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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.
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.
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.
ZIKV, an emerging flavivirus, shares common clinical symptoms with DENV and chikungunya virus (CHIKV). The outbreaks caused by these viruses present a large number of diagnostic challenges. The clinical manifestations of ZIKV involve similar clinical symptoms to DENV and CHIKV, which include fever, exanthema, conjunctivitis, retro-orbital headache, and arthralgia (Cardoso et al., 2015). The diagnosis of viral infection has specific management implications for medical personnel. The identification of DENV requires a routine follow-up to examine thrombocytes along with hematocrit, whereas for CHIKV, chronic arthralgia should be assessed due to its high prevalence. In the case of ZIKV, a detailed diagnosis of sexual and maternal-fetal transmission should be performed to confirm the risk of congenital microcephaly in newborn babies (Fauci and Morens, 2016). A variety of arboviral infections (arthropod-borne; DENV is the most common arboviral infection) may have similar clinical presentations; therefore, their circulation may be under-reported if specific diagnostic tools have not been implemented. However, there are several drawbacks in ZIKV diagnosis due to the lack of availability of diagnostic tools and the frequent cross-reactivity of antibodies between flaviviruses, which have resulted in several limitations in the use of serology (Musso et al., 2015). Commonly, no routine testing of virus cultures is performed, and an antigenic detection test is lacking at present (Musso et al., 2015; Saiz et al., 2016).
The symptoms of ZIKV infection usually tend to be mild, and the initial symptoms can escape notice, reducing the opportunity to collect a sample. Although the viremic period has not been completely defined, viral RNA has been detected in serum after the onset of symptoms up to day 10. In addition, RNA particles of ZIKV have been detected in urine over an extended period in the acute phase, leading to the possibility of considering an alternative sample type. Evidence suggests that serum samples should be taken during the first 5 days after the onset of symptoms supported in some more detailed studies (Musso et al., 2015). Symptoms of microcephaly associated with ZIKV during the development of newborns in the uterus have been reported (Oduyebo et al., 2016). For the diagnosis of infant microcephaly, a complete analysis of head circumference is requested (Kallen, 2014), as the diagnostic parameters for severe microcephaly include a head circumference more than 3 standard deviations below the mean (Von der Hagen et al., 2014). Testing should be performed in pregnant women with positive or inconclusive results from ZIKV testing. If diagnostic parameters confirm possibility of congenital ZIKV infection in an infant, further clinical evaluation should be performed in follow-up. Fever is a common presenting symptom in patients testing positive for arboviruses due to their association with multiple illnesses; hence, it is suggested to eliminate differential diagnoses (Kelser, 2016). Patients with DENV and ZIKV present with temperatures >40°C and <38.5°C, respectively. ZIKV is usually self-limiting, with symptoms lasting 2 to 7 days. Jaundice is a distinguishing clinical presentation of yellow fever virus and can aid in identifying patients with ZIKV virus. The presence of nausea, vomiting, and bleeding may be helpful in identifying DENV. Any of the above symptoms in an individual who has been exposed to ZIKV indicates the possibility of ZIKV infection, and immediate serum testing should therefore be performed (Centers for Disease Control and Prevention [CDC], 2015a,b).
OHFV distribution is restricted to western Siberia (Figure 1). The main vector of OHFV is the meadow tick, Dermacentor reticulatus, which can also transmit the virus to humans. However, humans are mainly infected after contact with infected muskrats (Ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection. Muskrats develop high viremia which can last for several weeks. Human infection occurs through contact with urine, feces, and blood. Secretion of OHFV in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed. The exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but 165 cases were reported between 1988 and 1997. OHFV may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. Thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. Case fatality rates range from 0.5 to 2.5%. No antiviral treatments are available against OHFV, instead treatment is focused on supportive care to minimize hemorrhage and other complications.
POWV is found in Russia and North America, and is the only TBFV present in America (Figure 1). It is transmitted by Ixodes scapularis, Ixodes cookei, and several other Ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. Milk-borne POWV transmission might also be possible since POWV virus has been found to be secreted in milk under experimental settings.
Although not much is known about POWV pathogenesis, recent studies in mice have found that tick saliva was important to enhance POWV transmission and the outcome of disease. Furthermore, it has been demonstrated that POWV infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected. Interestingly, macrophages were found to be the primary target for POWV in the spleen, and in the CNS, which is the main target site for POWV infection, neurons have been shown to be the primary target for POWV in mice and humans.
During the last 10 years there has been an increase of POWV in the USA with approximately 100 reported cases. The recent rise in incidence could be due to increased surveillance and diagnosis of POWV, or it may represent a true emergence of the disease in endemic areas, or both. The incubation period ranges from 1 week to 1 month. The symptoms of POWV infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of 10%. Approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www. CDC.gov). There are no antiviral treatments or vaccines available against POWV.
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.
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.
DENV is widespread in the temperate and tropical regions of the world, and each year approximately 50–150 million people are infected (Bhatt et al., 2013), with over 10 000 deaths (Stanaway et al., 2016). Symptoms of Dengue fever include a high fever, headache, vomiting, muscle and joint pains, and skin rash. Severe cases of disease is usually associated with secondary infection with heterologous types of DENV (Halstead, 1988), and can develop into Dengue hemorrhagic fever (with hemorrhage, thrombocytopenia and blood plasma leakage), or into Dengue shock syndrome, both of which are potentially fatal (Kularatne, 2015).
Infection of 129Sv/Ev mice with 108 pfu DENV-2 via the intravenous (IV) route resulted in 87% survival (26 out of 30 mice), and inoculation with 4.4×104 pfu DENV-1 via IV resulted in 93% survival (40 out of 43 mice) (Shresta et al., 2004). In their study, Shresta et al. challenged 5–6 week old Ifnar–/– mice (129Sv/Ev background) with DENV-2 strain PL046 (n=12) and DENV-1 strain Mochizuki (n=16) at the same doses and inoculation routes. While no lethality was observed, sera and major organs harvested from infected mice at 3 and 7 dpi showed the presence of virus in all sera, liver, spleen, and lymph node samples, as well as some brain and spinal cord samples (Shresta et al., 2004). Interestingly, mice deficient for the type I and II IFN receptors (AG129) showed uniform death from DENV-2 infection with animals dying between 7–30 dpi, and DENV-1 as well with mice succumbing to disease between 7–14 dpi (Shresta et al., 2004).
Occult hepatitis B virus infection (OBI) is an emerging type of HBV infection when HBV DNA is detectable among HBsAg negative infected patients (100). Attention to OBI has increased due to its potential role in accelerating the progression of liver fibrosis and cirrhosis, ultimately leading to hepatocellular carcinoma (HCC); It is transmitted via blood transfusion and transplantation (100).
Introduction of the occult infection was the consequence of improvement of HBV DNA detection and introduction of more sensitive methods which were not available earlier than 1985 (101). OBI seems to be highly prevalent in Asia (102). The first evidence of OBI in Iran refers to 2001–2002 when 22% of chronic liver patients were revealed to be positive for HBV genome tests (103). It has been detected amongst 30% of high risk groups as well as hemodialysis patients and is considered common in HIV patients (104–106).
As OBI has been found among a large proportion of HBcAb positive healthy blood donors, thus the blood of these people should be screened in blood transfusion centers to prevent HBV transmission (84, 107–109).
YFV is endemic in tropical areas of Africa and South America (WHO, 2016b), when the virus was introduced via the slave trade during the 17th century. Many infections are symptomatic, but if clinical symptoms appear, they include fever, chills, appetite loss, nausea, muscle pains, and headaches. A small percentage (~15%) of cases will go on to develop more severe disease including jaundice, dark urine, vomiting and abdominal pain. Hemorrhage from the mouth, nose, eyes or stomach may occur and 50% of patients with these symptoms succumb to disease (WHO, 2016b). YFV was responsible for ~127 000 severe infections and 45 000 deaths in 2013 (WHO, 2016b), with increased incidence over the past decades, and the risk of an outbreak in urban centers is a serious public health threat (Barrett & Higgs, 2007).
Inoculation of wild-type 129 mice SC in each rear footpad with 104 pfu of YFV did not result in any weight loss or death (Meier et al., 2009). In their study, Meier et al. (2009) challenged 3–4 week old Ifnar–/– mice (129 background) with YFV strains Asibi or Angola73 under the same conditions. The mice were shown to be susceptible to the challenge, with death occurring between 7–9 dpi. Additionally, the mice developed viscerotropic disease with virus dissemination to the visceral organs, spleen and liver, in which severe damage of the organs can be observed with gross pathological examination and hematoxylin/ eosin staining. Elevated levels of MCP-1 and IL-6 in these organs are suggestive of a cytokine storm (Meier et al., 2009).
The recent epidemic of Ebola virus in Africa as well as the emergence of a hitherto unknown virus known as Middle East respiratory syndrome coronavirus (MERS-CoV), Bas-Congo virus in central Africa or of severe fever with thrombocytopenia syndrome virus (SFTSV) in China have repeatedly shown the global impact of emerging infectious diseases (EIDs) on economics and public health. These EIDs, more than 60% of which are of zoonotic origin, are globally emerging and re-emerging with increased frequency. Surveillance and monitoring of viral pathogens circulating in humans and wildlife and the identification of EIDs at an early stage is challenging. Many potential emerging viruses of concern might already be infecting humans or wildlife but await their detection by disease surveillance. In remote and underdeveloped regions of the world, often no attention is paid towards possible infectious disease cases until a threshold of serious cases and deaths appears in a cluster and certain epidemic properties are reached. Some viruses might just be overlooked at population levels until they spread or re-emerge and become epidemic in another region or time. An effective strategy in virus surveillance would need to survey simultaneously a wide range of viral types in a large number of human and wildlife individuals in order to detect viruses before spreading. For example, the EcoHealth Alliance within the surveillance program PREDICT seeks to identify new EIDs before they emerge or re-emerge. Therefore, wildlife animals that are likely to carry viruses with zoonotic potential, e.g., bats, rodents, birds and primates, are sampled frequently. However, collecting swabs or blood from sufficient numbers of wildlife individuals and the subsequent identification of viruses is challenging. The solution for overcoming this challenge might be presented by the disease vector itself. Blood feeding arthropods feed on blood from a wide range of hosts including humans, mammals and birds. Therefore, they act as “syringes”, sampling numerous vertebrates and collecting the viral diversity over space, time and species. Xenosurveillance and vector-enabled metagenomics (VEM) are surveillance approaches that can exploit mosquitoes to capture the viral diversity of the animal, human or plant host the mosquito has fed on (Figure 1). Xenosurveillance, a term introduced by Brackney et al., refers to the identification of viral pathogens from total nucleic acids extracted from mosquito blood meals, either by next-generation sequencing (NGS) or conventional PCR assays. Recent developments in NGS and viral metagenomics, which is the shotgun sequencing of viral nucleic acids extracted from purified virus particles, offer great opportunities for the characterization of the complete viral diversity in an organism or a population. VEM, a technique used to sequence purified viral nucleic acids directly from insect vectors, has already been used to detect both animal and plant viruses circulating in vectors. This review summarizes findings from xenosurveillance efforts as well as VEM studies using mosquitoes, since both approaches combine sampling of multiple individuals of blood-feeding arthropods with the high-throughput properties of NGS.
Zika virus (ZIKV), the causative agent of the infectious disease Zika fever, is a positive-sense RNA virus that belongs to the family Flaviviridae, genus Flavivirus, and is similar to Dengue virus (DENV), yellow fever virus, Japanese encephalitis virus, and West Nile virus (Sikka et al., 2016). ZIKV was first isolated from Rhesus macaques in Uganda in 1947. Previously, only sporadic cases of negligible concern associated with human ZIKV infection were reported (Hayes, 2009). Now, ZIKV infections have become epidemic throughout the world (Charrel et al., 2016).
In the north-eastern states of Brazil, the public health authorities recently confirmed autochthonous transmission of ZIKV with the first known reported case of ZIKV infection in mainland South America (Campos et al., 2015; Zanluca et al., 2015), followed by 26 countries, including countries in the European Union and the outermost regions of the Americas, such as Barbados, Bolivia, Brazil, Colombia, Costa Rica, Curacao, Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala, Guyana, Haiti, Honduras, Jamaica, Martinique, Mexico, Nicaragua, Panama, Paraguay, Puerto Rico, Saint Martin, Suriname, the US Virgin Island, and Venezuela (Pan American Health Organization [PAHO], 2016; World Health Organization [WHO], 2016). An increased frequency of ZIKV infection among world travelers has been reported in European countries, including Austria, Denmark, Finland, France, Germany, Ireland, Italy, Portugal, the Netherlands, Spain, Sweden, Switzerland, and the UK (European Centre for Disease Prevention [ECDC], 2016).
The virion of ZIKV consists of an approximately 11 kb positive-sense RNA with a single capsid and two membrane-associated envelope proteins (M and E) (Leyssen et al., 2000; Daep et al., 2014; Charrel et al., 2016). Recent outbreaks of ZIKV infections have become fatal on a daily basis in the Americas, where this obscure viral candidate has been placed at the forefront of global healthcare. The reported occurrences of ZIKV infections are thought to be transmitted mainly by the mosquito species Aedes aegypti and Aedes albopictus. Infections have now dramatically increased in highly populated areas of South, Central, and North America due to the increased frequency of the international travel from Zika-infected areas (Bogoch et al., 2016). Considering the calamity of ZIKV infection, there is an urgent need to develop rapid detection methods for ZIKV along with DENV, which shares common clinical symptoms with ZIKV. The purpose of this review is to provide a complete update of the various analytical methods for virus detection, such as molecular, immunological, sensor-based and other detection assays, along with the advantages and limitations of these strategies. Furthermore, we suggest innovative hypothetical approaches for the development of liposome-based rapid detection assays for ZIKV detection, which will provide new insight to medical professionals for controlling this widespread epidemic virus candidate.
The incubation time for CHIKV is relatively short, requiring only 2–6 d with symptoms usually appearing 4–7 d post-infection. Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection. Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash,. The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims,.
Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM,,,. The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage. CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF,.
More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions,,. The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure,,,,,,.
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.
Bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. Virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance. However, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses.
Historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva). Haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. The significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated. In the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the Egyptian rousette bat (Rousettus aegyptiacus) as the reservoir for Marburg virus. Considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. Reviewed by Kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity. Further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal in order for vector-borne transmission to occur. Bats have long been suspected as reservoirs for arboviruses, but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. Here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs.
Chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus), is the causative agent of chikungunya fever. After first isolation of CHIKV in 1952 in present-day Tanzania, outbreaks and epidemics were limited to regions of Asia, Africa, and the Pacific Islands. In 2013, CHIKV emerged for the first time in the Americas, with sustained autochthonous transmission and rapid spread through the region [1–3]. The acute symptoms of CHIKV infection are similar to those of infection with other arbovirus species, including Dengue virus (DENV), Zika virus (ZIKV), and Mayaro virus (MAYV), each presenting with a constellation of symptoms including fever, headache, and myalgias/arthralgias. Long-term, CHIKV infections have been linked with persistent arthralgias in a subset of cases; it has also been reported that upwards of 90% of CHIKV-infected individuals are symptomatic, in contrast to findings with ZIKV, where it is estimated that only 20% of infected persons are symptomatic.
The similarity of the clinical presentation of acute-phase arbovirus infections is further complicated by the potential for simultaneous infection with multiple arboviruses. In a recent literature review, co-infections with CHIKV and DENV ranged from 1% to 34% of patients. However, virtually no data are available on frequency of co-infection with CHIKV and arboviruses other than DENV. Even where good laboratory diagnostic facilities are available, identification of co-infections often does not occur, as there is a tendency to cease investigation once an initial pathogen has been identified, and/or identification of a second pathogen may require facilities for virus isolation, which may not be available.
As part of ongoing studies of acute undifferentiated febrile illness in a cohort of school children in rural Haiti, we identified 82 children with RT-PCR-confirmed CHIKV infections during May-August 2014, corresponding with the time period when the Caribbean CHIKV epidemic was moving through Haiti. Specimens were also simultaneously screened by RT-PCR for DENV1-4, then additionally for ZIKV. Aliquots of the plasma specimens were then inoculated onto cell cultures for the isolation of additional pathogens of potential interest. We report here results of these studies, focusing on rates of arbovirus co-infection in our patient cohort and potential sources of origin of the co-infecting strains.
The median age of patients was 57.2 years (range, 23–88) and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5–14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.
The main clinical features in confirmed patients included sudden onset of fever (>37.5°C −40°C) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia (Table 1). A small number of cases suffered more severe complications, including hypotension, mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary hemorrhage, respiratory failure, disseminated intravascular coagulation, multiple organ failure, and/or death. Most patients had a good outcome, but elderly patients and those with underlying diseases, neurological manifestations, coagulopathy, or hyponatremia tended to have a poorer outcome.
Laboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Table 2). Biochemical tests revealed generally higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes, especially AST.
West Nile virus is the etiological agent of an emerging zoonotic disease whose impact on animal and public health is considerable, being the most widespread arbovirus in the world today (reviewed in Hayes et al., 2005a; Kramer et al., 2008; Brault, 2009). A percentage of WNV infections result in severe encephalitis, and it is a communicable disease both for human and animal health. WNV taxonomically belongs to the family Flaviviridae, genus Flavivirus. Virions are spherical in shape, about 50 nm in diameter, and consist of a lipid bilayer that surrounds a nucleocapsid that in turn encloses the genome, a unique single-stranded RNA molecule, which encodes a polyprotein that is processed to give the 10 viral proteins. Of them, three (C, E, and M) form part of the structure of the virion, and the rest (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) are so-called “non-structural” and play important roles in the intracellular processes of replication, morphogenesis, and virus assembly. Inserted into the lipid bilayer are two proteins, E (from “envelope”) and M (“matrix”), which participate in important biological properties of the virus, such as its host range, tissue tropism, replication, assembly, and stimulation of cellular and humoral immune responses. E protein contains the major antigenic determinants of the virus.
As far as we know, there are no serotypes of WNV, but two main genetic variants or lineages can be distinguished, namely lineages 1 and 2. While the former is widely distributed in Europe, Africa, America, Asia, and Oceania, the second is found mostly restricted to Africa and Madagascar, although it has recently been introduced in Central and Eastern Europe (Bakonyi et al., 2006; Platonov et al., 2008) and has further extended to southern Europe (Bagnarelli et al., 2011; Papa et al., 2011). In addition, other viral variants closely related phylogenetically to WNV have been described, which are different from lineages 1 and 2, and have been proposed as additional WNV lineages. One of them, known as “Rabensburg virus,” isolated form mosquitoes in the Czech Republic in 1997, shows low pathogenicity in mice (Bakonyi et al., 2005). Similarly, other viruses closely related to WNV have been isolated in India (Bondre et al., 2007), Russia (Lvov et al., 2004) Malaysia (Scherret et al., 2001), and Spain (Vazquez et al., 2010). All these viruses have been proposed to represent different genetic lineages of WNV. Except for the Indian variant, which has been involved in outbreaks of encephalitis in humans, the rest are of unknown relevance for animal and human health.
West Nile fever/encephalitis is a disease transmitted mainly by mosquitoes, while wild birds are its natural reservoir. WNV is capable of infecting a wide range of bird species. Nevertheless, birds were considered less susceptible to the disease until the recent epidemic of WNV in North America, affecting many species of birds lethally, made to re-examine this concept (Komar et al., 2003). Occasionally it may affect poultry species, mainly geese and ostriches. Other domestic birds like chickens and pigeons, are susceptible to infection but do not get sick, and are often used as sentinels for disease surveillance. In addition to birds, WNV can also affect a wide range of vertebrates species, including amphibians, reptiles, and mammals, and it is particularly pathogenic in humans and horses, which act epidemiologically as “dead end hosts,” that is, they are susceptible to infection but do not transmit the virus (McLean et al., 2002; Kramer et al., 2008).
The first case of WNF was described in Uganda (West Nile district, hence the name of the virus) in a feverish woman, from whose blood the virus was first isolated in 1937 (Smithburn et al., 1940). It was considered a mild disease, endemic in parts of Africa (an “African fever”). However, since around 1950s, the occurrence of disease outbreaks with neurological disease, lethal in some cases, caused by WNV, especially in the Middle East and North Africa, made necessary to rethink this concept. In humans, the majority of WNV infections are asymptomatic, about 20% may develop mild symptoms such as headache, fever, and muscle pain, and less than 1% develop more severe disease, characterized by neurological symptoms, including encephalitis, meningitis, flaccid paralysis, and occasionally severe muscle weakness (Hayes et al., 2005b). Advanced age is considered a risk factor for developing severe WNV infection or death. The mortality rate calculated for the recent epidemic of the disease in the U.S. is 1 in every 24 human cases diagnosed (Kramer et al., 2008).
In horses (reviewed in Castillo-Olivares and Wood, 2004) neurological disease is manifested by approximately 10% of infections, and is mainly characterized by muscle weakness, ataxia, paresis, and paralysis of the limbs, as a result of nerve damage in the spinal cord. They may also suffer from fever and anorexia, tremors and muscle stiffness, facial nerve palsy, paresis of the tongue, and dysphagia, as a result of affection of the cranial nerves. A proportion of horses infected with WNV die spontaneously or is slaughtered to avoid excessive suffering. The mortality rate can vary between outbreaks. For example, in the outbreak in 2000 in the Camargue (France), 76 horses were affected, of which 21 died (Zeller and Schuffenecker, 2004). In 1996 in Morocco, a WNV outbreak affected 94 horses, of which 42 died (Zeller and Schuffenecker, 2004). Severe equine cases do not seem to predominate in older horses, as occurs in humans (Castillo-Olivares and Wood, 2004). Other mammals may also suffer from the disease. Rodents such as laboratory mice and hamsters are highly susceptible, so they can be used as experimental model of WNV encephalitis. Lemurs and certain types of squirrels appear to be the only mammals capable of maintaining the virus in local circulation (Rodhain et al., 1985; Root et al., 2006). WNV can also infect other mammals, including sheep, in which it causes abortions, but rarely encephalitis (Hubalek and Halouzka, 1999). WNV has been isolated from camels, cows, and dogs in enzootic foci (Hubalek and Halouzka, 1999). The virus has been shown to infect frogs (Rana ridibunda), which in turn are bitten by mosquitoes, so that the existence of an enzootic cycle in these amphibians is postulated, at least for some variants of the virus (Kostiukov et al., 1986). Outbreaks of severe WNF with high mortality have been reported in captive alligators and crocodiles, presumably transmitted through feeding of contaminated meat (Miller et al., 2003). It has been shown experimentally that WNV can infect asymptomatically pigs (Teehee et al., 2005) and dogs (Blackburn et al., 1989; Austgen et al., 2004). However, guinea pigs, rabbits, and adult rats are resistant to infection with WNV (McLean et al., 2002). Among non-human primates, rhesus and bonnet monkeys (but not Cynomolgus macaques and chimpanzees), inoculated with WNV develop fever, ataxia, prostration with occasional encephalitis and tremor in the limbs, paresis or paralysis. The infection can be fatal in these animals.
The virus is propagated in the reservoir hosts, resulting in a viremic phase that usually lasts no more than 5–7 days (Komar et al., 2003). The duration and level of viremia depends on the species infected (Komar et al., 2003). The detection of the virus or its genetic material in serum or cerebrospinal fluid in a laboratory test is a proof of diagnostic value (De Filette et al., 2012). The virus is evidenced by virological (virus isolation) or molecular (RT-PCR-conventional and real-time, NASBA) techniques. In epidemiological surveillance it is useful to detect the presence of WNV in mosquitoes, for which they are homogenized and analyzed using the same methods mentioned above (Trevejo and Eidson, 2008). Specific antibodies against the virus are detectable in blood few days after infection (Komar et al., 2003; De Filette et al., 2012). Antibody detection is performed by serological tests (enzyme immunoassay or ELISA, hemagglutination inhibition or HIT) which can be confirmed by more specific serological techniques (virus-neutralization test; Sotelo et al., 2011c). Serological diagnosis of acute infection should be done by detection of IgM antibodies in serum and/or cerebrospinal fluid using an immunocapture ELISA together with the detection of an increase in antibody titer in paired sera taken one in the acute phase and the other, at least 2 weeks later (Beaty et al., 1989).
The fight against this disease is not straightforward because there are no vaccines licensed for human use, and even though there are some available for veterinary use, they are efficacious to prevent disease symptoms and outcome at the individual level but do not prevent the spread of the infection, mainly due to the establishment of an enzootic cycle among wild birds and mosquitoes (Kramer et al., 2008; De Filette et al., 2012). Control methods are mainly based on prevention and early detection of virus spread through epidemiological surveillance and targeted application of insecticides and larvicides (Kramer et al., 2008).
Rabies is an acute progressive encephalitis caused by viruses in the Genus Lyssavirus, Family Rhabdoviridae, with the highest fatality rate among conventional infectious diseases. Known in bats for well over a century, rabies is the best studied infection associated with the Chiroptera. Bats are the principal reservoirs for 10 of the 11 recognized lyssavirus species and are suspected as hosts of other putative species (5). Only one lyssavirus, Mokola virus (MOKV), has never been isolated from bats to date. However, the principal reservoir for MOKV is unknown (6). Another viral species, rabies virus (RABV), circulates in bats and other mammals (predominantly carnivores). Interestingly, RABV circulates in bats only in the Americas, whereas in carnivores, the disease circulates globally. In the Old World, bats maintain circulation of other lyssavirus species, such as Lagos bat virus (LBV), Duvenhage virus (DUVV), European bat lyssaviruses type 1 (EBLV-1) and 2 (EBLV-2), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), West Caucasian bat virus (WCBV), and Shimoni bat virus (SHIBV). For these viruses, bats are the principal hosts, with only a few spillover infections documented in other mammals. Isolation of RABV from Eurasian bats has been suggested several times, but never confirmed (reviewed in Kuzmin and Rupprecht (7)). Indeed, the surveillance data from developing countries is very limited. We do not know which lyssaviruses circulate in bats of northern Africa and southern Asia, although historical reports (8, 9) along with more recent serological findings (10–12) indicate that bats do maintain lyssavirus circulation in these territories.
A paralytic disease in cattle and sporadically in humans bitten by a vampire bat has been reported from the time of the Spanish first colonized Latin America. However, the diagnosis of rabies was first confirmed by the identification of Negri bodies in the brain of cattle during an outbreak in Brazil in 1911 (13). Vampire bats probably maintained rabies virus circulation for a long time prior to the arrival of Europeans in the Americas. The association between vampire bites and the disease was understood by natives, who cauterized or washed the bites to prevent the disease (14). However, historical antecedents might be some other progenitor virus, quite different from those ones that circulate in bat populations presently.
Economic losses due to vampire bat rabies in livestock are tremendous. In the enzootic area there is an at-risk population of more than 70million head of cattle. Vampire bats usually bite many animals in a herd. The proportion of animals bitten may vary from 6 to 52% (15). Significant outbreaks of vampire bat rabies were documented in Amazon area (Brazil, Peru) during recent years. Up to 23–55% of respondents interviewed had vampire bat bites during the last year. During the outbreaks, up to 15% of such bites caused rabies in humans (reviewed in Kuzmin and Rupprecht (7)).
An idea that vampire bats may be asymptomatic rabies carriers, shedding the virus in their saliva for months, was popular during initial studies of vampire bat rabies (16). However, in a well-documented experimental study by Moreno and Baer (17), the disease in vampire bats was similar to rabies observed in other mammals. The bats that developed signs of disease and excreted the virus via saliva soon died, whereas those that survived the inoculation without clinical signs never excreted the virus or had it in the brain as demonstrated upon euthanasia. More recently, the asymptomatic excretion of RABV in the saliva of experimentally infected vampire bats, which survived the challenge during at least 2 years of observation, was documented again (18). Clearly, this phenomenon requires additional investigation.
Rabies of insectivorous bats was first documented in 1953 in Florida. Later it was documented across the United States, in Canada, and Latin America. Several RABV lineages were documented, and in general, they correspond to particular host species (reviewed in Kuzmin and Rupprecht (7)). Moreover, widely distributed bat species, such as Mexican free-tailed bat (Tadarida brasiliensis) and big brown bat (Eptesicus fuscus), maintain circulation of several RABV variants across their geographic range. Insectivorous bats are the major source of human rabies in the United States and Canada, which became especially prominent after elimination of RABV circulation among dogs. During 1958–2009, a total of 49 naturally acquired human rabies cases caused by bat RABV variants were reported in the United States and Canada (excluding four rabies cases caused by organ transplantation from a donor who died of unrecognized rabies) (19, 20). In 19 of these cases the exposure was ‘cryptic’, as the patients did not recall any contact with animals or a bat was seen flying in the residence but no direct physical contact was reported.Appears that some bat bites, especially if they were inflicted by small bat species, may be ignored or not recognized as dangerous by people (such as a previously unattended child, mentally disabled, or intoxicated person).
Two closely related RABV variants (previously considered as one), associated with the silver-haired bat (Lasionycteris noctivagans) and eastern tri-colored bat (Perimyotis subflavus) have caused about 60% of human rabies cases, where the virus variant could be identified. These bats are relatively small, do not form large colonies, and usually do not roost in close proximity to human dwellings. In contrast, the big brown bat RABV variant and the Myotis RABV variant caused one human case each, even if these bats frequently occupy house attics and crevices in men-made constructions. Furthermore, big brown bats constitute about 90% of all rabid bats, submitted to diagnostic laboratories in the United States and definitely have more contacts with humans (21, 22). The Mexican free-tailed bat RABV variant caused several human rabies cases as well, including four cases that occurred in 2004 after transplantation of organs and vessel from a donor who died of rabies (23). Several versions were suggested to explain the disproportional prevalence of the silver-haired bat and eastern tri-colored bat RABV variant among human rabies cases. Investigations suggested that these viruses have enhanced pathogenicity to humans, for example they may have a greater ability to replicate in fibroblasts and epithelial cells, being delivered into a superficial bat bite (24).
In the Old World, the significance of bat rabies for veterinary and public health is well addressed only in the countries with developed surveillance systems, such as Western Europe and Australia. The EBLV-1 and EBLV-2 circulate in Europe among insectivorous bats Eptesicus fuscus and Myotis spp, respectively. These viruses caused at least three cases of human rabies, where the virus was characterized, in Finland, Russia, and in the UK (reviewed in Kuzmin and Rupprecht (7)). The IRKV, first identified in insectivorous bat Murina leucogaster in eastern Siberia during 2002 (25), was known by this only one isolate until 2007, when it caused a human death after a bite of unidentified insectivorous bat in the Russian Far East (26). Moreover, at least three other cases, where the viruses were not identified but the disease was compatible with rabies and developed after bat exposure, were reported from the Ukraine and China (27–29). A few cases of spillover EBLV-1 infections were documented in terrestrial mammals, including domestic cats (30), and they represent a potential exposure risk for humans.
The EBLVs, as well as IRKV, are covered by the commercially available rabies biologics (31, 32), therefore the disease can be efficiently prevented by administration of standard rabies post-exposure prophylaxis (PEP). This is not the case for WCBV. This virus, isolated from insectivorous bat Miniopterus schreibersii in south-eastern Europe, is the most divergent member of the Lyssavirus genus, and rabies biologics are incapable of providing significant protection against it (32). Because of lacking surveillance, there is only one isolate of WCBV available to date. Ecology of this virus and its significance for public health are unknown. However, laboratory animals and bats, infected with WCBV, developed typical rabies and died (33).
A variety of bat lyssaviruses have been documented in Africa. The LBV, first documented in Nigeria in 1956 (34), was further isolated in many sub-Saharan countries (35). Moreover, in 1999 it was imported into France with fruit bats Rousettus aegyptiacus captured in Togo or Egypt (36). Fruit bats of several species serve as reservoir hosts for LBV, with infrequent spillover infections documented in dogs, cats, and a mongoose (37). The viruses, currently included into LBV, represent several divergent lineages and there is a possibility that further taxonomic efforts may facilitate separation of these viruses into two or three species (5, 38, 39). Another divergent lyssavirus, SHIBV, was isolated from insectivorous bat Hipposideros commersoni in Kenya in 2009. The SHIBV demonstrates similarity to MOKV and LBV, but cannot be included into any of these species (5). Significance of these viruses for public health is unknown however, as in the case of WCBV, they are pathogenic for laboratory animals, which develop rabies and die after intracranial or peripheral inoculation (5, 35, 40). Furthermore, due to their antigenic differences, they are not covered by current rabies biologics (32, 41).
Recently, serologic reactivity to WCBV was detected in Miniopterus bats of several species from Kenya (42). Given that WCBV does not cross-react serologically with other known lyssaviruses, this seroprevalence indicates that WCBV or some other antigenically similar virus circulates in Africa as well (and probably more broadly, corresponding to the distribution range of Miniopterus bats).
Another African bat lyssavirus, DUVV, is covered by rabies biologics, but still kills people because of insufficient knowledge, either in general public and health professionals. The DUVV is perhaps the most mysterious African lyssavirus. Of four isolates available, three came from humans who died of rabies after bat exposures and only one was isolated from an insectivorous bat, presumptive Miniopterus sp (43). The most recent human case occurred in 2007 in Kenya, where a Dutch tourist was attacked by a bat in a campsite of Tsavo West national park. The patient applied for medical help, but a local physician assured that bat rabies does not exist in Kenya and PEP was not administered. Several weeks later, back in the Netherlands, the patient developed rabies and died. The virus was identified as DUVV (44).
The discovery of ABLV in 1996 in the ‘rabies-free’ Australia was surprising. Following the discovery that flying foxes were a reservoir of Hendra virus, surveillance of these animals was increased. During this activity, ABLV was identified first in a sick black flying fox (Pteropus alecto). The second case was diagnosed retrospectively in another bat of the same species, sampled in 1995 with signs of unusual aggressiveness (45). Later ABLV was documented in each of the four flying fox species, present in continental Australia. Furthermore, a genetically divergent variant of ABLV was discovered in insectivorous bats Saccolaimus flaviventris(46).
Two human cases of ABLV infection have been documented to date. Both were fatal and clinical symptoms were compatible with rabies. The first one was reported very shortly after the virus discovery in 1996. The patient was a 39-year-old female presumably infected by a S.flaviventris bat in her care. The virus that was isolated was compatible with this bat species (46, 47). The second case occurred in a 37-year-old female who developed rabies in 1998, approximately 27 months after presumable exposure from a bite by an unspecified flying fox. This isolate belonged to the pteropid ABLV variant (48, 49).
Flavivirus is a genus of the family Flaviviridae that contains a large number of viral agents capable of causing encephalitis and jaundice. Most flaviviruses are arboviruses and transmitted to the human population by a bite from infected mosquitoes or ticks. Flaviviruses typically contain a positive sense single-stranded RNA genome of approximately 10-11kb in length. The genome encodes 3 structural proteins (Capsid, prM, and Envelope) and 8 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS5B). The viruses are enveloped with a diameter of around 50nm, and appear icosahedral or spherical when observed under the electron microscope. Individual members such as dengue (DENV), yellow-fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) cause significant morbidity and mortality worldwide.
DENV is a major public health concern on a global scale with an estimated 400 million infections and 100 million clinical cases in 2010. Most of these patients will carry the disease asymptomatically. However, around 5% of infected individuals will progress to severe dengue, an illness characterized by plasma leakage leading to hypovolemic shock, hemorrhage, and potentially death. The case-fatality rate for individuals with severe dengue can be as high as 10% if untreated, or 0.1% with appropriate clinical management.
Alphaviruses are a diverse group of viruses that are classified as belonging to the group IV Togaviridae family of viruses. There are over thirty members in the alphavirus group that are able to infect a wide range of vertebrates including humans, rodents, fish, birds, and horses. At the genomic level alphaviruses consist of a positive sense, single-stranded RNA genome 11 to 12kb in length with a 5’ cap, and 3’ poly-A tail. Alphavirus particles are enveloped, have a size of around 70 nm in diameter under the electron microscope and appear to be spherical with a 40 nm isometric nucleocapsid. Like flaviviruses the main mode of transmission to the human population is via bites from infected mosquitoes. Notable viruses that infect the human population include chikungunya (CHIKV), Barmah Forest virus (BFV), Mayaro virus (MAYV), O'nyong'nyong virus (ONNV), Ross River virus (RRV), Una virus and Tonate virus.
Epidemics of flavivirus and alphavirus occur globally on an annual basis with different degrees of severity. Table 1 shows a small selection of recent flavivirus/alphavirus outbreaks worldwide.
The global distribution and severity of flavivirus and alphavirus infection requires accurate surveillance tools and timely diagnosis to ensure infected patients obtain the best medical treatment options and alert authorities to possible outbreaks of disease.
The most accurate method to diagnose viral agents is real time Polymerase Chain Reaction (RT-PCR). Primer and probe sequences complementary to the viral RNA are designed and cycled through a series of steps with positive samples seen as amplification curves on a RT-PCR instrument. This process can be completed in less than 1 hour, which significantly assists in patient management.
However, members of the flavivirus and alphavirus families are quite heterogeneous at the RNA level, therefore it can be difficult to design a single set of primers and probe sequences that can detect each of the families at the genus and species level. An example of this is DENV that contains four serotypes, each being quite diverse at the genomic level. Like most current dengue RT-PCR assays, the CDC DENV-1-4 RT-PCR Assay detects serotypes 1–4 using an individual primer pair and probe for each type. Assays that can universally detect all DENV serotypes have been described but these assays still employ more than 2 primers to detect all subtypes.
In order to simplify and improve the detection of alphaviruses and flaviviruses in clinical samples, we developed a commercially available 3base assay that is able to detect the presence of the target alphavirus or flavivirus using a single primer and probe set for each type. 3base assays use chemical modification to reduce the complexity of genomes from 4 to 3 base, which enable screening primers and probes with fewer mismatches to be developed so that bias in amplification efficiency across species is greatly reduced. (Fig 1).
The 3base protocol (Fig 2) deaminates all cytosine residues in nucleic acids to a uracil intermediate. This process makes closely related species more similar at the genomic level. This novel method ultimately means that primers and probe sets can be designed that have fewer mismatches and are able to hybridise to previously heterogeneous target regions with higher efficiency, thus improving PCR amplification of species that contain large numbers of individual pathogens.
The modification process of the genomic nucleic acids to a 3base form does not sacrifice specificity and individual typing primers can be constructed to detect the exact organism responsible for disease.
The method has been used to successfully detect the presence of high risk HPV in clinical samples and the presence of pathogens, including Norovirus, in patients with gastrointestinal disease.
We have utilised the method to produce pan-species assays for the detection of all flavivirus, alphavirus and dengue serotypes 1–4 and successfully applied these assays to screen samples in the 2016/17 Vanuatu dengue outbreak.
Hepatitis C virus (HCV) has affected about 175 million people worldwide and is considered as one of the leading cause of liver transplantation (77, 78). In Iran, the virus is introduced as an emerging viral infection amongst high risk populations like injecting drug users as this group has shown a higher prevalence of HCV in recent years (79).
A study conducted in 1994 on healthy blood donors, revealed 0.25% of seroconversion for HCV infection for the first time in Iran (79). HCV infection prevalence has a low rate in general population in Iran compared to the adjacent countries of Pakistan, Turkey and Iraq (80–82). The infection amongst blood donors is 0.1 to 0.5% in different cities of the country (83–86).
Different dialysis centers have had diverse frequency of HCV infection ranging from 5 to 23.9% (87, 88). The main route of HCV transmission among hemophilia and thalassemia patients is through blood products (89–93). In years 1999 and 2000, 0.59% of HCV antibody positive cases were confirmed in multi-transfused children with β-thalassemia in Shiraz blood bank (94). In 2005, a multicenter study pointed out that 19.3% of thalassemia patients suffered from HCV infection (91). In 2007, the infection rate varied between 15.7% and 63.8% (95). In recent years, other serological studies have shown that 15 to 91% of all patients with hemophilia have antibodies against HCV (96–99). This evidence emphasizes the importance of screening of hemophilic patients for HCV infection.
The HCV genome pattern has changed during recent years in Iran and it seems that such a change can be due to cross-border travels between Iran, Pakistan and Iraq (82).
To decrease the trend of infection, regular surveys and interventions should be done, focusing on high-risk groups such as IDUs, those who receive blood products and health care workers with occupational exposure (99).
Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.
Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually LR White). The ultrastructure of most common viruses is well documented in good atlases and book chapters and many classical publications of the 1960s, 1970s, and 1980s. Several excellent reviews were recently published on the use of TEM in the detection and identification of viruses.
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus within the Togaviridae family and was first isolated from horses in the end of the 1930s. These viruses have a natural transmission cycle between rodents and mosquitos. Millions of horses were affected by this arbovirus with a fatality rate of up to 80% in epidemics in Central and South America.
Several species of this family are pathogenic to humans and are recognized as potential biological warfare agent (BWA). VEEV is classified as Bioterrorism Agent Category B by the center of Disease Control (CDC). Alphaviruses do not only have the potential for illness and transmission, but they can also be produced in large quantities and are moderately easy to disseminate. Furthermore, these virus species have the capacity to cause human epidemics. VEEV causes disease symptoms ranging from mild febrile reactions to fatal encephalitic zoonoses. Outcomes are significantly worse for young and elderly patients, with case fatalities ranging from 4 to 35%. These viruses are highly infectious as aerosols and an intentional release of sufficient quantities as inhalable small-particle aerosol is expected to infect a high percentage of individuals within an area of a least 10,000 km2. They can replicate in cell culture to very high titers and are relatively stable to environmental influences.
For the surveillance of possible bioterrorism targets and endangered populations, rapid detection and diagnosis of VEEV are of crucial importance. In the past, the generation of monoclonal murine antibodies has improved the fast identification of VEEV infections to locate human and equine outbreaks of encephalitis. On the other hand, monospecific diagnostic monoclonal antibodies (mAbs) against VEEV are either hardly available on the market or too expensive for extensive use. In view of these current limitations the generation of specific high affinity recombinant antibodies may significantly improve the current situation and can make the rapid immunological detection widely available.
A promising method to generate recombinant antibodies against human pathogenic viruses like VEEV is the antibody phage display technology. Using antibody phage display, genotype and phenotype of an antibody fragment are linked by fusing the antibody gene fragment to the minor coat protein III gene of the filamentous bacteriophage M13. The resulting antibody fragment::pIII fusion protein is displayed on the surface of the phage particles. The most common antibody formats used for this technology are the Fragment antigen binding (Fab) and the single chain Fragment variable (scFv). In comparison to the Fab, that is consisting of the Fragment determining (Fd) of the heavy chain and the light chain linked by a disulphide bond, the scFv simply consists of the variable region of the heavy chain (VH) and the variable region of the light chain (VL), connected by a short peptide linker. The selection of antibody fragments from antibody gene libraries is performed by an in vitro selection process, that is also referred to as "panning".
In this study, we demonstrated the selection of human antibody fragments from a naïve antibody gene library specific for the detection of VEEV. We describe their immunological properties and discuss their possible application of these antibodies for diagnosis and detection of VEEV after a potential bioterrorism assault or natural outbreak of VEEV.
Members of the genus Orthonairovirus of medical and veterinary significance include Crimean Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV). CCHFV is transmitted by ticks in genera Rhipicephalus and Hyalomma. While neither live virus nor nucleic acid of CCHFV has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range. Further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries. A 2016 seroprevalance study by Müller and colleagues examining 16 African bat species (n = 1,135) found that the prevalence of antibodies against CCHFV was much higher in cave-dwelling bats (3.6%–42.9%, depending on species) than foliage-living bats (0.6%–7.1%). They also screened 1,067 serum samples by RT-PCR, but all were negative for CCHFV nucleic acid. Experimental studies to assess the ability of bats to support replication of CCHFV have not been published.