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Some patients suffer from maculopathy or retinopathy. Patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. One or both eyes could be affected [33–35], and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [34–39]. In addition, retinal detachment, uveitis, or arterial occlusion [35,36,39–41] was reported in some patients. In many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [34,35,37–41], while some patients show partial improvement in vision after several months of RVFV infection.
Maar et al. described a case of encephalitis in a RVF patient. The patient exhibited symptoms of sudden fever, rigor, and retro-orbital headache for two days. He had fever again at the 22nd day after the onset of illness and experienced neck rigidity lasting for five days from the 25th day. Subsequently, he was sometimes confused and otherwise mentally affected, and experienced temporal vision loss without detectable retinopathy. He also exhibited convulsive attacks, hyperflexia and fever until the 50th day. His serum contained anti-RVFV hemagglutination (HAI) antibodies of 1:160 at the 25th day and 1:640 at the 40th day, while his cerebrospinal fluid (CSF) contained 1:2 of HAI antibody at the 28th day and 1:64 at the 50th day. The CSF also contained an increased number of white blood cells consisting mainly of lymphocytes at the 28th day, indicative of the possible occurrence of viral meningitis or meningoencephalitis. The patient recovered after treatment with amantadine, rifampicin, and dexamethasone for two weeks, although the effect of therapy could not be evaluated precisely.
Another case with encephalitis and retinitis was described by Alrajhi et al.. The patient had a fever, ataxic gait, and bilateral retinal hemorrhage. She could not count fingers, and the CSF contained many leukocytes, including lymphocytes. Her consciousness level was decreased. She was discharged on day 30 of the illness to her home, at which time she was awake, blind, quadreparetic, and incontinent. Moreover, her neurologic conditions did not improve for the next year.
An additional report described a patient who had persistent hemiparesis for four months after the onset of illness, and another paper reported 12 RVF patients, who developed neurological signs and symptoms, including meningeal irritation, confusion, stupor and coma, hypersalivation, teeth-grinding, visual hallucinations, locked-in syndrome, and choreiform movement of upper limbs; in these patients, the histopathological lesions in brains were characterized by focal necroses associated with an infiltration of round cells, mostly lymphocytes and macrophages, and perivascular cuffing.
Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.
The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia–. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease.
Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.
Viral encephalitis is a relatively rare disease but with a potentially high morbidity and mortality. Advances in the diagnostic methods can lead to the identification of a large number of viruses that can affect the central nervous system.
Powassan virus (POWV) is the only North American member of the tick-borne encephalitis complex (TBE-C) of viruses, which are transmitted by the bite of an infected tick. Other members of the TBE-C include the following flaviviruses: tick-borne encephalitis virus (TBEV) in Eastern Europe and Western Asia, Omsk hemorrhagic fever virus in Siberia, Kyasanur Forest disease virus in India, Alkhurma virus in Saudi Arabia, and Louping ill virus in the United Kingdom. TBE-C viruses can cause a wide range of disease in humans, from mild febrile illness with biphasic fever to encephalitis or hemorrhagic fever (1). POWV is composed of two genetically and ecologically distinct lineages (2). Prototype POWV (lineage I) is transmitted primarily by Ixodes cookei, a tick with a narrow vertebrate host range that rarely feeds on humans. Powassan virus lineage II, also known as deer tick virus (DTV), is transmitted by Ixodes scapularis, a tick with a broad host range that also transmits the infectious agents that cause Lyme disease, anaplasmosis, ehrlichiosis, and babesiosis (3). Since the late 1990s, POWV infections have been reported in the Northeastern and Midwestern parts of the United States as well as in Canada, and incidence is increasing (4). Because the territory of I. scapularis is expanding and the prevalence of POWV in ticks and mammals is increasing, POWV poses an increasing threat (5, 6). In a recent survey, I. scapularis ticks collected from the northwest quadrant of Wisconsin from 2011 to 2012 demonstrated a POWV infection frequency of 4.67% (7). This is similar in frequency to a survey conducted in that same area in 1998 (8). Although POWV is rarely diagnosed as a cause of encephalitis, infections can be severe, and neurologic sequelae are common (9). Additionally, the potential for concurrent transmission with other tick-borne pathogens, particularly Borrelia burgdorferi, the causative agent of Lyme disease, has not been previously studied in North America.
Similarly to other arboviral infections, POWV diagnosis is complex, requiring review of clinical and travel history in addition to knowledge of and access to diagnostic testing (10). Serologic testing remains the primary method for diagnosis of POWV infection, with an emphasis on the detection of POWV-specific IgM antibodies in serum or plasma. Until recently, commercial laboratory testing has been unavailable for POWV in the United States. Prior to this, a positive POWV IgM enzyme immunoassay (EIA) result confirmed by plaque reduction neutralization test (PRNT), a 4-fold or greater increase in titers between acute- and convalescent-phase sera, or culture or direct identification of virus-specific nucleic acids at a state public health laboratory or the Centers for Disease Control and Prevention (CDC) (11) has been the mainstay of diagnostic testing.
We describe here a laboratory-developed, serologic test panel, commercially available at a reference laboratory, for the detection of IgG and IgM antibodies to POWV in serum and plasma samples. The first test in the panel is a highly sensitive, commercial TBE-C screen by EIA. Per the manufacturer, cross-reactivity with other flaviviruses is expected, particularly with West Nile virus (WNV) and dengue virus (DENV) antibody-positive samples. Samples that are screen positive are then confirmed for POWV by indirect immunofluorescence assay (IFA). Performance characteristics of the test panel were optimized, and validation studies were performed to assess the analytical sensitivity, reproducibility, and specificity/cross-reactivity of the serologic test panel for use in routine diagnostic testing.
We conducted a retrospective, comparative survey of the pediatric cases of viral encephalitis caused by respiratory viruses that were submitted at the National Institute for Infectious Diseases “Prof. Dr. Matei Balş” over the last 2 years (2011-2013).
West Nile virus (WNV) is a zoonotic Flavivirus belonging to the family Flaviviridae. The virus is transmitted by mosquitoes and causes fatal encephalitis in human, equines and birds.
WNV was first isolated and identified in 1937 from a woman presented with mild febrile illness in the Nile district of Uganda. It has been described in Africa, Europe, South Asia, Oceania and North America. Countries with incidence/serological evidence are presented in Fig. (1). In North America, more than 1.8 million people have been infected, with over 12,852 reported cases of encephalitis or meningitis and 1,308 deaths from 1999 to 2010. The mortality rate in human varies from 3-15% and can reach up to 50% in clinically affected horses. WNV in India has been confirmed by seroprevalence and by virus isolation on different occasions from mosquitoes bat and man [42, 45-47]. WNV infection has also been reported in animals and birds.
Horses and human are the main hosts. Animals other than horses may be susceptible to WNV, but rarely become ill. Antibodies have been found in serum samples from bats, horse, dogs, cats, racoons, opossums, squirrels, domestic rabbits, eastern striped skunks, cows, sheep, deer and pigs. The virus is transmitted to humans by mosquitoes. About 20% of the infected people develop fever with other symptoms. Fatal, neurologic illness occurs in less than 1% of infected people.
WNV is amplified by continuous transmission cycles between mosquitoes and birds. Generally Culex mosquitoes are the vectors and passerine birds are the vertebrate reservoirs in enzootic transmission cycles. The virus is carried in the salivary glands of infected mosquitoes and transmitted to susceptible birds during blood-sucking. Competent bird reservoirs sustain an infectious viraemia for 1 to 4 days subsequent to exposure, and then develop life-long immunity. Horses, human and most other mammals rarely develop the infectious levels of viraemia and are dead-end hosts. Few cases in human have been spread through blood transfusions, organ transplants, breast feeding and during pregnancy. The ticks observed to be infected naturally include Ornithodoros maritimus, Argas hermanni and Hyalomma marginatum and the virus has also been isolated from other species of hard ticks in Africa, Europe and Asia [36, 63, 64]. Swallow bugs (Oeciacus hirundinis) have been implicated as vectors in Austria. In most of the horses bitten by carrier mosquitoes, there is no disease. Approximately 33% of the infected horses develop severe disease and die or are affected severely. The time between the bite of an infected mosquito and appearance of clinical signs ranges from 3 to 14 days. The symptoms in horses may vary from none to trembling, skin twitching and ataxia. There can be sleepiness, dullness, listlessness, facial paralysis, difficulty in urination and defecation, and inability to rise. In some horses, there can be mild fever, blindness, seizures, and other signs.
There is no effective treatment for clinical WNV infection in humans, horses or any other animal. Vaccines are available for control of WNV in horses in the USA. Vaccination of horses protects valuable animals from a potentially fatal disease, but trade and competition practices make this undesirable as some countries use positive antibody tests and impose import restrictions. WNV encephalitis is so rare in human that vaccine development may not be feasible economically.
Epithelial cells that line the respiratory tract are the first cells that can be infected by respiratory viruses. Most of these infections are self-limited and the virus is cleared by immunity with minimal clinical consequences. On the other hand, in more vulnerable individuals, viruses can also reach the lower respiratory tract where they cause more severe illnesses, such as bronchitis, pneumonia, exacerbations of asthma, chronic obstructive pulmonary disease (COPD) and different types of severe respiratory distress syndromes. Besides all these respiratory issues, accumulating evidence from the clinical/medical world strongly suggest that, being opportunistic pathogens, these viruses are able to escape the immune response and cause more severe respiratory diseases or even spread to extra-respiratory organs, including the central nervous system where they could infect resident cells and potentially induce other types of pathologies.
Like all types of viral agents, respiratory viruses may enter the CNS through the hematogenous or neuronal retrograde route. In the first, the CNS is being invaded by a viral agent which utilizes the bloodstream and in the latter, a given virus infects neurons in the periphery and uses the axonal transport machinery to gain access to the CNS. In the hematogenous route, a virus will either infect endothelial cells of the blood-brain-barrier (BBB) or epithelial cells of the blood-cerebrospinal fluid barrier (BCSFB) in the choroid plexus (CP) located in the ventricles of the brain, or leukocytes that will serve as a vector for dissemination towards the CNS. Viruses such as HIV, HSV, HCMV, enteroviruses such as coxsackievirus B3, flaviviruses, chikungunya virus (CHIKV) and echovirus 30 have all been shown to disseminate towards the CNS through the hematogenous route. Respiratory viruses such as RSV, henipaviruses, influenza A and B and enterovirus D68 are also sometimes found in the blood and, being neuroinvasive, they may therefore use the hematogenous route to reach the CNS. As they invade the human host through the airway, the same respiratory viruses may use the olfactory nerve to get access to the brain through the olfactory bulb. On the other hand, these viruses may also use other peripheral nerves like the trigeminal nerve, which possesses nociceptive neuronal cells present in the nasal cavity, or alternatively, the sensory fibers of the vagus nerve, which stems from the brainstem and innervates different organs of the respiratory tract, including the larynx, the trachea and the lungs.
Although the CNS seems difficult for viruses to penetrate, those pathogens that are able to do so may disseminate and replicate very actively and will possibly induce an overreacting innate immune response, which may be devastating. This situation may lead to severe meningitis and encephalitis that can be fatal, depending on several viral and host factors (including immunosuppression due to disease or medications) that may influence the severity of the disease.
Recently, a very interesting manuscript produced by Bookstaver and collaborators underlined the difficulties of precisely deciphering the epidemiology and identifying the causal agent of CNS infections. These difficulties are mainly due to the tremendous variation in the symptoms throughout the disease process and to the myriad of viruses that can cause CNS infections. As stated in their report, these authors underlined that the clinical portrait of viral infections is often nonspecific and requires the clinician to consider a range of differential diagnoses. Meningitis (infection/inflammation in meninges and the spinal cord) produces characteristic symptoms: fever, neck stiffness, photophobia and/or phonophobia. Encephalitis (infection/inflammation in the brain and surrounding tissues) may remain undiagnosed since the symptoms may be mild or non-existent. Symptoms may include altered brain function (altered mental status, personality change, abnormal behavior or speech), movement disorders and focal neurologic signs, such as hemiparesis, flaccid paralysis or paresthesia. Seizures can occur during both viral meningitis and encephalitis. Furthermore, viral encephalitis may also be difficult to distinguish from a non-viral encephalopathy or from an encephalopathy associated with a systemic viral infection occurring outside the CNS. Considering all these observations, it is therefore mandatory to insist on the importance of investigating the patient’s history before trying to identify a specific viral cause of a given neurological disorder.
In humans, a long list of viruses may invade the CNS, where they can infect the different resident cells (neuronal as well as glial cells) and possibly induce or contribute to neurological diseases, such as acute encephalitis, which can be from benign to fatal, depending on virus tropism, pathogenicity as well as other viral and patient characteristics. For instance, 30 years ago, the incidence of children encephalitis was as high as 16/100,000 in the second year of life, while progressively reducing to 1/100,000 by the age of 15. More recent data indicate that, in the USA, the herpes simplex virus (HSV) accounts for 50–75% of identified viral encephalitis cases, whereas the varicella zoster virus (VZV), enteroviruses and arboviruses are responsible for the majority of the other cases in the general population. Several other viruses can induce short-term neurological problems. For example, the rabies virus, herpes simplex and other herpes viruses (HHV), arthropod-borne flaviviruses such as the West Nile virus (WNV), Japanese encephalitis virus (JEV), chikungunya virus (CHIKV), Zika virus (ZIKV), alphaviruses such as the Venezuelan, Western and Eastern equine encephalitis viruses and enteroviruses affect millions of individuals worldwide and are sometimes associated with encephalitis, meningitis and other neurological disorders. The presence of viruses in the CNS may also result in long-term neurological diseases and/or sequelae. Human immunodeficiency virus (HIV) induces neurodegeneration, which lead to motor dysfunctions and cognitive impairments. Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease associated with reactivation of latent polyoma JC virus (JCV). Progressive tropical spastic paraparesis/HTLV-1-associated myelopathy (PTSP/HAM) is caused by human T-lymphotropic virus (HTLV-1) in 1–2% of infected individuals. Measles virus (MV), a highly contagious common virus, is associated with febrile illness, fever, cough and congestion, as well as a characteristic rash and Koplik’s spots. In rare circumstances, significant long-term CNS diseases, such as post-infectious encephalomyelitis (PIE) or acute disseminated encephalomyelitis (ADEM), occur in children and adolescents. Other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (MIBE), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (SSPE) that appears 6–10 years after infection.
Yet, with the exception of HIV, no specific virus has been constantly associated with specific human neurodegenerative disease. On the other hand, different human herpes viruses have been associated with Alzheimer’s disease (AD), multiple sclerosis (MS) and other types of long-term CNS disorders. As accurately stated by Majde, long-term neurodegenerative disorders may represent a “hit-and-run” type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. Different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections.
Including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods. However, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of CNS viral infections often remains unknown. Indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases. Among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents.
Central nervous system (CNS) infections including meningitis and encephalitis are important causes of significant mortality and morbidity in the developing nations. Viruses are considered as important etiological agents of CNS infections, causing diseases ranging from febrile illness to myelitis to meningoencephalitis. However, in most cases, the etiology of CNS infection is not known due to lack of diagnostic capacity, standard clinical case definitions, or low levels of surveillance. Specific diagnosis for CNS infection is rarely made and usually categorized empirically as only “viral” or “bacterial”. There have been few reports on the viral etiologies of CNS infections in Indonesia except for Japanese encephalitis virus (JEV), a leading cause of acute encephalitis in children and young adults in the Southeast Asian region [2–5]. Still, JEV is significantly underreported in Indonesia. Furthermore, in endemic provinces like Bali where encephalitis is often suspected to be JEV, there is lack of laboratory capability to accurately determine the disease burden of JEV and other CNS viruses. The objective of this study was therefore to detect and identify the pathogens responsible for viral CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia.
Respiratory syncytial virus (RSV), a member of the Orthopneumovirus genus, infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old, making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide. Recent evidence also indicates that severe respiratory diseases related to RSV are also frequent in immunocompromised adult patients and that the virus can also present neuroinvasive properties. Over the last five decades, a number of clinical cases have potentially associated the virus with CNS pathologies. RSV has been detected in the cerebrospinal fluid (CSF) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems. Furthermore, RSV is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the CNS in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments.
An additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hMPV). Discovered at the beginning of the 21st century in the Netherlands, it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals. During the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. Viral material was detected within the CNS in some clinical cases of encephalitis/encephalopathy but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hMPV neuroinvasion and potential neurovirulence.
Hendra virus (HeV) and Nipah virus (NiV) are both highly pathogenic zoonotic members of the Henipavirus genus and represent important emerging viruses discovered in the late 1990s in Australia and southern Asia. They are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage. Although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for HeV and the pig for NiV as a first step before crossing the barrier species towards humans. In humans, it can lead to different types of encephalitis, as several types of CNS resident cells (including neurons) can be infected. The neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. Even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection. The use of animal models showed that the main route of entry into the CNS is the olfactory nerve and that the Nipah virus may persist in different regions of the brain of grivets/green monkeys, reminiscent of relapsing and late-onset encephalitis observed in humans.
Influenza viruses are classified in four types: A, B, C and D. All are endemic viruses with types A and B being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. They are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year. Associated with all major pandemics since the beginning of the 20th century, circulating influenza A presents the greatest threat to human health. Most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ARDS) and complications involving the CNS. Several studies have shown that influenza A can be associated with encephalitis, Reye’s syndrome, febrile seizure, Guillain–Barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (ADEM). Animal models have shown that, using either the olfactory route or vagus nerve, influenza A virus may have access to the CNS and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae. The influenza A virus has also been associated with the risk of developing Parkinson’s disease (PD) and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (EAE), which is reminiscent of the observation that multiple sclerosis (MS) relapses have been associated with viral infections (including influenza A) of the upper respiratory tract.
Another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the Enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (PV), coxsackieviruses (CV), echoviruses, human rhinoviruses (HRV) and enteroviruses (EV). This genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (PV, EV-A71 and -D68, and to a lesser extent HRV) can invade and infect the CNS, with detrimental consequences. Even though extremely rare, HRV-induced meningitis and cerebellitis have been described. Although EV infections are mostly asymptomatic, outbreaks of EV-A71 and D68 have also been reported in different parts of the world during the last decade. EV-A71 is an etiological agent of the hand–foot–mouth disease (HFMD) and has occasionally been associated with upper respiratory tract infections. EV-D68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes. Both serotypes have been associated with neurological disorders like acute flaccid paralysis (AFP), myelitis (AFM), meningitis and encephalitis.
Last but not least, human coronaviruses (HCoV) are another group of respiratory viruses that can naturally reach the CNS in humans and could potentially be associated with neurological symptoms. These ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses like PHEV (porcine hemagglutinating encephalitis virus), FCoV (feline coronavirus) and the MHV (mouse hepatitis virus) strains of MuCoV, which can all reach the CNS and induce different types of neuropathologies. MHV represents the best described coronavirus involved in short- and long-term neurological disorders (a model for demyelinating MS-like diseases). Taken together, all these data bring us to consider a plausible involvement of HCoV in neurological diseases.
This study was approved by the Medical Research Ethics Committee of R.D. Kandou General Hospital (Ethical Approval No. 066/EC-UPKT/III/2016) and Eijkman Institute Research Ethics Commission (Ethical Approval No. 78). Written informed consent for participating in the study was obtained from all of the patients, guardians, or accompanying close relatives.
Eastern equine encephalitis (EEE) commonly called triple E or, sleeping sickness is a rare but serious viral disease affecting horses and man. The disease is transmitted through mosquitoes and man and horses are dead-end hosts.
EEEV belongs to the genus Alphavirus of the family Togaviridae. It is closely related to Venezuelan equine encephalitis (VEE) virus and Western equine encephalitis (WEE) virus. This virus has North American and South American variants. The North American variant is more pathogenic. EEE is capable of infecting a wide range of animals including mammals, birds, reptiles and amphibians. The virus has been reported to cause disease in poultry, game birds and ratites. The disease has also been reported to occur in cattle, sheep, pigs, deer, and dogs though sporadically. The disease is present in North, Central and South America and the Caribbean. EEE was first recognized in the USA in 1831 from an outbreak where 75 horses died of encephalitic illness and EEE virus (EEEV) was first isolated from infection horse brain in 1933. The serological evidence and outbreaks of the disease have also been reported from horses in Canada and Brazil [119, 120]. Countries with incidence/serological evidence are presented in Fig. (3). EEEV infection in horses is often fatal. The human cases were identified first time in 1938 in the north-eastern United States. Thirty children died of encephalitis in this outbreak. The fatality rate in humans was 35%. The outbreaks of the disease also occurred in horses simultaneously in the same regions. A total of 19 human cases of the disease were reported in children between 1970-2010 in Massachusetts and New Hampshire. As per the CDC reports 220 confirmed human cases of the disease occurred in the U.S. from 1964 to 2004. In 2007, a citizen of Livingston, West Lothian, Scotland became the first European victim of this disease after infected with EEEV from New Hampshire. EEE has been diagnosed in Canada, the United States of America (USA), the Caribbean Islands and Mexico [122, 123]. Eighteen cases of Eastern equine encephalomyelitis occurred in six Brazilian states between 2005 and 2009.
Alternate infection of birds and mosquitoes maintains these viruses in nature. Culiseta melanura and Cs. morsitans species are primarily involved. Transmission of EEEV to mammals occurs via other mosquitoes which are primarily mammalian feeders and called as bridge vectors. Infected mammals do not circulate enough viruses in their blood to infect additional mosquitoes. The virus is introduced by mosquitoes, but feather picking and cannibalism also contribute towards the transmission of the disease within the flocks. Most people bitten by an infected mosquito do not develop any symptoms. The symptoms generally appear 3 to 10 days after the bite of an infected mosquito. The clinically affected patients may have pyrexia, muscle pains, headache, photophobia, and seizures. EEEV is one of the potential biological weapons. The disease in horses is characterized by fever, anorexia, and severe depression. Symptoms appear one to three weeks post-infection, and begin with a fever that may be as high as 106ºF. The fever usually lasts for 24–48 hours. In severe cases, the disease in horses progresses to hyper-excitability, blindness, ataxia, severe mental depression, recumbency, convulsions, and death. The nervous symptoms may appear due to brain lesions. This may be followed by paralysis, causing the horse to have difficulty raising its head. The horses usually suffer complete paralysis and die two to four days after symptoms appear. Mortality rates among horses range from 70 to 90%.
There is no cure for EEE. Severe illnesses are treated by supportive therapy consisting of corticosteroids, anticonvulsants, intravenous fluids, tracheal intubation, and antipyretics. Vaccines containing killed virus are used for prevention of the disease. These vaccinations are usually given as combination vaccines, most commonly with WEE, VEE, and tetanus. Elimination of mosquito breeding sites and use of insect repellents may help in control of the disease.
Despite the announcement of the successful eradication of smallpox in 1979, the last case of rinderpest in 2008 and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. These include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. Consequently, the World Health Organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future.1 Understandably, the high human fatality rates reported during the recent epidemics of Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome have attracted high levels of publicity. However, many other RNA viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases.2,3 Chikungunya virus (CHIKV), West Nile virus (WNV) and dengue virus (DENV) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome viruses. For instance, for DENV, the number of cases of dengue fever/hemorrhagic fever is between 300–400 million annually, of which an estimated 22 000 humans die.4 Moreover, in the New World, within 12 months of its introduction, CHIKV caused more than a million cases of chikungunya fever according to Pan American Health Organization/World Health Organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain.5 Likewise, within two months of its introduction, to Polynesia, the number of reported cases exceeded 40 0006 and is currently believed to be approaching 200000 cases. Alarmingly, this rapid dispersion and epidemicity of CHIKV (and DENV or Zika virus in Oceania) is now threatening Europe and parts of Asia through infected individuals returning from these newly endemic regions. This is an increasingly worrying trend. For example, in France, from 1 May to 30 November, 2014, 1492 suspected cases of dengue or chikungunya fever were reported.7 Accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses in the future.
Arboviruses are transmitted between arthropods (mosquitoes, ticks, sandflies, midges, bugs…) and vertebrates during the life cycle of the virus.8 Many arboviruses are zoonotic, i.e., transmissible from animals to humans.9,10 As far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals.9,10 The term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle.11,12 Humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. In contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. As of 1992, 535 species belonging to 14 virus families were registered in the International Catalogue of Arboviruses.12 However, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. Whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future.
A previously well 69-year-old Australian man traveled to Thailand in early May 2017. The planned duration of travel was 12 days, and he did not attend a travel clinic prior to departure. The patient did not take malaria chemoprophylaxis, nor did he have a prior history of Japanese encephalitis virus (JEV) vaccination. He flew to Phuket, before traveling north to the popular tourist destination of Khao Lak, where he stayed in a beachside holiday resort. Heavy rainfall occurred during the trip, which limited holiday activities. He did not travel to rural or remote areas, but did receive numerous mosquito bites. On the eighth day of travel, he became unwell with lethargy and generalized muscle aches. He flew to Bangkok on the ninth day of the trip, and over the following 3 days his symptoms included ongoing lethargy, poor appetite, and drenching sweats, but no headache, meningism, or confusion. He returned to Australia on day 12 of travel and was admitted to a regional hospital the following day, now the fifth day after symptoms commenced (Figure 1).
Shortly after admission, on day 7 of his illness, he became confused. Cerebrospinal fluid (CSF) obtained by lumbar puncture demonstrated a glucose of 3.5 mmol/L (reference interval, 2.2–3.9 mmol/L), protein of 1.3 g/L (reference interval < 0.45), polymorphs of 280 × 10^6/L, lymphocytes of 90 × 10^6/L, and red blood cells of 54 × 10^6/L. He was commenced on empiric broad-spectrum antibiotics with vancomycin, meropenem, benzyl penicillin, and acyclovir. Due to a deteriorating conscious state, he was intubated the following day and transferred to a tertiary center. Neurological examination upon arrival revealed a generalized flaccid paralysis. A magnetic resonance image of the brain demonstrated no abnormalities. Seizure activity developed on day 10 of his illness, for which anticonvulsant medication was commenced.
Diagnostic assays performed on the initial and a repeat CSF (on day 10) were negative using conventional gel-based and real-time multiplex polymerase chain reactions (PCRs) for herpes viruses (herpes simplex 1 and 2 and varicella zoster), enterovirus, pan-flavivirus (Murray Valley encephalitis [MVE], Kunjin, dengue, West Nile, Zika, yellow fever, JEV), respiratory viruses (influenza, respiratory syncytial virus, parainfluenza, human metapneumovirus, picornavirus, adenovirus, coronavirus), meningococcus, and pneumococcus. The conventional pan-flavivirus PCR and JEV real-time PCR performed on plasma were also negative (see the Supplementary Data for detailed methods). HIV and syphilis serology were negative, as were bacterial cultures and screening swabs for Burkholderia pseudomallel.
On the fourteenth day of illness, JEV immunofluorescence serology (Euroimmun) performed in parallel on plasma from day 7, day 12, and day 13 of illness, was positive. The JEV IgG titre increased across the serial bleeds from 160 to 1280 to >2560 (confirmed by neutralization), suggesting recent infection, and JEV IgM was detected in all 3 specimens. Serology performed on CSF from day 10 was also positive for JEV IgM and IgG. The presence of measles IgG in plasma but not CSF was consistent with local production of JEV antibodies in CSF rather than contamination. Serology for MVE (EIA—total antibody) was weakly positive on day 7 but negative on days 12 and 13 and thought to represent nonspecific cross-reactivity. Dengue virus IgM, IgG, and NS1 antigen were not detected.
Given the results of serological testing, serial samples of plasma, whole blood, and urine were tested using the conventional gel based pan-flavivirus PCR and JEV real-time PCR. Notably, both urine and whole blood specimens were found to be persistently positive for JEV (confirmed by sequencing of the NS5 region), while plasma was found to be persistently negative (Figure 1). Reproducibility of these findings was confirmed by re-extracting and retesting the 4 most recent urine and whole blood specimens directly from the primary samples, and repeat testing was performed on separate PCR runs. Whole-genome sequencing and subsequent phylogenetic analysis (Supplementary Figure 1) identified the isolated JEV strain (VIDRL_JEV aligned) as a member of Genotype I. Our patient’s isolate localized within a subclade of viruses isolated from Thailand, geographically aligning with the travel history.
Aliquots of urine and whole blood were inoculated onto cultured cells to assess viral infectivity (see the Supplementary Data for detailed methods). Cytopathic effects were observed 7 days postinoculation from the day 14 urine specimen. The cell culture supernatant was tested by JEV real-time PCR and was found to be positive with a high level of detection, suggesting efficient viral replication.
Japanese encephalitis viral RNA was detected in urine samples out to day 26 after the onset of symptoms and in whole blood up until the final specimen was tested on day 28.
Electromyogram and nerve conduction studies performed on day 22 were consistent with an acute motor-axonal neuropathy or anterior horn cell pathology. No clinical improvement was evident after 4 days of intravenous immunoglobulin (IVIG) administered at a dose of 1 g/kg daily, and the patient died 30 days after the onset of symptoms.
No difference in sensitivity was observed between the viral strains tested or between lots of slides. A sample dilution of 1:20 for IgM and 1:40 for POWV IgG IFAs demonstrated the optimal balance between sensitivity and nonspecific background staining (Fig. 1). Tick-borne disease (TBD) samples with titers of 1:320 and 1:160 in the plaque reduction neutralization test using a 90% reduction cutoff (PRNT90) were assayed at optimized screening dilutions to confirm. All but one of the POWV encephalitis samples obtained from the New York State Department of Health (NYSDOH) with PRNT90 titers of 1:20 were detected by the TBE-C EIA screen and confirmatory POWV IFA. A PRNT90 titer of 1:20 was determined to be the limit of detection (LOD) for the serologic test panel and was confirmed as such using known PRNT90-positive samples (Table 1). At these screening dilutions, the serologic panel showed an analytical sensitivity of 89% (Table 1). Reproducibility studies showed 100% accordance (k = 1.0).
Enteroviruses are non-enveloped single stranded RNA viruses of the genus Enterovirus within the family of Picornaviridae. Seven species of Enterovirus are associated with human disease: Enterovirus A-D and Rhinovirus A-C. While rhinoviruses commonly cause mild respiratory illness, enteroviruses A-D (EVs) are a significant cause of morbidity and mortality worldwide. Prior to being reclassified as EV A-D, EVs were originally classified as polioviruses (PV) 1–3, coxsackieviruses (CV) A1–24 and B1–6, echoviruses (E) 1–33 and numbered enteroviruses (68–121).
In addition to the surveillance system for poliomyelitis, some countries have established comprehensive surveillance programs for circulating non-polio EVs [2, 3]. In recent years, surveillance activities have been enhanced in response to the emergence of hand, foot, and mouth disease (HFMD) causing enteroviruses in the Asia-Pacific region and the global spread of EV-D68 causing respiratory infections [5, 6].
EV infections are often asymptomatic, but may also result in a diverse spectrum of clinical illness, varying from mild febrile illnesses to severe disease of the cutaneous, gastrointestinal, respiratory, cardiovascular, and central nervous system (CNS) [7, 8]. Generally, EV A is associated with herpangina and hand, foot and mouth disease (HFMD), EV B with herpangina and sporadic and epidemic viral meningitis or encephalitis, EV C with poliomyelitis and EV D with respiratory infections [2, 9–11]. In Viet Nam, since 2005, various serotypes of EV A, most commonly enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), CV-A10, and CV-A6 have been associated with outbreaks of HFMD [12, 13] and EVs have also been frequently detected in aetiological studies of CNS and respiratory infections [14–18].
In the majority of aetiological studies only generic RT-PCR is used for detection of enteroviruses [14–16, 18, 19]. Information about specific enterovirus serotypes circulation and their associated clinical phenotypes therefore remains sparse.
Here we report the clinical associations and serotyping results of EVs that were previously detected in our studies of CNS and respiratory infections in southern and central Viet Nam between 1997 and 2010.
The nervous system is a target for acute viral infections, as well as a reservoir of latent and persisting viruses. In general, the absence of overt neurological deficits or pathology indicates effective immune control of persisting viruses in immunocompetent individuals. However, this balance is highly tenuous, as indicated by cases of JC virus-mediated progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals with acquired immune deficiency syndrome or those receiving treatment for multiple sclerosis (MS) or lymphoma. Similarly, the activation of herpes simplex virus (HSV) and cytomegalovirus in the nervous system can be devastating in immunocompromised individuals. Moreover, senescent immune responses in an increasingly aging population enhance disease susceptibility to both reactivating persistent viruses in the central nervous system (CNS), as well as to acute encephalitic arboviral infections. Numerous human infections involving the CNS, including those caused by measles, rubella, polio, varicella zoster, mumps, HSV and Japanese encephalitis virus (JEV), as well as lyme neuroborreliosis are characterized by intrathecal antibody (Ab) in the cerebral spinal fluid (CSF) consistent with the presence of local Ab-secreting cells (ASC). Although the causative agent still remains unknown in many cases of suspected viral encephalitis, detection of virus-specific immunoglobulin (Ig) in the CSF can be a reliable diagnostic tool to confirm a suspected viral encephalitis indicated by molecular analysis. For example, acute poliomyelitis or encephalitis mediated by insect-borne viruses such as JEV are associated with virus-specific IgM and IgG in CSF within ~2 weeks of clinical presentation. While Ab persists over several months in the case of JEV, they appear more transient in poliomyelitis. Overall, Ab detection may be more transient in cases of acute encephalitis, while it persists during chronic disease such as measles virus-associated subacute sclerosing panencephalitis. A specific protective or detrimental role is often difficult to infer due to difficulties in obtaining longitudinal serum and CSF samples. Even when available, the role of serum versus intrathecal Ab cannot be readily distinguished. Overall, intrathecal humoral responses appear to be associated with protective rather than pathogenic functions. Thus, a beneficial outcome of JEV encephalitis is correlated with intrathecal IgG. Similarly, intrathecal Ab synthesis may be an indicator of protection during CNS retrovirus infection. Ab also correlates with reduced CNS viral load and milder clinical disease course in patients with tropical spastic paraparesis/HTLV-I-associated myelopathy. An inverse correlation between intrathecal-neutralizing Ab and macrophage-tropic SIV was also observed in the SIV encephalitis model of HIV. Lastly, the CSF of MS patients harbors Ab to multiple viruses prevalent in the Western population, e.g. varicella zoster, rubella, HSV-1 and JC viruses. These Ab appear to be markers of MS and are not indicative of active disease due to virus infection. Nevertheless, the potential danger of losing control of persisting CNS viruses became apparent by the development of PML following rituximab (anti-CD20 monoclonal Ab) reduction of circulating B cells during therapy for rheumatoid arthritis and MS.
During experimental CNS infections, particularly by RNA viruses such as Sindbis, rabies and corona viruses, ASC play a vital local protective role. The reliance on local ASC for sustained Ab output provides a potent complement-independent non-lytic mechanism of immune control within the CNS, potentially regulating a variety of neurotropic infections. Despite constituting a critical component controlling viral persistence, little is known about the origin and maintenance of ASC in the CNS or other specialized microenvironments. This review focuses on insights gained throughout the last decade on humoral immune responses within the CNS during encephalitis and persistent infections mediated by RNA viruses.
Viral encephalitis is defined as pathological inflammation of the brain parenchyma secondary to viral infection (1). This syndrome is a rare but clinically serious outcome of infection with a range of DNA and RNA viruses. Encephalitis is associated with appreciable mortality and high rates of permanent neurological impairment in survivors, and in most cases there is no available antiviral therapy (1). Annual healthcare expenditure associated with the acute care of patients with encephalitis was estimated in the region of $2 billion (2), although indirect costs are likely much higher. Furthermore, several emerging causes of viral encephalitis are considered by WHO to be a significant threat to global public health (3). Fundamental to addressing this unmet medical need is research to better understand the pathogenesis of viral encephalitis (4).
“When status epilepticus continues or recurs in spite of 24 hours of general anesthesia, it is defined as superrefractory status epilepticus” (8). Encephalopathies or another central nervous (CNS) infection should be considered when the patient has a group of symptoms of recent febrile illness with behavior, cognition, consciousness and personality changes or new focal neurological signs (6). The differential diagnosis of encephalopathy in children includes a wide range of etiologies such as metabolic, genetic, traumatic, infectious, para-infectious, toxic and malignancies (4). Inflammation caused by etiologies such as direct invasion of brain parenchyma, antibodies directed against components of CNS, has an important role in status epilepticus (SE) (2).
FIRES is an infection-related epileptic encephalopathy with undefined etiology. It has many names like, Devastating epileptic encephalopathy in school-aged children (DESC), Acute encephalitis with refractory repetitive partial seizures (AERRPS), Idiopathic catastrophic epileptic encephalopathy, and New onset refractory status epilepticus (NORSE). Severe refractory status epilepticus due to presumed encephalitis. Genetic susceptibility and inflammation have been proposed as the etiologies (3). In FIRES, children develop seizure that rapidly turns into SE. This situation occurs a few days to one week after a non-specific febrile infection.
The chronic phase continues after the acute phase characterized by pharmaco-resistent epilepsy. Seizures are usually generalized with focal onset. Weeks or months after the initiation of the illness, seizures decrease or stop, and intractable epilepsy starts a few weeks to 3 months after the end of SE (2). The prognosis of FIRES is poor (3).
All patients afflicted by FIRES have an infection one week before the beginning of the symptoms which is mostly respiratory tract infection (5).
Autoimmune encephalitis is a group of disorders associated with antibodies against the surface proteins of neuronal cells and synaptic receptors. They have a wide range of symptoms including seizure, behavioral changes, psychosis, catatonia, autonomic dysregulation and abnormal movements (10). Some patients afflicted by autonomic encephalitis may progress to a more generalized encephalopathy with movement disorder (4). Nonspecific viral infections may lead to breaking immune tolerance and increase the possibility of the antibodies to enter CNS by increasing the permeability of the blood-brain barrier. Occasionally a tumor that produces the target neuronal antigen likely contributes in triggering the immune response. However in many of autoimmune encephalitis disorders the blood brain barrier seems to be intact and the autoantibodies may be synthetized inside the CNS (10).
Perhaps HSV is the most frequent cause of sporadic encephalitis, and have a tendency to affect the temporal lobe (1).
In herpes encephalitis negative result of polymerase chain reaction for HSV, does not rule out the infection and treatment with acyclovir should be completed for 21 days (9). A cerebro-spinal fluid sample without abnormal cells has been described for varicella zoster virus, Epstein- Barr virus and cytomegalovirus and is more prevalent in immune-deficient patients (6).
Systemic infection with influenza virus can cause seizure provoked by fever and systemic illness, extrapyramidal syndromes, Guillain-Barre syndrome, encephalitis and transverse myelitis. H1N1 can lead to encephalopathy and focal findings and there has been no evidence supporting the benefits of antiviral treatment for neurologic course of the disease. A rare type of acute encephalopathy/ encephalitis associated with influenza, can begin with a fulminant neurologic disease and may be accompanied with any influenza viral serotype. This disease may be associated with a genetic disorder in releasing pro- inflammatory cytokines and hypercytokinemia (1).
Human herpes virus type 6 which is a common viral illness in childhood may invade CNS at the time of primary infection and rarely cause a clinical neurologic disease but long-term latent infection may occur, and in case of immunosuppression, acute limbic encephalitis, probably due to reactivation of the virus can occur (1).
The patient’s problem presented first with an infectious disease (upper respiratory tract infection) and fever that proceeded to vomiting, decreased level of consciousness and refractory seizure with lateralized features. He responded poorly to antiepileptic drugs and sustained a long course of hospitalization and complications of high doses of medications and longstanding stay in hospital. The neurologist consultant even tried ketamine, which has had promising effects in controlling refractory status epilepticus, when midazolam, propofol and phenobarbital, failed to control seizures (7). The differential diagnosis were as follows: Fever-induced refractory epileptic encephalopathy (FIRES), infectious and autoimmune encephalitis, however work-ups had not revealed any evidence of any specific diagnosis, at last we assume that he was afflicted by viral infectious encephalitis as he had, fever, vomiting, and prodromal symptoms of infectious (most probably viral) disease, prior to onset of the seizure attacks. Autoimmune encephalitis was considered because of the refractory course and abnormal movements occurred in the course of the illness and ruled out as the antibodies’panel of cerebro-spinal fluid (CSF) was negative and no response seen to high dose of methylprednisolone and intravenous immunoglobulin administration.
In Conclusion, we did not find abnormal results in CSF analysis and viral PCR work-ups, but as mentioned above occasionally this may happen and clinically we assume viral encephalitis as the first differential diagnosis of our patient.
Acute disseminated encephalomyelitis (ADEM) is an encountered immune-mediated demyelinating disorder usually associated with viral infections or post vaccination. The incidence of ADEM is 0.64 per 1,00,000 patients per year whereas central nervous system (CNS) complications is reported in up to 0.01% of the Mycoplasma pneumoniae infections. ADEM following Mycoplasma pneumoniae (M. pneumoniae) infection is a rare condition. It should be in the differential diagnosis whenever respiratory symptoms are followed by neurological manifestations and encephalopathic signs. We report a case of ADEM secondary to M. pneumoniae that failed to improve with intravenous methylprednisolone and immunoglobulin; successfully treated with plasmapheresis along with antimicrobials.
West Nile virus (WNV), a plus-sense, single-stranded neurotropic flavivirus, has been a public health concern in North America for more than a decade. The virus is maintained in an enzootic cycle that involves mosquitoes and birds, with humans and horses as incidental hosts. Infection in humans results from mosquito bites, blood transfusion, organ transplantation, breast feeding, and in utero or occupational exposure. WNV infection of the central nervous system (CNS, neuroinvasive disease) commonly presents as encephalitis, meningitis, or acute flaccid paralysis. The overall mortality rate in persons who develop WNV neuroinvasive disease is about 10%, although the mortality rate increases significantly in the elderly and immunocompromised. Recently, some WNV convalescent patients were reported to have significant long-term morbidity years after their acute illness; symptoms include muscle weakness and pain, fatigue, memory loss, and ataxia. At present, there is no specific therapeutic agent for treatment of the infection. No approved human vaccines are available for its prevention.
WNV has been studied in various animal models, including mice, hamsters, monkeys, and horses. The murine model is an effective in vivo experimental model to investigate viral pathogenesis and host immunity in humans. Following the initial subcutaneous or intraperitoneal inoculation in mice, WNV induces a systemic infection and eventually invades the CNS. Mice die rapidly when encephalitis develops, usually within one to two weeks. The severity and symptoms of lethal infection observed in the murine model mimic the symptoms caused by WNV infection in humans. Studies from experimental animal models, in vitro cell culture, and/or WNV patient samples have provided important insights into host immunity to WNV infection. Natural killer (NK) cells and γδ T cells are two innate lymphocytes that respond rapidly and non-specifically to viral infection. They are also known to form a unique link between innate and adaptive immunity. Moreover, the characteristics of these two cell types in adaptive immunity have been described in several disease models. In this review, we will discuss recent studies on these two unique cell types in both protective immunity and viral pathogenesis during WNV infection.
Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates. Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay.
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis (2-DE), protein chip, mass spectrometry, and suspension array technology. However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods. Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Zika virus (ZIKV) is a mosquito-borne virus from the genus Flavivirus in the family Flaviviridae and consists of two genetically distinct lineages: Asian and African. Other notable viruses within this genus include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and yellow fever virus (YFV). Like most flaviviruses, the ZIKV is an enveloped virus with a capsid 50 nm in diameter and an RNA genome of approximately 11 Kb in length. The genome is translated as a single long open reading frame (ORF) and encodes ten proteins. This includes seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), which mediate viral replication for synthesis of new viral particles, and three structural proteins (C, prM, and E), which comprise the capsid and play a key role in host immune evasion.
First identified in 1947 in the Zika Forest of Uganda, ZIKV infections in humans remained sporadic for 60 years, with very few cases reported, until April 2007 when the ZIKV caused an outbreak on Yap Island, Federated States of Micronesia. In 2013, the ZIKV was identified in French Polynesia and spread rapidly across the Pacific, including New Caledonia and Cook Islands. Coincidentally, during those outbreaks, the link between Guillain–Barré syndrome (GBS) and ZIKV was reported, raising concerns about the neurological tropism of the virus. In May 2015, the first case of ZIKV infection was reported in Brazil and the virus rapidly spread throughout the country and much of Latin America, causing the largest recorded epidemic of the virus to date. The Brazilian epidemic raised great international concern because of severe birth defects, including microcephaly, in neonates born to mothers infected by ZIKV during pregnancy.
ZIKV is transmitted mainly through the bite of infected mosquitoes from the genus Aedes, although other vectors may also be involved in the transmission. Additionally, other routes of ZIKV transmission have been identified, including blood transfusions, transplacental, perinatal, and sexual intercourse. ZIKV infection usually causes a self-limiting and a mild illness, where the majority of cases are asymptomatic and, when present, symptoms include fever, headache, rash, conjunctivitis, and arthralgia. In regions where there is a circulation of other arboviruses, such as DENV and chikungunya (CHIKV), the clinical diagnosis of ZIKV infection becomes extremely difficult because of common symptoms. Therefore, laboratory-based molecular diagnosis is of fundamental importance to correctly identify the etiologic agent.
Given the lack of approved vaccines and antivirals against ZIKV, a rapid and reliable point-of-care (POC) diagnostic test for detection of ZIKV is urgently required for control and prevention measures and to increase the diagnostic capacity of ZIKV-affected, mainly in low-resource areas. ZIKV infection is diagnosed in the laboratory by nucleic acid amplification tests or serological methods, including enzyme-linked immunosorbent assays (ELISA), plaque reduction neutralization tests (PRNT), and lateral flow assays (rapid tests). Currently, RT-qPCR is considered the gold standard method to detect ZIKV from patient and mosquito samples. Although RT-qPCR provides high-quality results, the test requires extensive sample preparation, RNA extraction, expensive equipment, and technical expertise to run and interpret the amplification of the viral RNA. Moreover, available serological methods are prone to produce false-positive results due to cross-reaction with other flaviviruses in circulation, such as DENV, and are therefore of limited value.
Loop-mediated isothermal amplification (LAMP) is a powerful alternative POC assay for the virus as it allows rapid, robust, and simple amplification of nucleic acid targets at a single and fixed temperature. The assay has many advantages over RT-qPCR, including rapidity, low cost, high sensitivity, and high specificity. LAMP results can also be easily read with the naked eye through color-based reporters that can be added to the reaction mixture. Importantly, the simple, single-temperature incubation allows LAMP reactions to be performed without expensive equipment, directly in the field. Since the 2015 emergence of the ZIKV in Brazil, many LAMP assays have been developed for diagnosis by research groups across the world. These diagnostic platforms based on LAMP have proven to be specific, sensitive and inexpensive POC tools that can be applied even in resource-limited regions of the world. Here we review the development and application of LAMP methods for the diagnosis of ZIKV and explore the next steps to bring this assay into mainstream use.
Samples from a total of 203 patients were included in this study, including from 79 patients with CNS infection and 124 with respiratory infection. When analyzing for the monthly distribution of cases, there was no clear peak among CNS cases, whereas two peaks of respiratory cases were found in April and November (Fig. 1).
Among patients with CNS infection (n = 79), 51 were children (median age 2 years, interquartile range [IRQ]: 1–5) and 28 were adults (20 years, IRQ: 17–29.5). All adults had EV detected in CSF and 93% (26/28) had meningitis as discharge diagnosis. Of the 51 children, 10 had EV detected in CSF, and the remaining (41/51) had EV detected in respiratory and/or rectal swabs. Encephalitis was the most common discharge diagnosis (30/41). Fifteen deaths were noted, and all were children with EV detected in swabs only (Table 1).
Among patients with respiratory infections, the median age was 12.7 months (IQR: 5.7–24.3). Inpatients (n = 65) were younger than outpatients (n = 59), due to study enrolment criteria (Table 2). Bronchiolitis (as assessed at the discretion of treating physicians) was the most common clinical diagnosis (57% in outpatients and 61% in inpatients). Thirty-five percent of inpatients had a clinical diagnosis of pneumonia (Table 2).