155
Herpes simplex virus (HSV) - Heparan sulfate; Hve A, B, and C; tumor necrosis factor receptor superfamily 14 (TNFSF14); HVEM; Prr1 and Prr2; and nectin-1 and nectin-2
Rabies virus - AChR, NCAM, and NGFR
Human immunodeficiency virus-1 (HIV-1) - CD4, CCR5/3, and CXCR4
JC virus - N-linked glycoprotein and alpha 2-6 sialic acid
West Nile virus - Cholesterol-rich membrane microdomains[5]
Table 1. Examples of Physiologic Roles of Known Viral Receptors
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Despite viral tropism, the pattern of distribution of lesions in the brain is rarely specific enough to permit identification of the infecting virus.
Recent studies have reported a Mendelian predisposition to some forms of encephalitis (especially herpes simplex encephalitis) due to defects in the following pathways: TLR-3 interferon, autosomal recessive STAT-1 deficiency and X-linked NEMO deficiency, UNC-93B deficiency, and autosomal dominant TLR3 deficiency.[7] The pathophysiology of viral encephalitis varies according to the viral family. Viruses enter the CNS through 2 distinct routes: (1) hematogenous dissemination and (2) retrograde neuronal dissemination.
Hematogenous dissemination is the more common path. Humans are usually incidental terminal hosts of many viral encephalitides. Arbovirus encephalitides are zoonoses, with the virus surviving in infection cycles involving biting arthropods and various vertebrates, especially birds and rodents. The virus can be transmitted by an insect bite and then undergoes local replication in the skin.
Transient viremia leads to seeding of the reticuloendothelial system and muscles. After continuous replication, secondary viremia leads to seeding of other sites, including the CNS. In fatal cases, little histopathologic change is noted outside the nervous system. St. Louis encephalitis is an exception, in that renal involvement is occasionally present.
On gross examination, variable degrees of meningitis, cerebral edema, congestion, and hemorrhage are observed in the brain. Microscopic examination confirms a leptomeningitis with round-cell infiltration, small hemorrhages with perivascular cuffing, and nodules of leukocytes or microglial cells. Demyelination may follow the destruction of oligodendroglias, and involvement of ependymal cells may lead to hydranencephaly. Neuronal damage is seen as chromatolysis and neuronophagia.
Areas of necrosis may be extensive, especially in eastern equine encephalitis (EEE) and Japanese encephalitis (JE). Recent experimental evidence has shown that arboviruses can induce apoptotic cell death in neurons in the brains of their hosts. Patients who survive the initial illness associated with viral encephalitis feature varying degrees of repair, which may include calcification in children.
Retrograde neural dissemination is the main route of spread for several important viral pathogens. Rabies virus usually spreads to the CNS through retrograde peripheral nerve dissemination. This virus tends to exhibit tropism for the temporal lobes, affecting the Ammon horns. One of the possible routes of CNS spread for herpes simplex virus (HSV) is through the olfactory tracts. Herpesviruses have tropism for the temporal cortex and pons, but the lesions may be widespread.
Herpes simplex encephalitis (HSE) in infants is usually part of a widespread infection that produces focal necrotic lesions with typical intranuclear inclusions in many organs. In adults and in some children, lesions are confined to the brain. Necrotic foci may be macroscopically evident as softening. Inclusion bodies are found readily in the margins of areas of necrosis; focal perivascular infiltration and neuronal damage are evident.
In addition to the direct effect of the viral pathogen, acute encephalopathy may be associated with viral infections and increased plasma concentrations of chemokine (CXC motif) ligand 8 (CXCL8; interleukin [IL]-8), chemokine (C-C motif) ligand 2 (CCL2; monocyte chemotactic protein-1 [MCP - 1]), IL-6, and CXCL10 (interferon gamma–induced protein 10 kd [IP-10], without viral neuroinvasion (hyperactivated cytokine response).
Accordingly, it is important to differentiate encephalitis from encephalopathy as a disruption of brain function that is not related to a direct structural or inflammatory process. To illustrate the difficulty of making this distinction in daily clinical practice, until recently it was not clear whether encephalopathy after dengue fever infection was due to direct CNS invasion or to viremia. Studies have now documented the presence of IgM and IgG for dengue virus in the cerebrospinal fluid (CSF) of patients with dengue fever and neurologic manifestations.
HSE is the most common form of encephalitis in the United States (see Herpes Simplex Encephalitis). Human herpesvirus (HHV)-6, the causative agent of exanthema subitum, has been associated with a wide spectrum of neurologic complications, including viral (focal) encephalitis. Numerous other viruses are known to cause encephalitis (see Tables 2 and 3 below). The viruses most commonly associated with acute childhood encephalitis are mumps virus, measles virus, and varicella-zoster virus (VZV).
Table 2. Common Viral Encephalitides: Part 1
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Table 3. Common Viral Encephalitides: Part 2
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Arthropod-borne viruses (arboviruses) are important causes of encephalitis worldwide. More than 20 arboviruses that can cause encephalitis have been identified. These arboviruses are enveloped RNA viruses from different families: Togaviridae (genus Alphavirus), Flaviviridae (genus Flavivirus), Bunyaviridae (genus Bunyavirus), and Reoviridae (see Table 4 below).
Table 4. Common Arboviral Encephalitides
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Important encephalitides caused by alphaviruses include EEE, western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE). EEE is endemic along the eastern and Gulf coasts of the United States, in the Caribbean region, and in South America. North American strains produce a fulminant disease (50-75% mortality) with a high incidence of neurologic sequelae.
WEE is most common in the western and midwestern United States but has a lower mortality rate (10%) than EEE. VEE occurs in South America and Central America as well as in the southwestern United States, typically causing mild disease and, rarely, neurologic impairment.
Flaviviruses are transmitted by ticks and mosquitoes and are found worldwide. The most common form of flavivirus is the Japanese B encephalitis virus. This flavivirus is one of the most important causes of viral encephalitis worldwide, with 50,000 new cases and 15,000 deaths annually. It has been found in China, Southeast Asia, the Indian subcontinent, the Philippines, New Guinea, Guam, and Australia.
West Nile virus is a flavivirus similar to the Japanese B virus. Its life cycle occurs between birds and mosquitoes. Culex mosquitoes, Anopheles mosquitoes, and Aedes mosquitoes are the primary vectors to humans. West Nile virus is endemic in Africa, the Middle East, Russia, India, Indonesia, and parts of Europe. It was detected for the first time in the Western hemisphere during an outbreak of encephalitis in the summer of 1999 in New York City (see West Nile Encephalitis).[8, 9]
Dengue fever is the most important arboviral infection of humans, with 100 million cases per year. It can now be seen in any country between the tropics of Capricorn and Cancer (placing an estimated 2.5 billion people at risk). Until recently, dengue fever was considered to be uncommonly associated with neurologic manifestations (except when dengue hemorrhagic fever is present). However, this view has changed; in endemic areas, dengue fever may be one of the most common forms of viral encephalitis.[10, 11, 12] It is important to emphasize that the geographic distribution of dengue virus has increased. In 2009, locally acquired disease was diagnosed in New York City and subsequently in Key West in Florida (no locally acquired disease had been reported in the US since 1945).
Before the 1999 outbreak of West Nile encephalitis (WNE), St Louis encephalitis was the most common disease caused by a flavivirus in the United States. Outbreaks of St Louis encephalitis occur from August to October throughout the country. Individual susceptibility to the St Louis virus increases with age, and encephalitis can be accompanied by hyponatremia due to the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Mortality is age related, ranging from 2-20%, and sequelae are present in 20% of survivors.
Other important flaviviral diseases include Far East tick-borne encephalitis (former eastern Russia), Central European tick-borne encephalitis (Central Europe),[13] and Powassan encephalitis (Canada and northern United States).[14]
Bunyaviruses are the largest group of arboviruses and include the viruses that cause La Crosse encephalitis, Jamestown Canyon encephalitis, and California encephalitis (CE). La Crosse virus is the most common cause of arboviral encephalitis in the United States and produces seizures and focal neurologic signs, manifested primarily in children, with a mortality of less than 1% and rare sequelae.[15] Toscana virus was identified in Central Italy in 1971. It has a larger geographic distribution over the northern border of the Mediterranean Sea, including Cyprus and Turkey. Most affected individuals are not symptomatic or mildly symptomatic, although severe cases of meningitis and meningo-encephalitis have been reported.
Orbivirus is transmitted by the tick Dermacentor andersoni and is seen in the Rocky Mountains of the United States.
Retroviruses are also a cause of encephalitis. Human T-cell lymphotrophic virus type 1 (HTLV-I) is associated predominantly with spastic paraparesis, not with causing encephalitis. Certain forms of encephalitis are observed almost exclusively in patients with HIV. Among those, cytomegalovirus (CMV) ventriculoencephalitis has emerged as a unique entity in patients with advanced HIV infection.
Measles and mumps viruses (paramyxoviruses) can also cause neurologic disease. Measles typically does not cause encephalitis in the acute phase, but 1 in 1000 cases can give rise to postinfectious autoimmune syndrome (ie, SSPE). Nipah virus (Paramyxoviridae family) was first detected after an outbreak of encephalitis in pig farmers in Malaysia. Nipah virus is a zoonosis and infects pigs. Subsequent outbreaks occurred in several countries in South Asia, including Bangladesh (2001 and 2003).[16]
Arenaviruses usually infect rodents. Thus, lymphocytic choriomeningitis most commonly occurs during the winter, when mice are indoors and humans have contact with their excreta. Meningitis or meningoencephalitis follows a 5- to 10-day incubation period. Recovery can be prolonged but is usually complete. Lassa fever is a West African disease that starts with gastrointestinal (GI) and respiratory complaints and progresses to hemorrhagic shock. Unilateral or bilateral deafness may follow the period of encephalitis. Mortality is in the range of 8-52%.
Enteroviruses are picornaviruses. The Picornaviridae family includes coxsackievirus A and B, poliovirus, echovirus, enterovirus (EV) 68 and 71, and hepatitis A virus (HAV). Enteroviruses are transmitted by the fecal-oral route, and CNS spread is through the hematogenous route. Infection is most common in summer and early fall. In 2014, the United States experienced a nationwide outbreak of EV-D68 associated with severe respiratory illness. From mid-August 2014 to January 15, 2015, CDC or state public health laboratories confirmed a total of 1,153 people in 49 states and the District of Columbia with respiratory illness caused by EV-D68. Almost all of the confirmed cases were among children, many whom had asthma or a history of wheezing.[17] One study reports that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7).[18] Outbreaks of enterovirus 71 have occurred in Japan, Malaysia, and Taiwan. Enterovirus 71 is typically associated with hand-foot-and-mouth disease, but up to 30% may develop neurological manifestations. In 2012, a severe encephalitis outbreak in Cambodia, with a 69% mortality rate in children, was secondary to enterovirus 71 serotype C4. The disease mainly affected children aged 3 years and younger.
Rabies is an important pathogen in developing countries, where endemic canine infection still exists. In Europe and the United States, rabies is present in wild animals (eg, skunks, foxes, raccoons, bats); however, it is controlled in domestic animals with vaccination. Rabies usually incubates for 20-60 days but can incubate for years.
In a 2007 outbreak of Chikungunya virus infection in Italy, 1 elderly patient developed encephalitis and died.[19] This reinforces the risk of new outbreaks of newer forms of encephalitis in Europe and other parts of the world.[20] Recent outbreaks of Zika virus in Brazil led to the development of CNS malformations in newborns of infected pregnant women, especially due to microcephaly.[21]
Epidemiologic studies estimate the incidence of viral encephalitis at 3.5-7.4 per 100,000 persons per year.
International
The annual incidence of viral encephalitis is most likely underestimated, especially in developing countries, because of problems with pathogen detection.[22] In a study from Finland, the incidence of viral encephalitis in adults was 1.4 cases per 100,000 persons per year.[23]
Epidemiologic studies estimate the incidence of viral encephalitis at 3.5-7.4 per 100,000 persons per year. Overall, viruses are the most common cause of encephalitis. The Centers for Disease Control and Prevention (CDC) estimates an annual incidence of approximately 20,000 new cases of encephalitis in the United States; most are mild in nature. (See the CDC Division of Vector-Borne Infectious Diseases, Arboviral Encephalitides.)
The 2 most common nonendemic causes of viral encephalitis in the United States are HSV and rabies virus. HSV encephalitis is the most common form of viral encephalitis and has an incidence of 2-4 cases per 1 million population per year and accounts for 10% of all cases of encephalitis in the United States.
Arboviral encephalitis affects 150-3000 persons per year, depending on occurrence and intensity of epidemic transmission. WNE affected 373 individuals in 2009, with 32 deaths. St. Louis encephalitis affected 3000 individuals in 1975; an outbreak with 24 confirmed or possible cases occurred in the state of Arkansas in 1991. La Crosse encephalitis usually affects 80-100 individuals per year. EEE was confirmed in 257 cases since 1964, and WEE was confirmed in 639 cases.
International statistics
The annual incidence of viral encephalitis is most likely underestimated, especially in developing countries, because of problems with pathogen detection.[22] JE affects at least 50,000 individuals per year.
In a study from Finland, the incidence of viral encephalitis in adults was 1.4 cases per 100,000 persons per year.[23] HSV was the organism most frequently identified as the cause (16%), followed by VZV (5%), mumps virus (4%), and influenza A virus (4%).
Age-, sex-, and race-related demographics
Children and young adults are typically the groups that are most often affected. However, severity is usually more pronounced in infants and elderly patients.[24] The clinical course in children may be considerably different from that seen in adults. HSE may be associated with a relapse in 25% of the cases, which may present as a movement disorder, most often choreoathetosis.[25]
Mumps meningoencephalitis affects men more often than women. Men working in areas infested by infected mosquitoes have a higher incidence of arboviral infections.
No racial predilection exists, although different genetic factors may predispose individuals to more severe forms of CNS involvement.
The mortality depends largely on the etiologic agent of the encephalitis. The severity of sequelae apparently varies according to the causative virus as well.[26] The average lifetime cost of the sequelae of encephalitis approaches US$3 million.
HSE carries a mortality of 70% in untreated patients, with severe sequelae among survivors.
After WEE, sequelae are uncommon in adults but are frequent in children. Recurring convulsions with motor or behavioral changes affect more than half of children who are infected when younger than 1 month.
With EEE, most adults older than 40 years who survive (10% mortality) do so unscathed; children younger than 5 years have crippling sequelae consisting of mental retardation, convulsions, and paralysis.
Permanent sequelae after St. Louis encephalitis are uncommon, except for elderly individuals; the mortality rate is 2% in young adults and 20% in elderly patients.
A large number of cases of newborns with microcephaly has been reported in pregnant women infected by Zika virus.[4]
La Crosse virus causes a relatively mild encephalitis with a low fatality rate.
Mortality is low in VEE, CE, and encephalitis due to Colorado tick fever virus. Neurologic sequelae in these conditions are not frequent and are usually mild.
JE has a mortality of almost 50% in patients older than 50 years and a mortality rate of less than 20% in children.
The Far East form of tick-borne encephalitis is more severe than the Central European form of tick-borne encephalitis, with mortalities as high as 20% and frequent sequelae. Epilepsia partialis continua may develop during the convalescent period or later. Residual weakness may also be present.
The 20-year risk of developing an unprovoked seizure is 22% for patients with viral encephalitis associated with early seizures and 10% for viral encephalitis without early seizures. Of patients with CNS infection, 18-80% develop epilepsy, which is usually refractory to medical treatment. A considerable number of such patients develop unilateral mesial temporal lobe epilepsy and can have a good outcome after surgery.[27]
Education helps in the early diagnosis of encephalitis, especially in areas of endemic disease. Control of the mosquito vector has been effective in several recent epidemics.
The belief that HSV-2 lesions initially appear 2 weeks after primary infection can lead to false accusations of infidelity. The physician should emphasize that the initial outbreak of lesions may occur at any time, possibly years, after infection.
For patient education resources, see the Brain and Nervous System Center, the Bacterial and Viral Infections Center, and the Bites and Stings Center, as well as Meningitis in Adults, Mumps, and Encephalitis.
Viral encephalitis is usually marked by acute onset of a febrile illness. Patients with viral encephalitis generally experience signs and symptoms of leptomeningeal irritation (eg, headache, fever, neck stiffness).
Patients with viral encephalitis also develop focal neurological signs; seizures[28] ; and alteration of consciousness, starting with lethargy and progressing to confusion, stupor, and coma. Behavioral and speech disturbances are common. Abnormal movements can be seen but are rare. Involvement of the hypothalamic/pituitary axis can lead to hyperthermia or poikilothermia.
Symptoms associated with specific viral infections
Specific clues taken from the patient’s history depend on the viral etiology. Clinical findings reflect disease progression according to viral tropism for different central nervous system (CNS) cell types.
Atypical presentations include a reversible frontal lobe and limbic syndrome without disturbances of consciousness or motor function. These presentations have been described in children with influenza virus infection.[29, 30]
Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) encephalitis have subacute forms, presenting with a psychiatric syndrome and an anterior opercular syndrome, known as benign recurrent meningitis. HSV-1 encephalitis may produce a brainstem encephalitis, and HSV-2 encephalitis may also produce a myelitis.
West Nile encephalitis (WNE) is usually asymptomatic in areas of endemic disease. In symptomatic individuals, an influenzalike illness occurs after incubation of 3-15 days; CNS involvement occurs in less than 15% of cases. Severe neurologic infection is more common when the virus is introduced in an area of nonendemic disease.[31, 32]
In 1999, during the New York City outbreak of West Nile virus infection, 62 patients developed encephalitis, and 7 died (a case fatality rate of 12%, with all deaths occurring in older patients). Axonal neuropathy, demyelinating polyneuropathy similar to that seen in Guillain-Barré syndrome, encephalitis with and without muscle weakness, and aseptic meningitis were described. Delayed weakness or recurrent clinical weakness after West Nile virus infection has been described.[33]
Japanese encephalitis (JE) typically affects children and young adults. Older adults are affected in epidemics. The clinical presentation includes a nonspecific prodrome and frequent seizures.[28]
Dengue fever[34] classically presents with a severe influenzalike illness or dengue hemorrhagic fever. Less commonly, dengue fever can lead to encephalitis or encephalopathy, transverse myelitis, and mononeuropathy or polyneuropathy similar to that in Guillain-Barré syndrome. The hemorrhagic form may also cause hepatic failure leading to a Reye syndrome–like illness.
Enteroviral encephalitis is usually associated with a good prognosis. However, enterovirus 71 has a high mortality and can present with herpangina or enteroviral hand, foot, and mouth disease. Complications include myocarditis and acute flaccid paralysis. Enterovirus 71 can cause a chronic meningoencephalitis in patients who are immunocompromised.
Mumps encephalitis typically starts 3-10 days after parotitis and usually resolves without sequelae, except for occasional hydrocephalus due to ependymal cell involvement. Measles does not usually cause acute encephalitis.
Rabies virus usually incubates for 20-60 days but is capable of incubating for years. Infection does not occur in all humans bitten by an infected animal but is uniformly fatal when clinical disease develops. After a prodrome of fever, headache, malaise, seizures, and behavioral abnormalities, hydrophobia and aerophobia supervene. Coma and death occur in 1 to several weeks. Once symptoms start, treatment is ineffective.
In southern Vietnam, a viral encephalitis that was caused by avian influenza A (H5N1) and did not involve the respiratory tract was diagnosed in 2 siblings: a 4-year-old boy, who presented with severe diarrhea, seizures, coma, and death, and his sister.[35] The boy’s cerebrospinal fluid (CSF) revealed only high protein levels, but H5N1 was isolated from CSF, fecal, throat, and serum specimens.
Newborns from mothers infected by Zika virus early during pregnancy exhibit higher risk of developping microcephaly and other several forms of CNS disease.
Findings from physical examination are not usually diagnostic. Focal neurologic deficits (eg, opisthotonos, pareses, tremors, ataxia, hypotonia, diplopia), accentuated reflexes, and extensor plantar responses may be observed. Abnormal movements and, rarely, tremor may be seen. Increased intracranial pressure (ICP) can also lead to papilledema and cranial nerve VI palsy.
Findings associated with specific viral infections
A minority of patients with arbovirus infections develop acute encephalitis (or encephalomyelitis), meningitis, or a combination of both. Focal signs are only occasionally prominent in arboviral encephalitis. Patients may also have evidence of spinal cord involvement.
JE can cause marked extrapyramidal manifestations, such as dull masklike face with wide staring eyes, tremor, choreoathetosis, head nodding, and rigidity. Flaccid paralysis, especially involving the lower extremities, has been described as being due to damage to the anterior horn cells.
Parkinsonism can be a sequela of JE, and von Economo encephalitis (encephalitis lethargica) is considered to be a sequela of influenza encephalitis.
Enterovirus 71 can cause rhombencephalitis with myoclonus, tremor, ataxia, cranial nerve involvement, neurogenic pulmonary edema, and coma.
Nipah virus, in addition to the classical encephalitis presentation, produces cerebellar and brainstem signs, as well as segmental myoclonus, significant hypertension, and tachycardia. Encephalitis delayed 4 months after exposure to the virus has been described, suggesting similarities to the subacute sclerosing panencephalitis (SSPE) phenotype.
Microcephaly may be seen in newborns from mothers infected by Zika virus early during pregnancy.
Secondary bacterial infections of the respiratory and urinary tracts are major complications of encephalitis. Complications depend on the severity of the encephalitis and generally decline in importance as the acute illness passes.
With recovery from acute viral encephalitis, evidence of neuronal injury and death becomes apparent as residual neurologic defects, impairment of intelligence, and psychiatric disturbances. The severity of these sequelae varies according to the causative virus.[36, 37, 29, 38]
Sequelae occur in 30-40% of patients aged 5-40 years and include extrapyramidal features (especially dystonia and occasionally parkinsonism), weakness, and seizure disorders. Sequelae are reported in only 3-10% of cases of JE in Japan. Yet 25-30% of young adult males serving in the armed forces of the United States during World War II had sequelae (including neuroses) 6 months after infection. In addition, 10 of 25 individuals who had JE in Guam in 1948 had neurological or intellectual defects 10 years later.
Hyponatremia due to the syndrome of inappropriate antidiuretic hormone secretion (SIADH) may be frequent in St Louis encephalitis. Dehydration, respiratory complications, nosocomial infections, and decubitus ulcers may also occur.
Usually, general laboratory studies are not helpful, except for identifying a viral infectious process (eg, a lymphocytic predominance in the complete blood count [CBC], rather than the polymorphonuclear predominance indicative of bacterial infection). The diagnostic evaluation should include a CBC, tests of renal and hepatic function, coagulation studies, and chest radiography.
During epidemics, viral encephalitis is diagnosed readily on clinical grounds. However, sporadic cases of viral encephalitis are often difficult to distinguish from other febrile illnesses (eg, gastroenteritis with dehydration and convulsions) or from intoxication. Although specific treatment for most causes of viral encephalitis is still not available, establishing the final diagnosis is important to avoid unnecessary treatments with potential side effects.[39]
In most instances, the currently available specific laboratory tests only help provide a retrospective diagnosis. Serologic tests depend on the occurrence of a rise in antibody titer. However, the early detection of specific immunoglobulin M (IgM) antibody may assist early diagnosis.
Analysis of cerebrospinal fluid (CSF), including polymerase chain reaction (PCR) testing, plays an important role. Reliance on magnetic resonance imaging (MRI) findings to make the diagnosis of encephalitis or to distinguish among the different viral etiologies is usually not advisable.
All patients with encephalitis should have blood cultures to rule out bacterial and fungal infections. Specific clinical findings should also guide the evaluation of other sites for culture (scraping of vesicles, sputum, nasopharynx, and stool). For most arboviral infections, the viremia is usually of low magnitude and short duration, so blood viral cultures are low yield tests most of the time.
Skin biopsies may be useful for diagnosis conditions such as Rocky Mountain spotted fever, and full-thickness skin biopsy from the neck with staining of sensory axons may be useful for the diagnosis of rabies. Viral cultures from throat, stool samples, and antigen detection for herpes and respiratory viruses are recommended during the first week.
Some causes of encephalitis can be diagnosed by detecting serum IgM antibodies (varicella and arboviruses).
Currently, IgM and immunoglobulin G (IgG) capture enzyme linked immunosorbent assays (ELISAs) are the most useful and most widely used tests for the diagnosis of arboviral encephalitis. However, there is significant cross-reactivity among flaviviruses (Japanese encephalitis virus, St Louis encephalitis virus, and West Nile virus). Anti-West Nile virus IgM is detectable in CS) and serum 10 days after infection onset.
A PCR-based test for rapid detection of West Nile virus has been developed in California. A diagnosis of Japanese encephalitis (JE) can be confirmed serologically with demonstration of IgM in the CSF (sensitivity and specificity >95%). The PCR test may detect the virus within 2 days, but its reliability is uncertain.
ELISAs for detection of dengue virus IgM and IgG are available for serum and CSF.[40] Antibodies to Borrelia burgdorferi and serologic testing for Rickettsia, Ehrlichia, and Anaplasma species should be checked in all patients coming from endemic areas. Blood from the acute phase should be saved for future comparisons with the titers from the convalescent phase.
Despite all major efforts, in a recent study from Spain, a significant number of cases of aseptic CNS infection (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) may still have no etiological diagnosis.[41]
Lumbar puncture should be performed immediately once a space-occupying lesion is ruled out. CSF examination is critical to establish the diagnosis and reveals, acutely, a typical viral profile: mildly to moderately elevated protein (60-80 mg/dL), normal glucose, and a moderate pleocytosis (up to 1000 leukocytes/µL). Mononuclear cells usually predominate, though early in fulminant encephalitis, polymorphonuclear leukocytes predominate. Persistent neutrophilic pleocytosis can occur in patients with West Nile encephalitis (WNE).
Viral cultures are rarely helpful for acute management. Findings from CSF cultures for enteroviruses, mumps, and certain arboviruses may be positive. Low CSF glucose is unusual with viral encephalitis and suggests infection by bacteria, fungal agents, or tuberculosis.
Herpes simplex encephalitis (HSE), as well as other forms of hemorrhagic encephalitis, may be associated with increased red blood cells (RBCs) and xanthochromia in the CSF. The fluid should be sent for PCR evaluation to detect herpes simplex virus (HSV) DNA; PCR is highly specific and remains positive for as long as 5 days after initiation of treatment (see below). Intrathecal antibodies can also be quantified.
Eosinophils can be present in infections with helminths, Treponema pallidum, Mycoplasma pneumoniae, Rickettsia rickettsii, Coccidioides immitis, and Toxoplasma gondii. They can be mistaken for neutrophils if cell count is done in automated cell counters or can be easily destroyed or distorted during processing.
Up to 10% of the patients with viral encephalitis may have completely normal CSF studies.
CSF findings in patients with acute disseminated encephalomyelitis (ADEM) are similar to those in patients with viral encephalitis, but pleocytosis is less marked or absent, and markers of intrathecal immunoglobulin synthesis may be present (less than in multiple sclerosis).
Polymerase chain reaction
PCR testing should be performed to detect viral nucleic acid in CSF. In undiagnosed cases, PCR should be repeated after 3-7 days, and blood tests should be performed after 2-4 weeks to show possible seroconversion or diagnostic increase in antibody levels.
PCR is especially useful for infections caused by herpesviruses and enteroviruses. In infants and neonates, the sensitivity and specificity for CSF PCR for HSV are more variable. In adults, the test may initially yield negative results, especially if the white blood cell (WBC) count in the CSF is lower than 10/µL. Results may turn positive 1-3 days after initiation of treatment.
PCR can also detect varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), JC virus, and West Nile virus (positive in < 60% serologically confirmed cases). Molecular testing of the saliva may establish the diagnosis of rabies.
PCR testing may also be important for the diagnosis of nonviral encephalitis (as in ehrlichiosis and Bartonella henselae infection).
Computed Tomography and Positron Emission Tomography
In HSV encephalitis, computed tomography (CT) scanning may show low-density lesions in the temporal lobes, which may not be present until 3-4 days after onset. Edema and hemorrhages may be found, and, after 1 week, contrast enhancement may be observed.
CT findings are usually not helpful in differentiating the different viral encephalitides. However, given its low cost and its ready availability in most institutions, CT scanning may be a good choice for evaluating acute disease progression and following up on complications. It scan can readily reveal important complications (eg, hemorrhage, hydrocephalus, and herniation) and can help guide neurosurgical interventions.
Positron emission tomography (PET) scanning may be useful for the evaluation of possible paraneoplastic disorders.
Relying on MRI findings to make the diagnosis of encephalitis or to distinguish among the different viral etiologies is usually not advisable.
MRI is more sensitive and specific than CT for identifying viral encephalitides, especially in the early phase. Diffusion-weighted imaging may be useful for the early diagnosis of HSV, enterovirus 71, and West Nile virus infections.
In HSE, MRI typically shows temporal lobe lesions, which may be hemorrhagic and unilateral or bilateral. Inferomedial temporal lobe and cingulate gyrus are the areas most commonly detected by MRI. In children and infants, a more widespread pattern may be observed.
MRI may help in differentiating Japanese encephalitis (JE) from Nipah virus encephalitis. JE is characterized by gray matter involvement, whereas Nipah virus encephalitis is associated with multiple, small, white matter lesions.
With flavivirus encephalitis and eastern equine encephalitis (EEE), MRI may show mixed intense or hypointense lesions in the thalamus, basal ganglia, and midbrain, being hyperintense on fluid attenuated inversion recovery (FLAIR) and T2.
The rhombencephalitis caused by enterovirus 71 can be visualized by T2-weighted MRI, which shows hyperintense signals in the brainstem.
A peculiar MRI pattern on diffusion-weighted imaging and magnetic resonance spectroscopy has been described in an acute and rapid form of subacute sclerosing panencephalitis (SSPE).[42]
In HSE, electroencephalography (EEG) shows abnormalities in four fifths of biopsy-proven cases. Focal temporal changes, diffuse slowing, and periodic complexes and periodic lateralizing epileptiform discharges (PLEDs) are commonly described. Frontal slowing and occasional frontal spikes have been described in encephalitis associated with influenza virus.
JE is commonly associated with 3 EEG patterns: (1) diffuse continuous delta activity, (2) diffuse delta activity with spikes, and (3) alpha coma pattern. In 1 study, the EEG pattern did not correlate with the Glasgow Coma Scale score and outcome.[43]
In St Louis encephalitis, EEG is characterized by diffuse delta activity, and spike and waves are not prominent in the acute stage.
Brain biopsies can yield definitive diagnosis of encephalitis, but at present they are rarely performed. A biopsy may be considered when a lumbar puncture is precluded or when the diagnosis is uncertain (eg, to rule out other conditions, such as vasculitis) and the patient’s condition is deteriorating despite treatment with acyclovir. If considered, it should be performed earlier in the course, rather than later, so that a potentially treatable condition can be identified.
In acute viral encephalitis, capillary and endothelial inflammation of cortical vessels is a pathologic hallmark occurring in the gray matter or at the junction of the gray matter and white matter. Lymphocytic infiltration of the gray matter and neuronophagia may also occur. Astrocytosis and gliosis become prominent with disease progression.
Some histopathologic features, such as Cowdry type A inclusion bodies in HSV infection and Negri bodies in rabies, are unique to viral infections. Arboviruses cause little histopathologic change outside the nervous system, with the possible exception of renal involvement in St Louis encephalitis.
Gross examination reveals varying degrees of meningitis, cerebral edema, congestion, and hemorrhage in the brain.
Microscopic examination confirms a leptomeningitis with round-cell infiltration, small hemorrhages with perivascular cuffing, and nodules of leukocytes or microglial cells. Demyelination may follow the destruction of oligodendroglias, and involvement of ependymal cells may lead to hydranencephaly. Neuronal damage is seen as chromatolysis and neuronophagia. Areas of necrosis may be extensive, especially in EEE, JE, and the Far East form of tick-borne encephalitis.
In patients who survive the initial illness, varying degrees of repair are observed, which may include calcification. The pattern of distribution of lesions in the brain is rarely sufficiently specific to enable identification of the infecting virus. Generally, in EEE, the lesions are concentrated in the cortex; in western equine encephalitis (WEE), they are concentrated in the basal nuclei; and in St Louis encephalitis, they are concentrated in the substantia nigra, thalamus, pons, cerebellum, cortex, bulb, and anterior horn cells.
HSE in infants is usually part of a widespread infection that produces focal necrotic lesions with typical intranuclear inclusions in many organs. In adults and in some children, lesions are confined to the brain. Necrotic foci may be macroscopically evident as softening. Hemorrhage and Cowdry type A inclusions bodies are found readily in the margins of areas of necrosis.
Herpesviruses have tropism for the temporal cortex and pons, but the lesions may be widespread. Rabies virus tends to exhibit a tropism for the temporal lobes, affecting the Ammon horns. Autopsy studies in individuals with West Nile virus have shown particular brainstem involvement, especially the medulla, with endoneural mononuclear inflammation of cranial nerve roots.
Medical care should be devoted to appropriate management of the airway, bladder function, fluid and electrolyte balance, nutrition, prevention of bedsores, secondary pulmonary infection, and hyperpyrexia. A multidisciplinary approach must be instituted as early as possible to start physical and cognitive rehabilitation and to minimize cognitive problems and long-term sequelae. Care in an intensive care unit (ICU) setting may be required, especially if seizure activity or increased intracranial pressure (ICP) is present.[44]
Delayed diagnosis of herpes simplex encephalitis (HSE) increases morbidity and mortality rates; failure to diagnose and treat early could result in litigation. With the wide availability of effective therapy, initiating antiviral treatment before a definitive diagnosis of HSE encephalitis (ie, during the workup) is now common practice.
The use of corticosteroids as an adjunctive therapy for viral encephalitis is controversial and currently being evaluated in a large clinical trial. Recent promising therapies with recombinant fully humanized antibody against Nipah and Hendra virus have been tested in experimental animals and in a compassionate basis in humans.
See the following for complete information on these topics:
Pharmacotherapy for HSE consists of acyclovir and vidarabine. Outcome is improved with either agent, but acyclovir is more effective and less toxic. Even if the final diagnosis of HSE has not been established, intravenous (IV) acyclovir should be initiated immediately. Acyclovir is also the drug of choice for varicella-zoster virus (VZV) encephalitis, although ganciclovir is also considered an alternative option.
Ganciclovir has been used for cytomegalovirus (CMV) encephalitis, but with therapeutic failures; consequently, the optimal therapy for CMV encephalitis is unknown. Ganciclovir combined with foscarnet has been used in the treatment of patients infected with HIV.
No specific treatment is available for the arbovirus encephalitides. Ribavirin seems to be effective for Lassa fever; its efficacy in other viral infections is being evaluated. Intraventricular ribavirin has been associated with clinical improvement in 4 patients with subacute sclerosing panencephalitis (SSPE) and apparently reduced mortality in an open-label trial in patients with Nipah virus encephalitis.
Results from a small series suggested that interferon alfa-2b reduced the severity and duration of the complications of St Louis encephalitis virus meningoencephalitis.[45]
Because specific therapy for encephalitis is limited and because potentially serious sequelae (or death) may result from HSE, early treatment with acyclovir should be started as soon as possible in all patients with suspected viral encephalitis, pending the results of diagnostic studies. Once an etiologic agent of the encephalitis is eventually identified, therapy should be targeted to that agent (if available).
Other antibacterial treatments (eg, for bacterial meningitis) should be administered on the basis of epidemiological and clinical factors or given until the diagnosis of bacterial meningitis is excluded. Doxycycline should be added if there is suspicion of rickettsial or ehrlichial infection during the appropriate season.
Increased ICP should be managed in the ICU setting with head elevation, gentle diuresis, mannitol, and hyperventilation. Surgical decompression may be necessary if there is impending uncal herniation or increased ICP that is refractory to medical management. Controlled studies are lacking, but there is some evidence that patients with life-threatening cerebral edema may benefit from craniectomy or other approaches to lower the increased ICP in neurocritical care units.[46]
Encephalitis causes a wide range of behavioral manifestations with limbic and frontal syndromes that can be difficult to distinguish from partial seizures.[47] Seizure activity can be closely observed using electroencephalography (EEG), and the threshold for administering temporary anticonvulsant therapy should be low.
Phenytoin and valproic acid can be administered intravenously. Phenytoin and carbamazepine can be administered when oral or intragastric drug administration is possible. Benzodiazepines are also important when used to abort status epilepticus.
If seizure activity persists after the acute phase, patients may need long-term anticonvulsant therapy. Accordingly, additional therapy may be necessary for extrapyramidal, motor, and behavioral complications.
Surveillance is important to predict outbreaks of arboviral infections. Mosquitoes can be sampled to estimate infection rates in mosquito pools. Protective clothing and repellents are useful in the prevention of arthropod bites. Avoidance of outdoor activities is also useful. Prompt removal of ticks may decrease the risk of transmission of a tick-borne virus.
Effective preventive measures include removing water-holding containers and discarded tires. Insecticides may be useful in the emergency control of infected mosquitoes. Control of the mosquito vector has been used with apparently good results in several recent epidemics.
Vaccines are available for eastern equine encephalitis (EEE), western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE) in horses. A live attenuated vaccine (TC-83) has been used to protect laboratory and field workers from the virus that causes VEE. Vaccines have also been developed for Japanese B virus encephalitis (JE) and tick-borne encephalitis.
Killed virus vaccines have been produced experimentally for several arboviruses. A live-attenuated Japanese B vaccine (SA 14-14-2) has been used widely in Asia. Since 1989, 120 million children have been immunized, and a recent report has demonstrated the efficacy of a single dose in preventing Japanese encephalitis (JE) when administered only days or weeks before exposure to infection. The only internationally licensed JE virus vaccine is a formalin-inactivated vaccine.
Limited use (eg, in exposed laboratory workers) has been made of vaccines for VEE and tick-borne viral encephalitis. Passive immunization of laboratory workers exposed to a known virus in a laboratory accident has been accomplished with immune (human) serum or gamma globulin.
Despite control efforts and disease surveillance, the 1999 outbreak of West Nile virus in New York, with subsequent spread to other states in the United States, showed that different viruses may be spread in the Western hemisphere because of increased international travel and trade. Massive culling of pigs in Malaysia decreased the incidence of Nipah virus infection.
Encephalitis is a neurological emergency. Consultation with a neurologist is recommended. Consultation with a neurosurgeon is helpful if a brain biopsy is considered. Consultation with an infectious disease specialist is also appropriate.
Given the high likelihood of long-term need for cognitive rehabilitation and physical rehabilitation, especially in moderately severe and severe forms of encephalitis, establishing a multidisciplinary approach early in the disease course is appropriate. A multidisciplinary approach includes consultations with physical, occupational, and speech therapists.
No dietary restrictions are necessary. The infectious process, especially with the presence of fever, increases nutritional requirements. Early assessment by a speech therapist and a dietitian helps prevent further body wasting and detects early behavioral manifestations that prevent adequate nutritional intake, such as placidity, apraxia, dysphagia, or agitation.
Pharmacotherapy for herpes simplex encephalitis (HSE) consists of acyclovir and vidarabine. Outcome is improved with either agent, but acyclovir is more effective and less toxic. Even if the final diagnosis of HSE has not been established, intravenous (IV) acyclovir should be initiated immediately. Acyclovir is also the drug of choice for varicella-zoster virus (VZV) encephalitis, although ganciclovir is also considered an alternative option.
Ganciclovir has been used for cytomegalovirus (CMV) encephalitis, but with therapeutic failures; optimal therapy for CMV encephalitis is unknown. Ganciclovir combined with foscarnet has been used in the treatment of patients infected with HIV.
No specific treatment is available for the arbovirus encephalitides. The efficacy of ribavirin in other viral infections is being evaluated.
Clinical Context:
Acyclovir has demonstrated inhibitory activity against both herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) and is taken up selectively by infected cells. Before the use of acyclovir, mortality from HSE was 60-70%; since acyclovir, it has been approximately 30%. Acyclovir may also be effective for VZV encephalitis.
Clinical Context:
Ribavirin is a synthetic guanosine analogue (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) that inhibits viral replication by inhibiting DNA and RNA synthesis. It is phosphorylated in vivo, and the active form may interfere with viral genomic synthesis.
Clinical experience in the treatment of arenavirus infections is primarily with Lassa fever, but anecdotal experience in South American arenaviruses also exists. Ribavirin is used clinically in combination with interferon for hepatitis C, in aerosol form for respiratory syncytial virus (RSV), and as potential prophylaxis and/or treatment of Congo-Crimean hemorrhagic fever, hantavirus infections, and arenavirus hemorrhagic fevers. In vitro evidence exists for activity against West Nile virus.
The IV form of the drug is not readily available, and the manufacturer should be contacted if the need arises.
Clinical Context:
Ganciclovir is a synthetic guanine derivative that is active against CMV. It is an acyclic nucleoside analogue of 2'-deoxyguanosine that inhibits viral replication in vitro and in vivo by competing with deoxyguanosine triphosphate for viral DNA polymerase, inhibiting DNA synthesis. Ganciclovir triphosphate levels are up to 100-fold greater in CMV-infected cells than in uninfected cells, possibly because of preferential phosphorylation in infected cells.
Clinical Context:
Foscarnet is an organic analogue of inorganic pyrophosphate that inhibits viral replication in vitro. It exerts its antiviral activity by selective inhibition at pyrophosphate-binding sites on virus-specific DNA polymerases at concentrations that do not affect cellular DNA polymerases, inhibiting DNA synthesis.
Viral resistance should be considered in patients with poor clinical response or persistent viral excretion. Patients who show excellent tolerance of foscarnet may benefit from initiation of a maintenance dosage (ie, 120 mg/kg/d) earlier in their treatment. Individualize the dosing according to the patient's renal function status. Foscarnet has been demonstrated to be effective against CMV encephalitis.
Antiviral agents shorten the clinical course, prevent complications, prevent development of latency and subsequent recurrences, decrease transmission, and eliminate established latency.
Clinical Context:
Phenytoin may act in the motor cortex, where it may inhibit the spread of seizure activity. The activity of brain stem centers responsible for the tonic phase of grand mal seizures may also be inhibited.
Individualize the dose. Administer a larger dose before retiring if the dose cannot be divided equally. The rate of infusion must not exceed 50 mg per minute to avoid hypotension and arrhythmia.
Clinical Context:
Diazepam depresses all levels of the CNS (eg, limbic, reticular formation), possibly by increasing the activity of gamma-aminobutyric acid (GABA). Alternatively, lorazepam can be used when indicated.
Clinical Context:
Carbamazepine is effective in treatment of complex partial seizures; it appears to act by reducing polysynaptic responses and blocking posttetanic potentiation.
Once a response is attained, attempt to reduce the dose to the minimum effective level or to discontinue the drug at least once every 3 months.
Clinical Context:
Valproic acid is chemically unrelated to other drugs that treat seizure disorders. Although its mechanism of action is not established, its activity may be related to increased brain levels of gamma-aminobutyric acid (GABA) or enhanced GABA action. It also may potentiate postsynaptic GABA responses, affect potassium channels, or have a direct membrane-stabilizing effect.
Clinical Context:
Mannitol may be used to decrease intracranial pressure. It may reduce subarachnoid space pressure by creating an osmotic gradient between CSF in the arachnoid space and plasma. This agent is not for long-term use.
Initially assess the patient for adequate renal function by administering a test dose of 200 mg/kg intravenously over 3-5 min. It should produce a urine flow of at least 30-50 mL per hour of urine over 2-3 hours.
In children, assess for adequate renal function by administering a test dose of 200 mg/kg intravenously over 3-5 min. It should produce a urine flow of at least 1 mL/kg/h over 1-3 hours.
Mannitol is recommended by some experts to help reduce intracranial pressure. Mannitol induces diuresis, which increases serum osmotic concentration. In the brain, this causes water to flow from brain cells into vascular space, thereby decreasing intracranial pressure.
Francisco de Assis Aquino Gondim, MD, PhD, MSc, FAAN, Professor Adjunto of Neurology and Clinical Skills, Department of Internal Medicine, Universidade Federal do Ceará, Brazil
Disclosure: Serve(d) as a director, officer, partner, employee, advisor, consultant or trustee for: Consultant for Pfizer, PTC Therapeutics and Alnylam<br/>Serve(d) as a speaker or a member of a speakers bureau for: Speaker for Shire, PTC Therapeutics and BSL Beringer<br/>Received travel grants from for: Aché, Biogen, Genzyme, Ipsen, Novartis, Baxter, Teva, Pfizer.
Coauthor(s)
Florian P Thomas, MD, PhD, MA, MS, Chair, Neuroscience Institute and Department of Neurology, Director, National MS Society Multiple Sclerosis Center and Hereditary Neuropathy Foundation Center of Excellence, Hackensack University Medical Center; Founding Chair and Professor, Department of Neurology, Hackensack Meridian School of Medicine at Seton Hall University; Professor Emeritus, Department of Neurology, St Louis University School of Medicine; Editor-in-Chief, Journal of Spinal Cord Medicine
Disclosure: Nothing to disclose.
Gisele Oliveira, MD, Resident Physician, Department of Neurology, St Louis University School of Medicine
Disclosure: Nothing to disclose.
Specialty Editors
Francisco Talavera, PharmD, PhD, Adjunct Assistant Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference
Disclosure: Received salary from Medscape for employment. for: Medscape.
Chief Editor
Michael Stuart Bronze, MD, David Ross Boyd Professor and Chairman, Department of Medicine, Stewart G Wolf Endowed Chair in Internal Medicine, Department of Medicine, University of Oklahoma Health Science Center; Master of the American College of Physicians; Fellow, Infectious Diseases Society of America; Fellow of the Royal College of Physicians, London
Disclosure: Nothing to disclose.
Additional Contributors
J Stephen Huff, MD, FACEP, Professor of Emergency Medicine and Neurology, Department of Emergency Medicine, University of Virginia School of Medicine
CDC. Zika Virus - For Pregnant Women. Centers for Disease Control and Prevention. Available at http://www.cdc.gov/zika/pregnancy/index.html. Accessed: February 12, 2016.
National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral Diseases. Non-Polio Enterovirus. Centers for Disease Control and Prevention. Available at http://www.cdc.gov/non-polio-enterovirus/about/ev-d68.html. March 23, 2015; Accessed: February 18, 2016.
CDC. Notes from the Field: Evidence of Zika Virus Infection in Brain and Placental Tissues from Two Congenitally Infected Newborns and Two Fetal Losses — Brazil, 2015. Morbidity and Mortality Weekly Report (MMWR). February 10, 2016. Available at http://www.cdc.gov/mmwr/volumes/65/wr/mm6506e1er.htm
Guillaume Le Flohic, Vincent Porphyre, Philippe Barbazan, Jean-Paul Gonzalez. Review of Climate, Landscape, and Viral Genetics as Drivers of the Japanese Encephalitis Virus Ecology. PLOS Neglected Tropical Diseases. 2013. 7:e2208.
Guillaume Le Flohic, Vincent Porphyre, Philippe Barbazan, Jean-Paul Gonzalez. Review of Climate, Landscape, and Viral Genetics as Drivers of the Japanese Encephalitis Virus Ecology. PLOS Neglected Tropical Diseases. 2013. 7:e2208.
