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Zika virus is a new emerging mosquito-borne virus belonging to the Flaviviridae family of viruses (9). This family is comprised of 4 genera: Flavivirus, Hepacivirus, Pegivirus, and Pestivirus (10). Zika virus belongs to the Flavivirus genus, which antigenically and phylogenetically is related to the Spondweni virus (9, 11). Many important human pathogens are included in this genus, for instance, Dengue, West Nile, Yellow fever, tick-borne encephalitis, Japanese encephalitis, Murray Valley encephalitis and St. Louis encephalitis viruses. These viruses are associated with a range of infections from asymptomatic or self-limiting febrile infections to some fatal diseases such as hemorrhage, shock, meningitis, and encephalitis (12).
In Flaviviridae family, all members have enveloped viruses with a single-stranded RNA genome of positive polarity (10) which contained one open reading frame (ORF) with two flanking noncoding regions (at 5′ and 3′ end) (13). The genomes are 5′ capped without a 3′ poly (A) tail.
A polyprotein is coded by the ORF then processed into three structural proteins (the envelope (E), the capsid (C) and the precursor of the membrane (prM)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1, 12). The ssRNA is held within an icosahedral capsid shaped from 12-kDa protein blocks; the nucleocapsid is surrounded by a host-derived membrane contained two viral glycoproteins (14).
Similar replication strategies are employed by the members of Flaviviridae family despite significant differences in tissue tropism, transmission, and pathogenesis (10).
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.
Zika virus RNA is detectable in different types of body fluids such as blood (serum or plasma) (47–50), urine (49, 51), saliva (52) semen (40), breast milk (49), conjunctival fluid (53) and amniotic fluid (54); and brain and placental tissues of congenitally infected fetuses (41, 42).
Reverse transcriptase PCR (RT-PCR) is highly sensitive and specific and known as the “gold standard” for ZIKV diagnosis (55). Regarding Zika virus-specific RT-PCR, several conventional and real-time assays targeting prM, E, NS1, NS3, NS4, and NS5 genes have been developed (50, 56–59). However, to the best of our knowledge, the Food and Drug Administration (FDA) has approved only one commercial assay e.g. Cobas Zika test (Roche) which is a qualitative nucleic acid test for screening Zika virus RNA in blood donors. Additionally, FDA has authorized the use of several molecular assays under an Emergency Use Authorization (EUA) (60) all of them are based on RT-PCR (such as Triplex Real-Time RT-PCR, a multiplex assay for detection of Zika virus, Dengue virus, and Chikungunya virus (CDC) (61), Zika virus RNA qualitative real-time RT-PCR (Quest Diagnostics Infectious Disease, Inc.) and RealStar Zika virus RT-PCR kit (Altona Diagnostics, GmbH)) expect Aptima Zika virus assay based on Transcription-Mediated Amplification (TMA) Technology (60).
Molecular diagnosis of Zika virus infection in human usually performs on plasma or serum specimens within the first week after onset of clinical symptoms (60). Although, there are several lines of evidence for advantage of urine for Zika virus RNA detection because of the long duration of viral shedding in this easily collectable specimen (55
, 12, 62), interesting findings indicating the shorter persistence of ZIKV RNA in urine versus serum have been observed (60, 63, 64).
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.
West Nile virus (WNV) is an enveloped spherical, single-stranded RNA flavivirus that is transmitted to birds by ornithophilic mosquitoes, mainly belonging to the genus Culex and is occasionally transmitted to mammalian hosts. The geographical area of WNV circulation encompasses most of Africa, Israel, North America and South America, Australia and scattered areas in southern and central Europe, including Russia, Czech Republic, Hungary, Greece, Romania, Italy, Southern France, Portugal, Turkey and Spain. At least two distinct genetic lineages have been identified among the WNV isolates in diverse areas: lineage 1 includes most of the American, European and some African strains, whereas lineage 2 contains mainly sub-Saharan African isolates, although some lineage 2 strains have been detected in humans and mosquitoes outside Africa. Additional lineages have been proposed: lineages 3, 4 and 5 include viruses isolated from Czech Republic (Rabensburg strain), Caucasus and India, respectively. A sixth lineage was recently described in Indonesia and a putative seventh lineage has been identified in Spain.The strains belonging to lineages 1 and 2 have up to 30% nucleotide divergence. This wide diversity together with the elevated threat for human health posed by WNV infections resulted in the development of a variety of methods for laboratory diagnosis. It is important to emphasise that about 80% of the human infections caused by WNV remain asymptomatic and therefore approximately 20% of cases become clinically evident. The clinical syndromes associated with WNV human infections are predominantly mild flu-like fevers (WNV fever) of which less than 1% develops severe neuroinvasive disease. For detail about the case classification of the WNV infection and for proposals concerning laboratory diagnosis of this virus please refer to the conclusion of this paper.
This paper reviews the currently available techniques for the identification of WNV infection in humans. Four External Quality Assessment (EQA) studies have been performed: two for the molecular diagnostics of WNV infections, and two for the serological methods. The results of these studies are presented at the end of this review, focusing on the main diagnostic problems.
Currently, the laboratory methods for the diagnosis of infection by WNV belong to two main categories: serology and viral detection.
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.
The Flaviviridae is a large family of positive-strand RNA viruses, that comprises four genera: Flavivirus, Pegivirus, Pestivirus, and Hepacivirus. The Flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens that cause a variety of clinical diseases, ranging from asymptomatic to mild fever to more severe diseases including encephalitis and hemorrhagic fever [1, 2] Most flaviviruses are transmitted through the bite of an infected arthropod vector, mainly Aedes genus (Aedes aegypti and to a lesser extent, Aedes albopictus) and Cluex mosquitos, and most were once maintained by animal reservoirs in sylvatic transmission cycles. Many flaviviruses, however, such as dengue virus, yellow fever and Zika virus, are now principally maintained by mosquito-borne transmission with a possible human-to-human transmission through transfusion of infected blood or transplantation of infected tissue.
Some flaviviruses can cause globally significant vector-borne diseases with a substantial public health impact such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV) and yellow fever virus (YFV). Other members of the Flaviviridae family that have a more regional impact include Murray Valley encephalitis virus (MVEV) in Oceania, St. Louis encephalitis virus (SLEV) in North America, and tick-borne encephalitis virus (TBEV) in Europe. Over the past few decades, many of these flaviviruses have re-emerged for a range of reasons including decreases in mosquito control efforts, rapid changes in climate and vector's demography, dense urbanization, population growth and globalization with increased transportation and trade activities. Examples include the geographic spread of DENV throughout the tropical world; JEV throughout south Asia, Australasia and the Pacific; ZIKV into South and Central America; YFV into the Americas and the invasion of WNV into much of North America [1, 5].
It is estimated that there are over 390 million DENV infections per year, of which 96 million manifests clinically with varying degrees of severity and 3.9 billion people in 128 countries are at risk of infection. Similarly, high incidence rates for symptomatic cases of JEV were reported over the past three decades to reach 2.4 per year per 100,000 population. Epidemic waves of YFV are projected to result in 30,000 to 200,000 clinical cases per year with case-fatality rates ranging from 2 to 15% [9–12]. WNV, first appeared in the northeastern USA in 1999, are spread presently across much of the USA and southern Canada. For example, in 2015, the CDC reported 2,175 cases of WNV, of which 1,616 (74%) were hospitalized and 146 (7%) died. In the developing world, WNV incidence is likely to be underestimated due to political, psychological, and economic barriers to reporting [12, 14].
Although most human flavivirus infections are asymptomatic or have an undifferentiated febrile illness, a small percentage of affected individuals develop acute fever that can progress to severe clinical manifestations such as hemorrhage, vascular leakage and encephalitis. Currently, our knowledge of the host-related factors that influence the pathogenesis of severe disease is inadequate to allow prediction of who will develop severe clinical illness. However, some mechanisms and etiological factors underlying inter-individual variations in response to flavivirus infections have been identified. Interactions of virus-encoded proteins with human innate immune pathways; the effect of host-cell surface molecules in virus binding and entry; the role of viral protein nuclear localization in the host cell response; and the flavivirus replication dynamics within multiple immune systems have all been considered as host-pathogen interaction events that may regulate viral virulence or attenuation and the subsequent disease severity. Over the past few years, however, host-related factors such as preexisting chronic conditions, e.g., cardiovascular diseases, diabetes, obesity, and asthma have received attention as predictors for increased risk of progression to severe flavivirus infection [19–21]. Recent studies have raised the proposition that cardiovascular disease, stroke, diabetes, respiratory diseases and renal disorders may contribute, together with old age, to severe clinical manifestations of dengue [19, 20]. A few studies of WNV and JEV infections, and responses to YFV vaccination, have also explored the role of chronic comorbidities in the prognosis of infections. Given the lack of specific medical treatment for flavivirus diseases, effective public health surveillance for vector-borne infections together with continuing vector control efforts will be critical to preventing infection. However, elucidating the impact of comorbidities to the severity of disease when infection occurs will be critical to identifying vulnerable populations, to whom effective interventions protocols and individually-tailored clinical monitoring practices should be particularly targeted.
The objective of this study is to systematically review the existing literature on the prevalence of the most common non-communicable comorbidities related to the cluster of metabolic syndromes-associated diseases, such as diabetes mellitus, heart diseases, hypertension, asthma, stroke and obesity in flavivirus infections and to evaluate the difference of their prevalence in severe vs. non-severe clinical outcomes to infection. Identifying and characterizing associations between comorbidities and severity of flavivirus infections will be significant factor in designing public health measures that aim to prevent the severe outcomes of infection.
Arthropod-borne flaviviruses (genus Flavivirus, family Flaviviridae) include human pathogens of global medical importance such as dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Kyasanur Forest disease virus (KFDV), Omsk hemorrhagic fever virus (OHFV) and tick-borne encephalitis virus (TBEV). These viruses are causative of many serious diseases with a broad spectrum of clinical symptoms ranging from near asymptomatic or mild flu-like infections through neurological diseases (WNV, TBEV) to viscerotropic (DENV), hemorrhagic (KFDV, OHFV) or even terratogenic manifestations (ZIKV). Up to 200 million new cases of infection caused by arthropod-borne flaviviruses are reported annually. These viruses can be widespread, as exemplified by the epidemiological outbreaks of WNV infection across North America, Mexico, South America, and the Caribbean during 1999–2002, or ZIKV infection in Oceania and Latin America during 2014–2016. At present, there is no effective antiviral therapy directed against these viruses, and therefore, search for small molecule-based inhibitors represents an unmet medical need.
Arbidol, also known as umifenovir, is a broad-spectrum antiviral compound developed at the Russian Research Chemical and Pharmaceutical Institute about 25 years ago and licensed in Russia and China for the prophylaxis and treatment of human influenza A and B infections, plus post-influenza complications. Subsequently, arbidol was shown to be active against numerous DNA/RNA and enveloped/non-enveloped viruses. The predominant mode of action of arbidol is based on its intercalation into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes. In influenza virus, arbidol was observed to interact with virus hemagglutinin (HA), causing an increase in HA stability thereby preventing the pH-induced transition of HA into its a functional fusogenic state. In the case of hepatitis C virus (HCV) arbidol interacts with HCV envelope protein to cause various degrees of inhibition to critical membrane fusion events. Alternatively, arbidol may also be immunomodulatory, and as such be capable of interferon induction and/or macrophage activation. Due to such broad-spectrum antiviral activities, arbidol represents a promising candidate for treatment of viral infections in humans.
Accordingly, we describe here using standardized in vitro assay systems the cytotoxicities and antiviral activities of arbidol against three representative flaviviruses; ZIKV and WNV as emerging mosquito-borne pathogens, and TBEV as an important tick-borne pathogen. Since antiviral compounds are extensively inactivated/metabolized in the intracellular environment, different cell lines were utilized to assess simultaneously both the antiviral and corresponding cytotoxic effects of arbidol.
Zika infection is asymptomatic in 80% of cases. When symptomatic, it is typically described as a mild dengue-like illness, manifested by two or more of the following: sudden onset fever, conjunctivitis, arthralgias, and maculopapular rash (Figure 2). Myalgias and headache are also common. The WHO interim case definition describes suspected, probable and confirmed cases.12 Unlike in dengue, death from acute Zika virus infection is rare, but has been reported in a child with sickle cell disease in Colombia.13 The incubation period for Zika virus after a person is bitten by an infected mosquito ranges from 2–7 days, and may be up to 14 days. This is the reason for screening for risk factors within 14 days of symptom onset. The incidence of GBS or microcephaly appears to be similar between symptomatic and asymptomatic cases.
Japanese encephalitis virus (JEV), a member of the genus Flavivirus in the family Flaviviridae, is a mosquito-transmitted and zoonotic pathogen that causes 50,000 cases and 10,000 deaths per year. There are >70 arboviruses in the genus Flavivirus including JEV, dengue virus (DENV), West Nile virus (WNV), and yellow fever virus. JEV can cause severe central nervous disorders such as poliomyelitis-like paralysis, aseptic meningitis, and encephalitis in humans. The fatality rate caused by JEV is 10–50% and half of the survivors have severe neurological sequelae, including persistent motor defects and severe cognitive and language impairments. The geographic range of JEV is still expanding with an enhanced threat, and JEV infections have been reported in Australia,, Pakistan, and Saipan in the past 30 years. Therefore, JEV is still an important pathogen that has global health significance.
Inactivated and live-attenuated vaccines have been used for prevention of JEV infection for many years,. Although vaccines have reduced the incidence of JE in some countries, they seem not to be effective against all the clinical isolates. In August 2006, there was an outbreak of JEV in Shanxi Province, China, which caused 66 cases and 19 deaths. There is an urgent need for antiviral agents that can reduce the death toll and neurological sequelae of JEV infection. Two anti-hepatitis C virus drugs targeting viral protease, telaprevir VX-950 (developed by Vertex) and boceprevir SCH503034 (developed by Merck), won approval in 2011. Various effective inhibitors against DENV and WNV have also been identified as drug candidates,. In recent studies, some agents were found to have good antiviral effects against JEV. Indirubin, derived from Isatis indigotica extract, was proved to have inhibitory effects on JEV in vitro with less cytotoxicity. Dehydroepiandrosterone (DHEA) suppressed the replication and virus-induced apoptosis in neuroblastoma cells by acting on the extracellular signal-regulated protein kinase. N-nonyl-deoxynojirimycin affected the interaction between calnexin (endoplasmic reticulum chaperone) and JEV glycoproteins (premembrane, envelope, and non-structural protein 1), and thus had anti-JEV effects both in vitro and in vivo
. SCH 16, a derivative of N-methylisatin-β-thiosemicarbazone, inhibited 50% of the plaques produced by JEV at a concentration of 16 µg/mL (0.000025 µM). However, there are currently only a small number of JEV inhibitors available for drug development.
In this study, a cytopathic-effect-(CPE)-based, high-throughput screening (HTS) assay was developed for discovery of JEV antiviral inhibitors. It was used to screen 1280 pharmacologically active compounds and three compounds were identified to have antiviral effects against JEV.
Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), and is one of the most important mosquito-borne diseases with a mortality rate as high as 20% to 50%, and is widely distributed in most of East and South-east Asia and parts of Oceania. Up to 50,000 human cases of JE are reported annually in Asian countries, of which 10,000–15,000 result in fatality. A high proportion (nearly 50%) of survivors, especially young children and those greater than 65 years of age, exhibit permanent neurologic and psychiatric sequelae. A wide range of animals including swine, equines and birds can also be infected. Pigs, as well as birds, serve as amplifying and reservoir hosts,. Further, JEV infection has accounted for significant economic losses in the pig industry due to fetal encephalitis and reproductive failure in pregnant sows and hypospermia in boars. There is no specific treatment available for JE, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. JEV non-structural protein 1 (NS1) has been shown to induce both humoral and cell-mediated immunity against JE,. Further, like other flaviviruses, NS1 is able to elicit protective immunity without the risk of antibody-dependent enhancement. These characteristics make NS1 an attractive alternative immunogen. As such, much research is currently being devoted to NS1-based vaccine development,,. Although NS1 is not present in the virion, NS1-induced antibodies can protect against infection in vivo by an undetermined mechanism, which presumably depends on the Fc portion of the antibody since they kill their target cells through a complement-dependent pathway,.
JEV is a member of the family Flaviviridae, and the genus Flavivirus, and is primarily transmitted by Culex mosquitoes. Besides JEV, the Japanese encephalitis virus serocomplex of Flaviviridae includes the West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEM). JEV serogroup viruses and Dengue virus (DENV) have a similar ecology; it is very common that two or more of these flaviviruses co-circulate in some regions of the world–, and cross-reactivity can be demonstrated among these flaviviruses in serological tests. These cross-reactive responses could confound the interpretation of results during serological testing, including neutralization tests and enzyme-linked immunosorbent assays (ELISA). This serves to emphasize the utility of virus-specific epitopes for the differential diagnosis of disease and epidemiological surveys. The serological cross-reactivity is primarily caused by cross-reactive epitopes on the structural protein E,. In contrast, NS1 is more specific in serological testing of flavivirus infections, and it has been reported that NS1 can induce antibodies without cross-reactivity among flaviviruses, and even among different serotypes of DENV,, therefore the development of an NS1-based specific serological diagnosis is of great interest,,. First, it is necessary to precisely identify the B-cell epitopes on NS1. In this study we have identified and characterized five JEV NS1-specific epitopes with monoclonal antibodies. This work demonstrates progress toward the development of a specific serological diagnostic test for JEV infection, extends our understanding of the antigenic structure of JEV NS1, and could help inform vaccine design.
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).
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).
In planning for vaccine efficacy studies, we developed challenge models of POWV infection in adult mice. Although others have described murine models of POWV infection, these have been in juvenile mice (4–5 weeks old), which are not ideal for experiments requiring iterative immunization and resting periods (Hermance and Thangamani, 2015, Holbrook et al., 2005, Mlera et al., 2017, Santos et al., 2016, Wang et al., 2013). Accordingly, we tested the ability of two POWV strains, LB (a lineage I strain) and Spooner (a lineage II DTV strain) to cause morbidity and mortality in wild-type (WT) C57BL/6 mice at 7 or 14 weeks of age. Following subcutaneous inoculation of 102 focus-forming units (FFU) of POWV LB, lethal infection ensued in 93% of 7- and 14-week-old mice, with a mean time to death of 8.6 and 9.5 days post-infection (dpi), respectively (Figure 1
A). All mice inoculated with POWV LB lost approximately 15%–25% of their body weight (Figure 1B). POWV LB-infected mice exhibited ruffled fur, hunched posture, lethargy, and partial paralysis, although ∼50% of mice did not exhibit these signs before death (Figure 1C). In comparison, POWV Spooner resulted in lower mortality rates; 57% and 60% of 7- and 14-week-old mice succumbed to infection with mean times to death of 13.0 and 12.7 days, respectively (Figure 1D). Because of the partial lethality caused by POWV Spooner in WT mice, we created a more susceptible host by transiently blocking type I interferon (IFN) signaling with an anti-IFN receptor (Ifnar1) monoclonal antibody (mAb), MAR1-5A3 (Sheehan et al., 2006). When 0.5 mg of anti-Ifnar1 mAb was administered one day before infection, POWV Spooner resulted in 100% lethality, with a mean time to death of 9.7 days (Figure 1D). Whereas POWV Spooner-infected WT mice typically displayed ∼15%–20% body weight loss, infected mice treated with anti-Ifnar1 mAb sustained greater weight loss of ∼30% (Figure 1E). Most POWV Spooner-infected WT mice (70%–85%) displayed ruffled fur, hunched posture, and lethargy, with a minority developing limb paralysis (Figure 1F). In comparison, anti-Ifnar1 mAb-treated, POWV Spooner-infected mice uniformly displayed signs of morbidity before death.
Patients should be assessed for pregnancy as well as for neurological symptoms and signs indicating possible GBS. While the situation is rapidly evolving, as of March 2016, laboratory testing is only available through the Centers for Disease Control and Prevention (CDC) and several state and territory health departments. Generally, testing is not recommended for asymptomatic persons, with the exception of pregnant women 2 to 12 weeks after travel to areas with ongoing Zika virus transmission.17 In symptomatic patients, serum should be obtained for polymerase chain reaction (RT-PCR), IgM, and IgG if within seven days of symptom onset. If more than seven days has elapsed, only IgM and IgG should be assessed; RT-PCR is not indicated.18 Note that serological cross reactivity may occur between Zika and other flaviviruses (e.g., dengue, yellow fever, St. Louis encephalitis, Japanese encephalitis, West Nile). Public health departments may decline to test if the clinical scenario is not suggestive of Zika virus or associated complications. In particular, due to the high cross-reactivity with related flaviviruses, serologic test interpretation is complex and can be difficult, leading to false positive results. Conversely, a negative Zika IgM or RT-PCR test result does not rule out Zika virus infection. Emergency clinicians should consult with public health experts to assist with interpretation of test results.
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) is responsible for the most important viral encephalitis affecting humans, with a yearly estimation of 68,000 cases and with a mortality rate of 14% to 21% (1). Importantly, about 30% to 50% of the surviving encephalitis patients can suffer severe long-term neurological sequelae (2). JEV is a positive single-stranded RNA virus belonging to the genus Flavivirus and is currently endemic in the rice-growing areas of North, Southeast, and South Asia. JEV is predominantly maintained kept in an enzootic cycle between mosquitoes of the genus Culex and vertebrate hosts, in particular certain waterbirds and pigs as maintenance or amplifying hosts (2–5).
Pigs are highly susceptible to JEV infection and rapidly develop viremia, which can last for a few days, during which they can transmit the virus to mosquitoes (4, 6). Infected pigs normally present mild neurological symptoms, but the infection of pregnant sows can cause abortions and stillbirth (7). Although JEV infection is typically mosquito borne, contact transmission and high susceptibility to oronasal infection have been described (8). Several in vivo experiments demonstrated that JEV can be excreted oronasally in infected pigs, independently of the route of inoculation, for a period of 5 days, with a maximum level of virus shedding reached after the end of viremia (8–12). Furthermore, mathematical modeling using data from outbreaks in Cambodian pig farms supported the possibility that direct transmission also occurs under field conditions (13). These findings represent a warning that JEV could have the potential to continue to circulate in the pig population by direct transmission in temperate regions during cold mosquito-free periods. In fact, a reemergence of JEV in pig farms located in temperate areas after the winter season without the presence of vectors has been reported in Hokkaido Island in northern Japan (14). It is also important to note that vector-free transmission of mosquito-borne flaviviruses is not restricted to JEV and pigs. Several species, including macaques, mice, hamsters, guinea pigs, rats, and squirrel hamsters, are susceptible to oronasal infection with JEV (15–17). In addition, non-vector-borne transmission has been described for other flaviviruses (18, 19). Examples are Zika virus (ZIKV) (20, 21), West Nile virus (WNV) (22, 23), St. Louis encephalitis virus (SLEV) (24), Bagaza virus (BAGV) (25), Tembusu virus (TMUV) (26), and Wesselsbron virus (WESSV) (27).
Considering these observations, the present study addressed the potential role of the nasal epithelium in contact transmission. Our hypothesis was based on the observation that after oronasal JEV challenge of immune pigs, it is possible to detect the challenge virus in oronasal swabs for several days in the absence of detectable viremia indicating local virus replication (9). While JEV is also found in tonsils (8, 9, 12), immune animals’ tonsils were found to be protected from infection, indicating that this organ is infected only following viremia (9) and would therefore not represent the point of JEV entry. On the other hand, a recent study demonstrated the presence of viral RNA in the nasal epithelium as well as in the olfactory neuroepithelium (11). We therefore investigated whether the nasal epithelium can be infected from the apical surface and whether this infection would cause both apical and basolateral virus shedding. This would indeed represent a prerequisite for the epithelium of the upper respiratory tract to play a role in direct transmission of JEV. The present work included ex vivo and in vitro experimental models as porcine nasal explants and well-differentiated porcine primary nasal epithelial cells (NEC) cultured at the air-liquid interface (ALI). In fact, our data demonstrate that JEV has the ability of infecting apically, resulting in both apical and basolateral virus shedding in swine nasal epithelial cells and indicating that the porcine nasal mucosa could represent a gateway for JEV entry and exit in pigs. Furthermore, we demonstrate that epithelial cells release soluble factors favoring subsequent infections of macrophages known to be present in the lamina propria of the mucosa.
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.
Influenza caused by highly-pathogenic avian H5N1 virus has been one of the most important zoonotic viral infections of humans during the last decade [1–3], with human fatality rates more than 50 percent in some areas of 15 affected countries, where outbreaks continue. Influenza viruses belong to the family Orthomyxoviridae, of which the H5N1 type has a broad range of hosts. Although H5N1 naturally infects poultry and wild birds, transmission occurs to mammalian species, including humans [6–9]. H5N1-infected humans often develop severe clinical respiratory and gastrointestinal symptoms and signs; in some cases, symptoms of the central nervous system (CNS) ensue [10–13]. Infection of the CNS by H5N1 may be severe, causing encephalopathy and other serious neurological complications and sequelae [14–16]. Recent reports found that mice experimentally infected with H5N1 virus developed encephalitic lesions during the acute phase, which was associated with a degree of neuronal degeneration and necrosis. Interestingly, CNS inflammation has not generally been observed in the chronic phase of infection, despite the detection of virus in such lesions. Furthermore, Jang et al. (2009) reported protein aggregation mediated by the influenza virus within degenerated and necrotic neurons. These findings suggest the hypothesis that pathogenic influenza virus might induce a “hit and run mechanism”, in which virus infection triggers a cytokine storm resulting in acute CNS inflammation, with consequent chronic Parkinson-like disease, encephalitis lethargica, or other neurodegenerative diseases. Nevertheless, the exact pathologic mechanism(s) of H5N1-induced encephalopathy remains unclear.
Neurogenesis in the adult CNS is regulated by the neural stem/progenitor cells residing in the subventricular zone (SVZ) of the lateral ventricles, and subgranular zone (SGZ) of the hippocampus. This neurogenesis was shown to be de-regulated by infection with neurovirulent viruses causing Borna disease and Varicella-zoster. Thus, it would be essential to determine whether infection by H5N1 virus occurs in neural progenitor cells, and if so, whether such infection plays a role in the pathogenesis of H5N1-induced encephalopathy. In order to shed light on these questions relevant to the pathogeneses of influenza virus-induced neuropathology, we infected human neural progenitor cells (hNPCs) with H5N1 avian influenza virus and examined the resulting virus-cell interactions, the induction of cellular mediators, and the phenotypic characterizations of hNPCs following H5N1 virus infection in vitro.
We carried out a systematic literature search that conforms to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guidelines (See S1 Table), in PubMed, Ovid MEDLINE(R), Embase and Embase Classic databases from inception to the last week of November 2016 (November 25, 2016). The date for searching database is the same date for the upper limit of the period considered. A grey literature search was also conducted in the American Society of Tropical Medicine and Hygiene and Open Forum Infectious Diseases—Infectious Diseases Society of America for the two most recent years. Using the PICO format (acronym for “population or problem”, intervention or exposure of interest”, “comparison” and “outcome”), the research questions were: "what is the frequency of chronic comorbidities in flavivirus infections?" and “is the severity of flavivirus infection associated with higher prevalence of comorbidities?” (S2 Table). The search related terms (MeSH), inclusion and exclusion criteria and synonyms are shown in Table 1. Non-English language reports were excluded from this study. To identify relevant studies, we used the comprehensive four-step search strategy of PRISMA (Fig 1), i.e., (i) identification, (ii) screening, (iii) eligibility and (iv) inclusion of studies. We evaluated 47 studies in DENV [26–72], 18 in WNV [73–90] and two in JEV [22, 91] for inclusion (see Fig 1). Given the small number of studies in JEV, the two articles were excluded from further quantitative analysis. No studies met the inclusion criteria for YFV and ZIKV infections.
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.
The Japanese encephalitis virus (JEV) is the main pathogen that causes severe encephalitis in humans. JEV belongs to the genus of Flavivirus, which also includes other arboviruses, such as the Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV is a positive-sense single-stranded RNA virus. The genome of JEV is approximately 11 kb in length, containing a single open reading frame (ORF) flanked by the 5′- and 3′-untranslated regions (UTRs). The ORF encodes a long polyprotein that is cleaved into three structural proteins (capsid [C], pre-membrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins make up the infectious viral particle and the nonstructural proteins participate in multiple steps of viral life cycle including viral replication, virion assembly, and immune evasion.
Since the first record of the virus in the late 1800s, JEV has posed a significant threat to global health. It is reported that there are 69,000 cases of JEV infection per year. The average mortality rate caused by JEV can be as high as 30% in the past 30 years, and the proportion of survival with permanent neurological or psychiatric sequelae is approximately 44%. With its epidemic area expansion, JEV affects approximately 25 countries in Asia, and approximately 60% of the population lives with a risk of JEV infection. At present, vaccination is the most effective way to prevent JEV infection. The common vaccines include the inactivated mouse brain-derived vaccine (JE-VAX), inactivated BHK-21 cell-derived vaccine, live-attenuated vaccine (SA14-14-2), inactivated Vero cell-derived vaccine, and the chimeric attenuated vaccine. However, approximately 80% cases of the JEV infection occur in areas covered by the JEV vaccination program due to the failures of immunization strategies or the limitation of vaccines themselves. To date, no clinically approved antiviral agents have been available for the treatment of JEV infection. Furthermore, few randomized clinical trials have tested treatments for JEV. In the past 30 years, only six agents for the treatment of JEV infection have been tested by clinical trials, but none of them have been found effective. Therefore, it is essential and urgent to find a safe and effective treatment.
Drug repurposing has recently become a very popular method for drug discovery; drug repurposing provides old drugs (including approved drugs, under research drugs, and withdrawn drugs) with new indications by exploring new molecular pathways and targets. With this strategy, finding an alternative agent to treatment JEV infection will be fast and safe. During the past decades, the traditional method for drug repurposing depends on high-throughput screening of small-molecule libraries consisting of approved and developing drugs. However, the success rate of high-throughput screening for effective repurposed drugs has dropped dramatically. With the development of computational methods, the high-throughput omics data, virtual screening, and text mining have been used for drug repurposing. One of the computational methods for antiviral drug repurposing is to target pathogen to block its lifecycle. Using the crystal structure of the E protein and the strategy of structural-based virtual screening (SBVS), Leal et al. identified a compound exhibiting marked antiviral activity against DENV with its EC50 being 3.1 µM. The other methods for antiviral drug repurposing are targeting host genes to inhibit pathogen infection. Identifying the proteins participating in the pathogen infection process is the basis of host-targeted drug repurposing approaches. Quan et al. identified 170 Mycobacterium tuberculosis (Mtb) infection-associated genes by theoretical genetic analysis, and obtained high potential anti-Mtb drugs by targeting these genes. Therefore, it is possible to rapidly identify effective therapeutics for JEV infection using the method of drug repurposing through targeting JEV-susceptible genes.
Systems biology has been used to identify the pathogenic mechanisms of complex human diseases by integrating genetic variation, genomics, pathways, and molecular networks. The advent of systems biology provides a powerful method for facilitating drug development and drug repurposing. The representative algorithms used in the systems biology field include GeneRank and HotNet2. In this study, we applied the methods of HotNet2 and GeneRank to identify the genes essential in JEV infection (Figure 1). Additionally, we analyzed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) athway enrichment of these genes to validate our results. Using the information of the drug-target, we obtained the agents that have a potential treatment effect on JEV infection. We found that multiple targets of bortezomib play critical roles in the progress of JEV infection based on the analysis of the PheWAS data of encephalitis and of the gene expression data of human microglial cells after JEV infection. Furthermore, we investigated the effect of bortezomib using a JEV-infected mouse model. Overall, our research provided a novel agent for the treatment of JEV infection.
Powassan virus (POWV) is a tick-borne flavivirus (TBFV) that was first described following its isolation from the brain of a child who died of encephalitis in Powassan, Ontario, in 1958 (McLean and Donohue, 1959). Human cases of POWV have been reported in the United States, Canada, and Russia (reviewed in Ebel, 2010, Hermance and Thangamani, 2017). Though POWV infections are relatively rare, they can cause severe or even fatal neuroinvasive disease, including encephalitis, meningoencephalitis, and meningitis. Approximately 10% of neuroinvasive POWV cases are fatal, and 50% of survivors suffer long-term neurological sequelae (Ebel, 2010, Hermance and Thangamani, 2017). Unfortunately, POWV is emerging; increasing numbers of cases have been diagnosed in the United States over the past decade (Hermance and Thangamani, 2017, Krow-Lucal et al., 2018), and up to 5% of Ixodes scapularis ticks isolated in parts of New York, Connecticut, and Wisconsin now test positive for POWV (Aliota et al., 2014, Anderson and Armstrong, 2012, Knox et al., 2017).
Two genetic lineages of POWV circulate in North America, lineage I and lineage II (also called deer-tick virus [DTV]), although they are serologically and clinically indistinguishable and share at least 96% amino acid identity in their envelope (E) proteins (Ebel et al., 2001). POWV lineage I strains are predominantly maintained in Ixodes cookei ticks and include isolates from New York and Canada, whereas lineage II strains are found in Ixodes scapularis deer ticks and include strains from regions infested by these ticks (Ebel et al., 2001). Because deer ticks are more aggressive at biting humans, lineage II viruses may have greater epidemic potential. Although POWV has been found predominantly in north-central and northeastern parts of the United States in Ixodes species ticks, POWV also has been isolated from Dermacentor andersoni ticks in Colorado (Thomas et al., 1960), indicating the vector and geographical range may be larger than previously estimated.
The TBFVs are divided into three groups: the mammalian group, the seabird group, and the Kadam virus group (Grard et al., 2007). The mammalian TBFV group includes POWV and several other human pathogens, including tick-borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus (OHFV), Kyasanur forest disease virus (KFDV), and Alkhurma hemorrhagic fever virus (AHFV). Within their E proteins, the mammalian TBFV group shares ≥70% amino acid identity but only about 54%–60% identity with seabird TBFVs. One exception is Gadgets Gully virus (GGYV), which is more closely related to the mammalian TBFVs even though it causes infection of seabirds (Grard et al., 2007). Vaccines have been developed against several mammalian TBFVs, although only TBEV vaccines have proven efficacy (Ishikawa et al., 2014). TBEV vaccines induce antibodies capable of neutralizing closely related TBFVs, including OHFV, KFDV, and AHFV (McAuley et al., 2017). However, TBEV immune sera had limited cross-neutralizing activity against the more distantly related POWV. No approved vaccines for POWV exist.
The lack of a POWV-specific vaccine, limited induction of cross-neutralizing antibodies by other TBFV vaccines, and epidemic potential for POWV prompted us to design a vaccine and test its immunogenicity and efficacy. We selected a vaccine platform that we developed for a distantly related flavivirus, Zika virus (ZIKV): a lipid nanoparticle (LNP)-encapsulated modified mRNA encoding the structural premembrane (prM) and E protein genes (Richner et al., 2017a). Co-expression of the flavivirus structural proteins prM and E results in the secretion of subviral particles (SVPs) that share many functional and antigenic features with infectious virions and elicit neutralizing antibodies. SVPs are heterogeneous in size and likely display E proteins in distinct chemical environments, with the potential to affect epitope display (Allison et al., 1995, Heinz et al., 1995, Kiermayr et al., 2009, Konishi and Fujii, 2002, Konishi et al., 1992). In addition to selecting the POWV prM-E structural gene components, we used an mRNA platform, because some modified mRNA have been reported to enhance follicular helper T cell and germinal center B cell responses that are essential for inducing memory B cells and durable levels of neutralizing antibody (Pardi et al., 2018).
Here, we describe an LNP-encapsulated modified mRNA vaccine encoding the prM-E of POWV that induces a potent neutralizing antibody response in mice and protects against lethal viral challenge by strains of both POWV lineages. Furthermore, the POWV mRNA vaccine induced cross-neutralization antibody titers against other TBFVs and conferred protection against disease following challenge of mice with the distantly related Langat virus (LGTV).
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.