TY - JOUR
AU1 - Wiley, Clayton, A
AB - Abstract Biological evolution of the microbiome continually drives the emergence of human viral pathogens, a subset of which attack the nervous system. The sheer number of pathogens that have appeared, along with their abundance in the environment, demand our attention. For the most part, our innate and adaptive immune systems have successfully protected us from infection; however, in the past 5 decades, through pathogen mutation and ecosystem disruption, a dozen viruses emerged to cause significant neurologic disease. Most of these pathogens have come from sylvatic reservoirs having made the energetically difficult, and fortuitously rare, jump into humans. But the human microbiome is also replete with agents already adapted to the host that need only minor mutations to create neurotropic/toxic agents. While each host/virus symbiosis is unique, this review examines virologic and immunologic principles that govern the pathogenesis of different viral CNS infections that were described in the past 50 years (Influenza, West Nile Virus, Zika, Rift Valley Fever Virus, Hendra/Nipah, Enterovirus-A71/-D68, Human parechovirus, HIV, and SARS-CoV). Knowledge of these pathogens provides us the opportunity to respond and mitigate infection while at the same time prepare for inevitable arrival of unknown agents. Emergent viral infection, Evolution, Microbiome, Viral encephalitis INTRODUCTION From the perspective of a 2019 denizen of the developed world, it is difficult to appreciate the importance of infectious disease in shaping our society and even our species. While COVID19 may change that, one need reflect back a mere 100 years to observe how infectious agents have affected the course of human history. The war to end all wars was in fact ended, to a significant degree, by the emergence of a new strain of influenza and a 1918 pandemic. With a worldwide mortality estimated to be 50 million (disproportionately affecting young adults), nations ran out of cannon fodder to throw at their mechanized weapons, so a short 2-decade truce was declared to recover from the decimation of flu. Perhaps the first prehistorical attempt to arrest the scourge of infectious disease was the introduction of quarantine. This had some level of success despite no clear understanding of infectious disease. The earliest modern medical intervention could perhaps be pinned to as recently as the beginning of the 19th century, with the advent of vaccination for smallpox. The advent a century later of Koch’s postulates vastly improved our understanding of infectious agents and their transmission. This led to improved sanitation followed by the miracle of antibiotics in mid-20th century and more recently antivirals. Thus, it is perhaps forgivable that modern man has become complacent believing he has won the war on infectious disease and can rest on the laurels of his scientific accomplishments. But biological evolution is a ceaseless process. New species are constantly arising on a timescale directly related to their size, abundance, and environmental pressure. Without history and science, humans can not appreciate this constant evolutionary process. New species of large animals take hundreds of generations (i.e. thousands of years) to evolve. But microbes are a different story. We are blissfully oblivious to the constant microbial genetic evolution we live amongst. We actively anticipate evolution of existing pathogens like tuberculosis, designing new therapies as resistant strains appear, but this review will focus on the emergence of new viral pathogens that surprise us and disrupt our complacent equilibrium. Where is the human brain in this story of emergent viral infections? As the evolutionarily most sophisticated and complex organ in the known universe, the human brain may be the most susceptible organ to emergent infections. Sequestered from the environment within a boney calvarium and guarded by elaborate and interlinked innate and adaptive immune systems, infectious agents nevertheless discover a means to invade our brains and tap into our highest energy depot to further their replication. But the human brain has its own emergent property called consciousness that can combat emergent infections through understanding their pathogenesis and designing effective therapies. A recent notorious viral infectious disease to emerge and plague mankind is HIV. Its rapid multiplication kinetics coupled to slow disease onset facilitated its rapid spread through broad transmission before symptomatic recognition of infection. Little appreciated is the fact that before treatment was introduced, the largest reservoir of HIV in terminally ill patients was the brain (1), with virus surviving in the long-lived macrophage elements, microglia (2). But as notorious as HIV is, it is by no means the only agent stalking mankind. Unfortunately for many known and unknown reasons perhaps attributable to the complexity of the human brain, some of the worse viral infections selectively target the CNS. In this review, I will examine a dozen viral infections that have emerged within the last 50 years, highlighting the vulnerability of the human brain to new infectious diseases. What most of these agents have in common is a sylvatic reservoir, an ecosystem wherein the agent can experiment with replication strategies best suited to branch out into mankind. As viral replication is asexual, viral evolution mostly requires environmental selection of rare mutations made more common by poor nucleic acid polymerase fidelity. However, viruses with the capacity for multiple strains to infect individual hosts and individual cells can undergo a “pseudosexual” recombination of genes that rapidly accelerates their evolution. In the case of influenza such periodic recombinations are responsible for the periodic pandemics of the past and future. Emergence of a viral infection is not the result of a single process, but rather disruption of a complex ecosystem of competing hosts and pathogens. In the molecular era, we are naturally predisposed to focus on molecular mutation of a viral genome causing increased pathogenesis, but this is a less common cause of emergent disease than disruption of ecosystems. Interposing mankind into established sylvatic cycles leads to new human disease with little to no molecular mutation in the pathogen genome. General Virology Peter Medawar succinctly defined a virus as, “a piece of bad news wrapped in protein” (3). Said another way, a virus is a nucleic acid capable of replicating itself only when introduced into a living organism. The number of extant viruses exceeds enumeration. Because most viruses are so small they cannot be resolved by light microscopy, their initial categorization was based on the host infected and disease produced. The advent of electron microscopy provided a means of categorizing viruses by ultrastructure but their sheer number naturally gave way to the current accepted classification system based upon nucleic acid sequence (4). Viruses can be coded by either RNA or DNA and are categorized into 79 families with genetic trees that elucidate their evolutionary past. The advent of molecular biology has now permitted man to expand nature’s work by artificially designing and creating new viruses. Viral categorization is based upon nucleic acid sequence and structure. The nucleic acid can be DNA or RNA, linear or circular, segmented or nonsegmented, double- or single-stranded, and for those that are single-stranded: sense or antisense. To stabilize the nucleic acid and assure its transmission, it is coated with protein, the nucleocapsid. The self-assembling nucleocapsid performs many functions including protection from environmental degradation, targeting to cellular receptors and facilitating intracellular transcription and translation. Some viruses supplement the protein coat with an additional lipid bilayer coat by budding through modified host membranes. To facilitate their replication some viruses will package additional host or viral proteins within the virion. Viral infection can be broken into half a dozen steps. To facilitate entry into the host cell, the virus must survive in the extracellular environment and bind to the cell surface (5). While naked nucleic acids can enter cells, the process is so inefficient that such a strategy is almost unheard of in nature. Instead the virus utilizes its nucleocapsid or envelope to bind proteins, lipids, or sugars on the cell surface. The co-opted normal host surface molecule is branded the “viral receptor.” Affixed to the cell surface through a lock and key mechanism of capsid or envelope/receptor interaction, the viral nucleic acid has multiple means of entering the cell cytoplasm. The capsid binding might trigger a molecular conformational change that physically injects the viral nucleic acid into the cytoplasm. Alternatively binding to the surface might aggregate cellular “receptors” eliciting their endocytosis followed by fusion with lysosomes, vacuole acidification and different forms of nucleic acid release into the cytoplasm (6). Once in the cytoplasm, the viral nucleic acid needs to access the cells replication machinery. Depending upon whether the viral nucleic acid is DNA or RNA and if the latter, positive- or negative-stranded, this will require targeting different regions of the cell. Viral nucleic acid sequences or proteins can direct it to the appropriate cytoplasmic or nuclear compartment to be transcribed or translated. With synthesis of thousands of new viral genome copies and capsid proteins, the virus undergoes self-assembly and is ready for new transmission. This could entail a broad spectrum of strategies from; simply bursting the cell, releasing new virions in the local environment, polarized budding from a select cell surface (e.g. the respiratory epithelium), latently infecting a cell for later release when the environment is more propitious or, as in the case of HIV, when the cell (the Trojan Horse) migrates into a new organ or host. Spread within the host is most readily achieved by hematogenous dissemination; however, some viruses infecting the nervous system have co-opted neuronal axoplasmic transport to carry the virus from the periphery into the CNS. The final step in viral replication is moving from host to host. Perhaps the simplest strategy is to kill the host and wait for a new noninfected host to contact or consume the corpse. Such a strategy is incredibly inefficient and seldom used (e.g. Ebola) (7). Instead viruses have adopted a broad range of strategies adapted to the specific host they infect. Enteroviruses utilize a simple mass action strategy of replicating to astronomical levels in the host gut and relying upon hand to mouth contact to infect a new alimentary canal. Arboviruses employ a similar strategy of achieving a high viral expression but limited to the blood and taking advantage of hematophagous arthropods to transmit virions from infected to noninfected hosts (8). While effective, this strategy has notable limitations of requiring extended periods of elevated host viremia and capacity of virus to replicate in both mammalian and arthropod cells. Despite these hurdles, numerous viral lifecycles have successfully adopted this strategy. With respect to the nervous system, an even more ingenious strategy has been adapted by the rabies virus. This virus achieves high viral titer in the salivary secretions permitting efficient transmission to the new host through a bite. After inoculation, rather than requiring a high viral titer, the virus uses neuronal uptake and axonal transport into the CNS escaping detection and destruction by the host immune system. Within the host brain the virus travels trans-synaptically to eventually achieve simultaneous infection of neurons innervating the salivary glands and rage centers in the brain to force the host to repeat the transmission cycle by biting new noninfected hosts. Which of these strategies is adopted by the virus, will impact how the virus is maintained in the host population. Viruses have adopted 4 modes of persistence. Some viruses can cause chronic persistent infection frequently by evading or exhausting the host immune response. Others, like herpesviruses, have the capacity to go latent, hiding dormant in host cells escaping immunological detection. Newer emergent infections have taken advantage of massive human population growth and concentration through advent of cities with populations of 100 000–500 000. Here, the virus can hop from uninfected patient to uninfected patient moving as a wave through the population with young naïve hosts being born in sufficient numbers to support future viral infection and persistence. The fourth mode of persistence is to establish a chronic infection in sylvatic populations and only periodically emerge in human dead-end hosts. Zoonotic viruses that infect numerous mammalian hosts and only occasionally get passed into humans (e.g. influenza) are unaffected by human population size and density for their survival. Viruses that infect only a single mammalian host have achieved an almost symbiotic relationship with the host (e.g. herpesviruses) surviving in very low population densities, such as those seen pre-agriculture, while other viruses have adapted to high population densities only achieved after introduction of farming and modern cities (e.g. measles). General Immunology Viral nucleic acids compete for the hosts replicative energy and machinery. This competition has led to evolution of intricate defense mechanisms to prevent the virus from co-opting host metabolism. These defenses are divided into 2 main classes: innate and adaptive immunity. While evolutionarily the oldest branch of immunity, mechanisms of innate immunity have been the most recently discovered. Specialized membrane and cytosolic receptors evolved to detect pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPS). These receptors detect minute amounts of pathogen specific molecular patterns (e.g. nucleic acid sequences, polysaccharides) and activate cellular metabolism to destroy the pathogen. Building upon the innate immune response’s capacity to detect and destroy primary invaders, most multicellular hosts have developed adaptive immune responses that remember initial pathogen infections and, with subsequent encounters, mount a robust and effective protective response at minimal cost to the host. The adaptive immune response requires educating the host to nonhost molecules and priming it so that a second exposure quickly neutralizes the pathogen. The adaptive immune response is divided into humoral and cellular arms. The humoral arm consists of a wealth of cytokines and antibodies released by specialized immune cells that either prevent viral replication or neutralize infectious particles. The cellular arm can help the humoral arm or indirectly defend the host by killing infected host cells, thus arresting viral replication. The human brain offers an additional set of barriers to viral infection. For the most part, the CNS is surrounded by a calvarium blocking direct environmental access; however, orifices through the skull are present to permit axonal entry and exit. Axoplasmic transport in these fibers permits active rapid transport of viruses from the periphery into the brain. A second barrier immediately beneath the skull is the pachymeninges which provides another physical means of excluding pathogens and allowing the brain to float in a sea of cerebrospinal fluid (CSF). On one level, being encased in fluid filled sac of leptomeninges would seem to provide a third barrier to infection; however, paradoxically the leptomeninges appear to be uniquely susceptible to infection. Whether this relates to some to-be-defined sentinel system for mounting an immune response to pathogen threats remains to be determined. The brain vasculature offers a final physical barrier to viral entry. Unlike the systemic vasculature, the endothelial cells of brain vasculature have tight junctions between individual cells and absence of transcellular fenestrae, permitting passage of only the smallest molecules and excluding virions. Once in the brain environment, tissue histiocytes (i.e. microglia) patrol the tissue capable of responding to invading pathogens (9). General CNS Pathogenesis Natural selection has created an amazing sanctuary to sequester the CNS from pathogens. Numerous physical barriers prevent environmental agents from contacting the brain. This begins with hair on the scalp forming an insulating layer of air above an impermeable skin surface. Beneath the skin and subfascial connective tissue layers lies the cranial vault which prevents traumatic introduction of infectious agents. Of course, modern life has introduced stresses not envisioned by mother nature (e.g. high-speed bullets and cars) that can disrupt this pristine sanctuary, but thankfully for most of us these stresses are not common in our daily activities. So, if direct introduction of infectious agents is so difficult, how do they gain access to the CNS? The key principle of brain infection is that it only occurs in the presence of active infection elsewhere in the body. No agent is so abundant in the environment as to permit an adequate inoculum to enter the CNS directly. Instead, an agent must first colonize some part of the body and then multiply many fold before it can spread to the CNS. Dry and dead skin surfaces defy colonization particularly by viruses, so an agent must either land on a mucosal surface or be artificially introduced into the body through the bite of an animal or insect. Once introduced, the infectious agent is in a life or death struggle to replicate and gain access to the nervous system before being cleared by the immune system. The 2 principal conduits for viruses to get from the periphery into the nervous system are via blood or axons. Hematogenous dissemination is by far the most common means for nonviral infectious agents to reach the CNS. Rapid replication of the agent, at the site of inoculum or secondarily after transport to and replication in lymphoid organs, creates massive numbers of infectious particles that spill over into the blood as a viremia. The final hurdle a virus must clear to initiate CNS infection is transgressing the blood-brain barrier (BBB). It is not entirely clear how viremia alone permits crossing the BBB. Junctions between endothelial cells are sealed, fenestrae that characterized systemic endothelial cells do not exist in most CNS vessels and endothelial cell pinocytosis is uncommon in CNS. Despite these barriers, successful pathogens achieve entry prior to the host before neutralizing antibodies develop. Massive viremia can seed the CSF and leptomeninges followed by acute meningitis. If virus achieves further entry to the CNS parenchyma, close contact between host cells results in rapid transcellular spread and severe acute encephalitis. The cellular and humoral arms of the immune system will attempt to abrogate viral spread to the CNS. Cytotoxic T-cells will kill infected host cells prior to their release of infectious particles while antibodies will attempt to neutralize any virion that escapes. Unfortunately, evolution has selected viruses that can evade these immunological strategies either by escaping antibody-mediated neutralization (or worse exploiting antibodies to enhance infectivity) or hiding inside circulating white blood cells as in the case of HIV, where virus circulates inside cellular Trojan horses (monocytes) capable of migrating into the CNS. The other principal conduit into the CNS, axonal transport, delivers infectious agents, particularly viruses, via active intracellular transport systems critical in maintaining neurons with extended processes. First demonstrated with rabies, where after viral replication at the inoculation bite site, there is active uptake of virion into peripheral motor nerve terminals and transport back to central motor neurons. This form of targeted delivery and spread is utilized by many viruses. Mucosal surfaces are well innervated providing quick uptake of infectious agents before they have time to contact the immune system. Infection of the respiratory or gastrointestinal mucosae can lead to either a viremia or axonal transport through innervating fibers. In practice, it can be very difficult to dissect the roles of hematogenous from axonal transport of virus into the CNS. General Diagnostics There are approximately a quarter of a million hospitalizations for encephalitis per year in the United States (10). Of these, a third to half are infections with the rest evenly split between autoimmune or unknown causes. So, diagnosis from what is clinically an ambiguous presentation remains an important problem (11). The first step in diagnosing suspected viral encephalitis is generating a broad differential based upon host characteristics and clinical setting. Newborns are subject to a different set of infections than adults or the aged. Immunocompromised individuals experience different infections based upon the degree and etiology of immunosuppression. While some viruses are ubiquitously present throughout the year, others vary seasonally, such as arthropod born viruses present in the summer and fall, and enteric viruses present more in the winter during inside crowding. Noninvasive imaging (e.g. CT or MRI) offers a ready and convenient means to assess the brain, but is limited to detecting regional changes in X-ray density or water content. Hemorrhage from necrotic infections can be sensitively detected by CT. For some viruses, like herpes simplex, localization to the temporal and frontal lobes, or JC virus localization to white matter, regional involvement is almost pathognomonic. However, many viruses cause diffuse pan-encephalitis resulting in generalized nonspecific edema. More invasive than imaging, but more sensitive and specific for diagnosis is CSF analysis (12). Opening pressures sensitively detect brain edema, while simple biochemical analysis of elevated protein and normal glucose point toward viral infection. Historically, to achieve a more specific diagnosis required tissue culture, but <2% of CSF cultures detect an infectious agent. Culture has given way to more rapid and sensitive PCR techniques. Beginning with targeted single agent PCR, these techniques have expanded to multiplex probes capable of assessing entire panels of potential pathogens, to high-throughput-sequencing with metagenomic analysis where, in theory, an assay can detect all possible infectious agents (13). These latter techniques require intensive bioinformatics and are early in their validation. An alternate approach, building upon prior experience with serology, using sensitive array technology has been deployed to examine host immune response as an indirect means of defining infectious agents (14). This latter technique takes advantage of biological amplification when B-cells are activated and individually produce up to 109 agent specific antibody molecules. Of course, the CSF can be only an indirect marker of what is going on directly in the tissue, so occasionally brain biopsy is used to get a tissue diagnosis of infection. The invasiveness of this procedure is minimized through the use of stereotactic protocols. Paradoxically, open biopsy, where craniotomy is performed, in addition to rendering diagnostic tissue can have a therapeutic effect through relief of intracranial pressure. Once tissue is procured, it can be subjected to the same tests used in CSF analysis without the dilutional effects associated with CSF study. Tissue allows individual cell by cell analysis using immunohistochemistry (IHC) or in situ hybridization or, more rarely, electron microscopy. For some viral infections, like herpes simplex virus, brain biopsy has proven to be 99% specific and 95% sensitive (15). Unfortunately, in many cases where brain biopsy for encephalitis is performed, no specific diagnosis beyond “encephalitis” is rendered. General Therapeutics For most of history infections of the nervous system were uniformly lethal as there were no effective therapeutics. The advent of antibiotics was accompanied by dramatic improvements in survival from most bacterial infections. Therapy for viral encephalitis is a much more recent advent (16). Therapies for viral and nonviral encephalitis are founded on the same principle: Disrupt metabolic pathways that are unique to the infectious agent but not (or less) involved in host metabolism. Perhaps the earliest and most successful therapy for viral encephalitis was developed over 50 years ago to treat herpesvirus infections. Taking advantage of a viral thymidine kinase (TK) that is essential to the pathogen’s replication strategy, antagonist drugs were developed. Drugs like acyclovir are selectively metabolized by the viral TK leading to inhibition of viral DNA polymerase. Since only infected cells possess the viral TK, acyclovir is selectively concentrated only in infected cells with minimal toxicity to host noninfected cells. Early treatment with acyclovir reduces risk of death due to herpes simplex encephalitis from 70% to <20% (17). The success of this therapy facilitated development of derivatives that could penetrate the BBB and be administered orally. Unfortunately, acyclovir is not as effective with other members of the herpesvirus family (e.g. cytomegalovirus), so different drugs, based on similar principles, have been developed. The AIDS epidemic provides one of the most dramatic examples of targeting viral metabolic Achille’s heels to suppress viral infection (18). Over the decades, HIV’s metabolic strategy has been extensively probed and dissected. Drugs have been designed to inhibit: Viral reverse transcriptase (both nucleoside and nonnucleoside based), viral protease, viral integrase, and maturation enzymes. Highly effective at suppressing viral replication, combinations of these drugs continue to be refined to limit off-target effects on host cell metabolism. Initial concern about getting these drugs into viral sanctuaries like the brain proved moot when peripheral viral suppression permitted immune reconstitution and control of host brain infection by the host immune system. Success in the field of pharmacological therapy for herpes encephalitis has not been matched in other viral encephalitides. As one of the most common causes of viral encephalitis, enteroviruses have been extensively studied. Unfortunately, inhibition of replication with the nucleoside analog Ribavirin or inhibition of the nucleocapsid binding protein VP1 have had limited success. Less agent-specific therapies for viral encephalitis have also been tried. Given the malignant outcome of brain swelling in an enclosed space caused by inflammation, steroids are frequently used to suppress brain edema. Effective as steroids are in reducing brain edema, they obviously come at the cost of diminishing the immune system’s capacity to fight infection. With the development of recombinant technology, new drugs like synthetic interferon-alpha have been explored as a means of using the hosts natural defense to restrict viral replication. Unfortunately, the temporally and spatially nonphysiological use of these host defenses comes at a high cost and limited targeted benefit. Perhaps our best therapy for treating viral encephalitis is to prevent it from occurring in the first place. A substantial amount of effort has been put toward designing safe and effective vaccines. Mankind’s successes on this front have been astounding with the purging of infectious agents like smallpox, to the near complete curtailment of infections, such as polio. But vaccine development is not a lucrative business and it comes with high financial risks (e.g. low but real incidence of Guillain-Barre syndrome in vaccinated individuals). Nevertheless, our experience in vaccine development continues to grow and offers great hope in many viral infections. Beyond vaccination, more global ecosystem control is critical to curtailing emergent viral infections. In cases like Ebola, we can simply quarantine the caves that serve as reservoir for infections. If an infectious viral agent cannot be controlled it may be most effective to control the vector (e.g. mosquitoes, ticks, dogs) and arrest infection at the ecosystem level. VIRAL INFECTIONS OF THE CNS EMERGENT WITHIN THE LAST 50 YEARS Infections of the CNS are frequently lethal (19). Viruses, such as Rabies, have a long history of acute transmission from infected wild and domestic animals into humans. On the other end of the time scale, herpesviruses have evolved for millennia and establish a stable symbiotic relationship with their mammalian hosts. The following discussion will be focused on viruses that have emerged with the past 50 years that mediate severe neurological disease (Table). TABLE. Viral Infections of the CNS Emergent Within the Last 50 Years Name . Flu . West Nile Virus . Zika . Rift Valley Fever Virus . Hendra/Nipah . EV-A71/EV-D68 . HPeVA . HIV/SIV . SARS-CoV . Family Orthomyxoviridae Flaviviridae Flaviviridae Bunyaviridae Paramyxoviridae Picornavirinae Picornaviridae Retroviridae Coronaviridae Genus Influenza A Flavivirus Flavivirus Phlebovirus Henipavirus Enterovirus Parechovirus Lentivirus Coronavirus Approximate date of emergence of human neuropathogenic strain 1997 1999 in new world 2015 in Brazil 1970s 1990s 2000s 2000s 1980s 2000s Viral structure  Nucleic acid RNA RNA RNA RNA RNA RNA RNA RNA RNA  Genome size (kb) 13.5 11 11 11.5 18 7.5 7.3 9.2 (dimer) 27–32  Sense/antisense Antisense Antisense Antisense Ambisense Antisense Sense Sense Sense Sense  Segmented 8 segments Nonsegmented Nonsegmented 3 segments Nonsegmented Nonsegmented Nonsegmented Nonsegmented (dimer) Nonsegmented  Enveloped Yes Yes Yes Yes Yes No No Yes Yes  Viral receptor Hemagglutinin E protein E protein N protein G protein VP1/VP2 ? gp160 & gp41 S & HE proteins  Host receptor Sialic acid residues Several Several Caveolin? EFNB2 SCARB2 ? CD4 and chemokine receptors ACE2  Normal mode of transmission Aerosol Culex Mosquito Aedes Mosquito Aedes/Culex Mosquito oral exposure to bat excreta or infected livestock oral-oral, fecal-oral oral-oral, fecal-oral Blood products, secretions: for example, milk, semen Oral-oral, fecal-oral  Neuropathogenic mode of transmission Aerosol Mosquito, transfusion, transplantation Mosquito to naïve mom followed by transplacental Aerosol Oral Oral Presumed oral Sexual, transplacental, perinatal, blood products Presumed oral  Neurotropic strain H5, H7 & H9 Lineage 1 > lineage 2 NA NA NA NA HPeVA3 Macrophagetropic strains ?  Seasonality Winter Late spring to early fall Late spring to early fall Rainy season Nonseasonal Summer Bi-annual variation No Winter  Reservoir Waterfowl Birds Birds Livestock Bats Humans Humans (voles?) Primates Bats  Human to human transmission Yes Minimal to No Minimal to No No No Yes Yes Yes Yes  Host immune status at time of infection Immune intact Elderly or immunosuppressed First trimester naive mom Immune intact Immune intact Late exposure of naive host? Heterotypic immunity? Newborn, ? maternal naive? Immune intact Intact and aged  Route to brain ? Axonal versus hematogenous HEMATOGENOUS? Hematogenous ? Hematogenous ? Axonal versus hematogenous Hematogenous? Trojan horse within infected monocytes ? Axonal versus hematogenous  Neurological disease Lethal Pan-encephalitis Encephalitis involving gray matter (esp. Substantia nigra); occasionally limited to spinal cord gray matter Microencephaly secondary to pan-encephalitis in first trimester; cerebral infarction in later stage gestation Lethal pan-encephalitis Meningitis, encephalitis Meningitis, flaccid paralysis Meningitis Meningitis and encephalitis Meningitis + ?  Cells infected in brain Neurons Neurons Neuronal progenitor cells of first trimester; meningeal cells at later gestational ages Neurons Neurons and endothelium Neurons vascular smooth muscle macrophages ?  Mode of pathogenicity Genetic mutation and reassortment, Interspecies transmission High viremia leading to rare CNS infection primary infection of naive mom during early pregnancy abnormal route of infection (aerosol) Animal husbandry Oral-fecal ? Depletion of systemic CD4 T-cells followed by abundant replication in macrophages with disruption of microglia physiology ?  Emergent neuropathogenicity issues Neurotropic strains bind Alpha2-3 sialic acid linkage are highly pathogenic but limited aerosol transmission Sylvatic reservoir, immunosuppressed patients Transplacental Aerosol exposure; Weaponization Control of aerosol exposure; animal husbandry New assortment leading to emergent neurotropic strain Infection postnatal, role of transplacental IgG? ? Role of macrophages in normal CNS physiology Uncertain  Vaccine Must be designed seasonally None None Animal vaccine under development No current human vaccine No current human vaccine ??? Maternal immunization? None None  Drug therapy Multiple drugs available all must be given early in infection None None None Ribavirin, monoclonal antibody Under preclinical evaluation None Combined highly effective anti-retroviral therapy None  Neurological disease threat potential Currently low no evidence of significant human to human transmission of neurotropic Flu Low occasional case in immunosuppressed Low except for primary infection during pregnancy and occasional case in immunosuppressed High if weaponized for aerosol transmission Low unless livestock not vaccinated Uncertain could develop mutant neurotropic strains but no zoonotic reservoir Low presumed protective maternal immunoglobulin High no vaccine if drug resistance develops Low rare case reports Name . Flu . West Nile Virus . Zika . Rift Valley Fever Virus . Hendra/Nipah . EV-A71/EV-D68 . HPeVA . HIV/SIV . SARS-CoV . Family Orthomyxoviridae Flaviviridae Flaviviridae Bunyaviridae Paramyxoviridae Picornavirinae Picornaviridae Retroviridae Coronaviridae Genus Influenza A Flavivirus Flavivirus Phlebovirus Henipavirus Enterovirus Parechovirus Lentivirus Coronavirus Approximate date of emergence of human neuropathogenic strain 1997 1999 in new world 2015 in Brazil 1970s 1990s 2000s 2000s 1980s 2000s Viral structure  Nucleic acid RNA RNA RNA RNA RNA RNA RNA RNA RNA  Genome size (kb) 13.5 11 11 11.5 18 7.5 7.3 9.2 (dimer) 27–32  Sense/antisense Antisense Antisense Antisense Ambisense Antisense Sense Sense Sense Sense  Segmented 8 segments Nonsegmented Nonsegmented 3 segments Nonsegmented Nonsegmented Nonsegmented Nonsegmented (dimer) Nonsegmented  Enveloped Yes Yes Yes Yes Yes No No Yes Yes  Viral receptor Hemagglutinin E protein E protein N protein G protein VP1/VP2 ? gp160 & gp41 S & HE proteins  Host receptor Sialic acid residues Several Several Caveolin? EFNB2 SCARB2 ? CD4 and chemokine receptors ACE2  Normal mode of transmission Aerosol Culex Mosquito Aedes Mosquito Aedes/Culex Mosquito oral exposure to bat excreta or infected livestock oral-oral, fecal-oral oral-oral, fecal-oral Blood products, secretions: for example, milk, semen Oral-oral, fecal-oral  Neuropathogenic mode of transmission Aerosol Mosquito, transfusion, transplantation Mosquito to naïve mom followed by transplacental Aerosol Oral Oral Presumed oral Sexual, transplacental, perinatal, blood products Presumed oral  Neurotropic strain H5, H7 & H9 Lineage 1 > lineage 2 NA NA NA NA HPeVA3 Macrophagetropic strains ?  Seasonality Winter Late spring to early fall Late spring to early fall Rainy season Nonseasonal Summer Bi-annual variation No Winter  Reservoir Waterfowl Birds Birds Livestock Bats Humans Humans (voles?) Primates Bats  Human to human transmission Yes Minimal to No Minimal to No No No Yes Yes Yes Yes  Host immune status at time of infection Immune intact Elderly or immunosuppressed First trimester naive mom Immune intact Immune intact Late exposure of naive host? Heterotypic immunity? Newborn, ? maternal naive? Immune intact Intact and aged  Route to brain ? Axonal versus hematogenous HEMATOGENOUS? Hematogenous ? Hematogenous ? Axonal versus hematogenous Hematogenous? Trojan horse within infected monocytes ? Axonal versus hematogenous  Neurological disease Lethal Pan-encephalitis Encephalitis involving gray matter (esp. Substantia nigra); occasionally limited to spinal cord gray matter Microencephaly secondary to pan-encephalitis in first trimester; cerebral infarction in later stage gestation Lethal pan-encephalitis Meningitis, encephalitis Meningitis, flaccid paralysis Meningitis Meningitis and encephalitis Meningitis + ?  Cells infected in brain Neurons Neurons Neuronal progenitor cells of first trimester; meningeal cells at later gestational ages Neurons Neurons and endothelium Neurons vascular smooth muscle macrophages ?  Mode of pathogenicity Genetic mutation and reassortment, Interspecies transmission High viremia leading to rare CNS infection primary infection of naive mom during early pregnancy abnormal route of infection (aerosol) Animal husbandry Oral-fecal ? Depletion of systemic CD4 T-cells followed by abundant replication in macrophages with disruption of microglia physiology ?  Emergent neuropathogenicity issues Neurotropic strains bind Alpha2-3 sialic acid linkage are highly pathogenic but limited aerosol transmission Sylvatic reservoir, immunosuppressed patients Transplacental Aerosol exposure; Weaponization Control of aerosol exposure; animal husbandry New assortment leading to emergent neurotropic strain Infection postnatal, role of transplacental IgG? ? Role of macrophages in normal CNS physiology Uncertain  Vaccine Must be designed seasonally None None Animal vaccine under development No current human vaccine No current human vaccine ??? Maternal immunization? None None  Drug therapy Multiple drugs available all must be given early in infection None None None Ribavirin, monoclonal antibody Under preclinical evaluation None Combined highly effective anti-retroviral therapy None  Neurological disease threat potential Currently low no evidence of significant human to human transmission of neurotropic Flu Low occasional case in immunosuppressed Low except for primary infection during pregnancy and occasional case in immunosuppressed High if weaponized for aerosol transmission Low unless livestock not vaccinated Uncertain could develop mutant neurotropic strains but no zoonotic reservoir Low presumed protective maternal immunoglobulin High no vaccine if drug resistance develops Low rare case reports Open in new tab TABLE. Viral Infections of the CNS Emergent Within the Last 50 Years Name . Flu . West Nile Virus . Zika . Rift Valley Fever Virus . Hendra/Nipah . EV-A71/EV-D68 . HPeVA . HIV/SIV . SARS-CoV . Family Orthomyxoviridae Flaviviridae Flaviviridae Bunyaviridae Paramyxoviridae Picornavirinae Picornaviridae Retroviridae Coronaviridae Genus Influenza A Flavivirus Flavivirus Phlebovirus Henipavirus Enterovirus Parechovirus Lentivirus Coronavirus Approximate date of emergence of human neuropathogenic strain 1997 1999 in new world 2015 in Brazil 1970s 1990s 2000s 2000s 1980s 2000s Viral structure  Nucleic acid RNA RNA RNA RNA RNA RNA RNA RNA RNA  Genome size (kb) 13.5 11 11 11.5 18 7.5 7.3 9.2 (dimer) 27–32  Sense/antisense Antisense Antisense Antisense Ambisense Antisense Sense Sense Sense Sense  Segmented 8 segments Nonsegmented Nonsegmented 3 segments Nonsegmented Nonsegmented Nonsegmented Nonsegmented (dimer) Nonsegmented  Enveloped Yes Yes Yes Yes Yes No No Yes Yes  Viral receptor Hemagglutinin E protein E protein N protein G protein VP1/VP2 ? gp160 & gp41 S & HE proteins  Host receptor Sialic acid residues Several Several Caveolin? EFNB2 SCARB2 ? CD4 and chemokine receptors ACE2  Normal mode of transmission Aerosol Culex Mosquito Aedes Mosquito Aedes/Culex Mosquito oral exposure to bat excreta or infected livestock oral-oral, fecal-oral oral-oral, fecal-oral Blood products, secretions: for example, milk, semen Oral-oral, fecal-oral  Neuropathogenic mode of transmission Aerosol Mosquito, transfusion, transplantation Mosquito to naïve mom followed by transplacental Aerosol Oral Oral Presumed oral Sexual, transplacental, perinatal, blood products Presumed oral  Neurotropic strain H5, H7 & H9 Lineage 1 > lineage 2 NA NA NA NA HPeVA3 Macrophagetropic strains ?  Seasonality Winter Late spring to early fall Late spring to early fall Rainy season Nonseasonal Summer Bi-annual variation No Winter  Reservoir Waterfowl Birds Birds Livestock Bats Humans Humans (voles?) Primates Bats  Human to human transmission Yes Minimal to No Minimal to No No No Yes Yes Yes Yes  Host immune status at time of infection Immune intact Elderly or immunosuppressed First trimester naive mom Immune intact Immune intact Late exposure of naive host? Heterotypic immunity? Newborn, ? maternal naive? Immune intact Intact and aged  Route to brain ? Axonal versus hematogenous HEMATOGENOUS? Hematogenous ? Hematogenous ? Axonal versus hematogenous Hematogenous? Trojan horse within infected monocytes ? Axonal versus hematogenous  Neurological disease Lethal Pan-encephalitis Encephalitis involving gray matter (esp. Substantia nigra); occasionally limited to spinal cord gray matter Microencephaly secondary to pan-encephalitis in first trimester; cerebral infarction in later stage gestation Lethal pan-encephalitis Meningitis, encephalitis Meningitis, flaccid paralysis Meningitis Meningitis and encephalitis Meningitis + ?  Cells infected in brain Neurons Neurons Neuronal progenitor cells of first trimester; meningeal cells at later gestational ages Neurons Neurons and endothelium Neurons vascular smooth muscle macrophages ?  Mode of pathogenicity Genetic mutation and reassortment, Interspecies transmission High viremia leading to rare CNS infection primary infection of naive mom during early pregnancy abnormal route of infection (aerosol) Animal husbandry Oral-fecal ? Depletion of systemic CD4 T-cells followed by abundant replication in macrophages with disruption of microglia physiology ?  Emergent neuropathogenicity issues Neurotropic strains bind Alpha2-3 sialic acid linkage are highly pathogenic but limited aerosol transmission Sylvatic reservoir, immunosuppressed patients Transplacental Aerosol exposure; Weaponization Control of aerosol exposure; animal husbandry New assortment leading to emergent neurotropic strain Infection postnatal, role of transplacental IgG? ? Role of macrophages in normal CNS physiology Uncertain  Vaccine Must be designed seasonally None None Animal vaccine under development No current human vaccine No current human vaccine ??? Maternal immunization? None None  Drug therapy Multiple drugs available all must be given early in infection None None None Ribavirin, monoclonal antibody Under preclinical evaluation None Combined highly effective anti-retroviral therapy None  Neurological disease threat potential Currently low no evidence of significant human to human transmission of neurotropic Flu Low occasional case in immunosuppressed Low except for primary infection during pregnancy and occasional case in immunosuppressed High if weaponized for aerosol transmission Low unless livestock not vaccinated Uncertain could develop mutant neurotropic strains but no zoonotic reservoir Low presumed protective maternal immunoglobulin High no vaccine if drug resistance develops Low rare case reports Name . Flu . West Nile Virus . Zika . Rift Valley Fever Virus . Hendra/Nipah . EV-A71/EV-D68 . HPeVA . HIV/SIV . SARS-CoV . Family Orthomyxoviridae Flaviviridae Flaviviridae Bunyaviridae Paramyxoviridae Picornavirinae Picornaviridae Retroviridae Coronaviridae Genus Influenza A Flavivirus Flavivirus Phlebovirus Henipavirus Enterovirus Parechovirus Lentivirus Coronavirus Approximate date of emergence of human neuropathogenic strain 1997 1999 in new world 2015 in Brazil 1970s 1990s 2000s 2000s 1980s 2000s Viral structure  Nucleic acid RNA RNA RNA RNA RNA RNA RNA RNA RNA  Genome size (kb) 13.5 11 11 11.5 18 7.5 7.3 9.2 (dimer) 27–32  Sense/antisense Antisense Antisense Antisense Ambisense Antisense Sense Sense Sense Sense  Segmented 8 segments Nonsegmented Nonsegmented 3 segments Nonsegmented Nonsegmented Nonsegmented Nonsegmented (dimer) Nonsegmented  Enveloped Yes Yes Yes Yes Yes No No Yes Yes  Viral receptor Hemagglutinin E protein E protein N protein G protein VP1/VP2 ? gp160 & gp41 S & HE proteins  Host receptor Sialic acid residues Several Several Caveolin? EFNB2 SCARB2 ? CD4 and chemokine receptors ACE2  Normal mode of transmission Aerosol Culex Mosquito Aedes Mosquito Aedes/Culex Mosquito oral exposure to bat excreta or infected livestock oral-oral, fecal-oral oral-oral, fecal-oral Blood products, secretions: for example, milk, semen Oral-oral, fecal-oral  Neuropathogenic mode of transmission Aerosol Mosquito, transfusion, transplantation Mosquito to naïve mom followed by transplacental Aerosol Oral Oral Presumed oral Sexual, transplacental, perinatal, blood products Presumed oral  Neurotropic strain H5, H7 & H9 Lineage 1 > lineage 2 NA NA NA NA HPeVA3 Macrophagetropic strains ?  Seasonality Winter Late spring to early fall Late spring to early fall Rainy season Nonseasonal Summer Bi-annual variation No Winter  Reservoir Waterfowl Birds Birds Livestock Bats Humans Humans (voles?) Primates Bats  Human to human transmission Yes Minimal to No Minimal to No No No Yes Yes Yes Yes  Host immune status at time of infection Immune intact Elderly or immunosuppressed First trimester naive mom Immune intact Immune intact Late exposure of naive host? Heterotypic immunity? Newborn, ? maternal naive? Immune intact Intact and aged  Route to brain ? Axonal versus hematogenous HEMATOGENOUS? Hematogenous ? Hematogenous ? Axonal versus hematogenous Hematogenous? Trojan horse within infected monocytes ? Axonal versus hematogenous  Neurological disease Lethal Pan-encephalitis Encephalitis involving gray matter (esp. Substantia nigra); occasionally limited to spinal cord gray matter Microencephaly secondary to pan-encephalitis in first trimester; cerebral infarction in later stage gestation Lethal pan-encephalitis Meningitis, encephalitis Meningitis, flaccid paralysis Meningitis Meningitis and encephalitis Meningitis + ?  Cells infected in brain Neurons Neurons Neuronal progenitor cells of first trimester; meningeal cells at later gestational ages Neurons Neurons and endothelium Neurons vascular smooth muscle macrophages ?  Mode of pathogenicity Genetic mutation and reassortment, Interspecies transmission High viremia leading to rare CNS infection primary infection of naive mom during early pregnancy abnormal route of infection (aerosol) Animal husbandry Oral-fecal ? Depletion of systemic CD4 T-cells followed by abundant replication in macrophages with disruption of microglia physiology ?  Emergent neuropathogenicity issues Neurotropic strains bind Alpha2-3 sialic acid linkage are highly pathogenic but limited aerosol transmission Sylvatic reservoir, immunosuppressed patients Transplacental Aerosol exposure; Weaponization Control of aerosol exposure; animal husbandry New assortment leading to emergent neurotropic strain Infection postnatal, role of transplacental IgG? ? Role of macrophages in normal CNS physiology Uncertain  Vaccine Must be designed seasonally None None Animal vaccine under development No current human vaccine No current human vaccine ??? Maternal immunization? None None  Drug therapy Multiple drugs available all must be given early in infection None None None Ribavirin, monoclonal antibody Under preclinical evaluation None Combined highly effective anti-retroviral therapy None  Neurological disease threat potential Currently low no evidence of significant human to human transmission of neurotropic Flu Low occasional case in immunosuppressed Low except for primary infection during pregnancy and occasional case in immunosuppressed High if weaponized for aerosol transmission Low unless livestock not vaccinated Uncertain could develop mutant neurotropic strains but no zoonotic reservoir Low presumed protective maternal immunoglobulin High no vaccine if drug resistance develops Low rare case reports Open in new tab The enterovirus polio mediated a severe encephalomyelitis in a small subset of infected individuals but because intensive vaccination efforts have led to its near eradication, it will not be discussed. Sadly, other enteroviruses have begun to emerge and are capable of causing severe neurological disease so these new agents will be reviewed. INFLUENZA A VIRUS Influenza A Virus Virology Influenza A virus (IAV) is a member of the Orthomyxoviridae family consisting of enveloped viruses with single-stranded segmented genome (4). IAV is an RNA virus with an antisense genome meaning that, upon infection, the viral genome must first be copied to a sense strand before it can be translated by the host cellular machinery. Based upon molecular analysis of a key envelope coat gene, hemagglutinin (HA), Influenza A, B, and C viruses were derived from a common ancestor diverging ∼2000, 4000, and 8000 years ago, respectively (20). So, these viruses are relatively “new” yet important human pathogens that are a major cause of morbidity and mortality in man. First isolated in 1933, the molecular and physical ultrastructure of IAV has been thoroughly described identifying numerous targets to disrupt viral replication and spread (21). In the past, serology was used to subtype IAV strains. This technology utilized the host immune system’s recognition of 2 of the surface proteins: HA and neuraminidase (NA) to define “serological types.” Today molecular sequencing of HA and NA is employed to distinguish viral isolates and decipher viral evolution. Modern nomenclature distinguishes 18 different HA and 11 different NA subtypes that can occur in any combination (i.e. 198 possible different subtypes of IAV). Since these surface proteins are key to binding the host cell surface and permitting infection, to a high degree their combination determines which host species can be infected. Molecular steps of IAV replication are thoroughly elucidated but only key features that pertain to pathogenesis as an emerging threat to the CNS are reviewed here. HA in the surface envelope binds to neuraminic acids (also known as sialic acids) that decorate proteins on the surface of host cell membranes. These sugar polymers chemically modify extracellular portions of cell membrane proteins. Subtle differences in HA amino acid sequence determine its capacity to bind different terminal sugar polymers on host proteins. Biochemical modification of HA by host proteases is key to binding to the cell surface and thus infecting the host. The virulence of different IAV strains is in part determined by how readily the HA molecule is processed by cellular specific extracellular proteases. HAs that require trypsin-like proteases for processing and activation are of low virulence, while HAs that can be processed by ubiquitous proteases are readily activated and thus highly pathogenic (22). One key to understanding IAV infection is recognizing that viral HA paired with host protein sialic acid residues determines in which species and in which organs a particular viral strain can replicate. Proteins in the avian gut epithelium are decorated with alpha2, 3-linked sialic acids, while proteins in the mammalian epithelium are decorated with alpha2, 6-linked sialic acids. Not surprisingly, avian and human influenza strains have evolved HA molecules that bind different sialic acids, thus avian IAVs for the most part cannot infect humans and vice versa. After viral HA binds the cell surface, it is endocytosed followed by fusion with low pH endosomal compartment required to release of the viral genome into host cell cytoplasm (21). Knowing this pathway permits designing drugs to inhibit viral replication (e.g. blocking acidification of the endosomal compartment). In the cytoplasm, the virion’s nucleocapsid is uncoated, and viral RNA genome with attached polymerases are transported to the nucleus. From here sense-strand mRNA templates are synthesized while host protein synthesis is blocked. Newly synthesized viral genomes are transported into the cytoplasm for viral assembly. Given the segmented nature of the viral genome, only virions with all 8 viral RNA segments are infectious (23). The viral genome then buds from a modified cell surface studded with viral HA and NA undercoated by viral matrix protein 1 (M1). The assembly and budding of IAV occurs from the host cell surface in a polarized fashion concentrating newly synthesized virions within the lung or gut, improving chance of transmission. After virion assembly, NA is crucial for viral release and virion movement through an environment filled with mucous and replete with sialic acid binding sites. IAV Immune Response The host mounts a variety of innate and adaptive immune responses to block IAV infection and replication. Mucosal surfaces are thick with soluble proteins, such as mucins, gp-340, pentraxins, collectins, natural IgM, complement, and defensins, all of which can promote virion aggregation, clearance and inactivation. Virions that elude the extracellular surface defenses, enter endosomal and cytoplasmic compartments rich with sensors to detect molecular patterns of infectious agents. Single-stranded viral RNA is detected by TLR7 in the endosomal compartment where it initiates secretion of type I interferons (IFN alpha/beta). In turn, type I IFNs induce antiviral responses that augment innate and adaptive immunity (e.g. natural killer and B cell proliferation, dendritic cell maturation, T cell survival and activation), induce antiviral genes and initiate a cascade of cytokine secretion (IL6, IL8, TNFalpha, amongst others) (24, 25). To combat these cellular defenses IAV has evolved a nonstructural protein 1 (NS1) that blocks IFN synthesis in host cells. Beyond arresting primary IAV infection, the innate immune response is critical to initiating an adaptive immune response that will clear virus more quickly and effectively block viral entry upon future exposure. The adaptive immune response has 2 arms: humoral and cellular. The humoral arm targets viral surface envelope proteins HA and NA (26). Development of vaccines is based on the observation that resistance to subsequent infection and illness correlates with HA and NA antibody titers. Mutations in HA or NA genes accounts for IAV’s capacity to reinfect host populations and periodically cause epidemics, while reassortment of HA and NA genes accounts for pandemics (see below). We know less about the role of cellular immunity in combating IAV infection. CD4 T cells that recognize IAV help B cells produce neutralizing antibodies. Additionally, they secrete cytokines that also directly control IAV infection. Importantly, the major targets of T cell immunity in IAV are different from the humoral arm targeting epitopes in M1 and nucleoprotein. Through cytotoxic effector delivery of proinflammatory cytokine secretion (e.g. TNFalpha, IFN-gamma), expression of FasL and TRAIL death domain receptors, specific CD8 T cells also help clear IAV. Epidemic Flu A common strategy by which pathogens elude immune detection is to change their protein coat. Malaria does this so successfully that it defies our efforts to create efficacious vaccines. The ability of IAV to modulate its HA and NA glycoproteins accounts for much of its capacity to elude immune detection and stay in constant circulation amongst a host population. The diversity of HAs and NAs is also key to maintaining sylvatic avian viral species. In the aquatic bird host, IAV causes limited to no disease and thus is under limited to no selective pressure from an immune response. But when transmitted to land based birds (e.g. chickens) or mammals (e.g. pigs or humans), adaptive immune pressure leads to slow antigenic drift through selection of mutations within HA and NA genes. Multiple viral strains are in constant circulation in the ecosystem. Periodically the “drift” in amino acid mutations mediates enough antigenic change that every few years the virus escapes neutralization by antibodies specific to previous strains, leading to a new epidemic. Significant public health resources are committed to identify newly emergent strains and create vaccines to mitigate the severity of these epidemics. Pandemic Flu While antigenic drift can lead to significant epidemics, the introduction of a new HA subtype not previously circulating in the host population, causes pandemics. This much rarer event termed “antigenic shift,” occurs when 2 different IAVs infect the same host (mixing vessel). When 2 different IAVs infect the same cell at the same time, gene segments can be exchanged (e.g. HA of one strain is exchanged with HA of another strain leading to a new HA-NA combinations). Entirely new viral subtypes or reassortments are generated that, with some minor adaptations of transmission efficiency, can mediate pandemics in immunologically naive populations. For IAV, swine are proposed to function as the “mixing vessel.” Pigs and avian species (such as ducks or geese) cohabit the barnyard, permitting the mixing of IAVs between the 2 species to create novel potentially pandemic IAVs. When pandemics of IAV emerge, they kill large percentages of the human population and can change the course of history (27). Besides infecting a greater percentage of the population than epidemic flu (e.g. 50%), a pandemic flu infection is also more severe. It is neigh impossible for contemporary citizens of the developed world to imagine what the next pandemic flu could do to humans as a species. The “Spanish Flu” (H1N1) killed more people (25–50 million) in 1 year, than the world war that raged for 4 years. In the U.S. the average life expectancy decreased by 10 years. During the usual IAV epidemic 0.1% of infected individuals (usually the very old or very young) die but the mortality rate during the Spanish flu was 25 times that and perversely, preferentially killed young adults. We have had subsequent pandemics (1957, 1968, and 2009) caused by reassortments generating novel H2N2, H3N2, and H1N1 viral strains, but for a variety of reasons, including immunization, these pandemics were more benign. Avian Flu If IAV mostly causes pulmonary disease with rare systemic spread, what is its relevance to neurological disease? The outbreak of avian flu in humans in Hong Kong demonstrated the potential for H5N1 to cause severe encephalitis (28). After direct avian-to-human transmission, the virus had limited capacity to spread human to human and thus was unable to support an epidemic. In the past 2 decades avian H5N1 became endemic in animal populations of Asia, the Middle East, Europe, and Africa. There have been fewer than a thousand confirmed human cases but half of these were fatal, demonstrating broad dissemination throughout the body and brain (29). Transmission of avian influenza viruses to humans is not limited to H5N1 and has been seen with H9N2 and H7N9 (30). Fortunately, none of these avian strains appears capable of supporting efficient spread between human contacts. To support human to human transmission, an emergent IAV must: (1) replicate at lower temperatures of the upper airways, (2) achieve adequate titer, and (3) evolve an HA that binds to human epithelial glycosylation pattern of sialic acid with alpha2, 6 linkage (31). Avian Influenza Neurological Disease Given the paucity of human H5N1 infections, we have a limited understanding of its pathogenesis. Paradoxically, H5N1 causes more gastrointestinal than pulmonary disease. While this may limit aerosol dissemination, it has also been associated with systemic infection in the individual host. As described in general principles above, systemic viral spread sets up CNS infection (i.e. encephalitis) which in the case of H5N1 is uniformly lethal. Three conditions are necessary for IAV to directly mediate neurological disease: (i) Escape local innate or adaptive immune control of replication at site of inoculum. (ii) Possess an HA that can be readily cleaved and activated by ubiquitously expressed proteases to permit maturation of infectious viral particles. (iii) Discover a route to enter the CNS, that is, enter nerve endings and use axoplasmic transport or achieve ingress via hematogenous routes. Like other neurovirulent viruses (e.g. polio and rabies) H5N1 IAV has been shown to spread to the CNS by axoplasmic transport through olfactory, vagus, trigeminal, and sympathetic nerves (32). But many RNA viruses can utilize hematogenous dissemination and transgress the BBB by infecting brain microvascular endothelium. We have observed patterns of both hematogenous and olfactory epithelium entry in brains of ferrets infected with H5N1 IAV (33). Because most neurons have abundant expression of alpha2,3-linked sialic acid glycosylation pattern (34, 35), once in the CNS, IAV efficiently binds to neuronal cells. After entry, retrograde transport of IAV brings it in proximity of neuronal replication machinery. The brain connectome then supplies a myriad of opportunities for dissemination. The CNS appears to have a more limited capacity to clear IAV infection. Innate immunity shutting down RNA replication may be more muted given the importance of RNA processing in normal brain functioning. Adaptive immunity (e.g. antibodies and T-cells) is less abundant in the CNS than in systemic organs. The final result is that once in the CNS, there are few barriers to lethal disease. New Neuropathology of Avian Influenza H5N1 virus can infect the brains of many avian and mammalian species (33), but the complexity of host pathogen interactions limits the utility of trying to model human disease in animals. The simplest example of the impact of the myriad of infection variables on modeling human disease is viral entry. Viral entry requires binding of IAV to the host cell surface. The gut and respiratory epithelium of different avian and mammalian species varies widely in terms of glycosylation. The most common laboratory mammal, mice, have different glycosylation patterns than humans and ferrets. This simple difference requires that ferrets be used for most IAV pathogenesis studies. This is a severe handicap because we know so much less about the ferret than the mouse immune system. After binding and entry, the innate immune system responds (see above) with expression of cytokines that in addition to inhibiting viral replication, produce the early clinical symptoms of fever and malaise prior to the development of adaptive immunity. Viral replication is robust in the first day and the host rapidly disseminates the virus through coughs and sneezes. Peak viral replication in the respiratory system is achieved within 2–3 days and this is rapidly suppressed by 7–8 days when infected cells are difficult to detect (33). Pathogenic strains of high or low virulence are in part distinguished by how quickly virus is cleared. Lung damage is more prolonged and severe after infection with highly pathogenic strains leading to worse secondary bacterial infection complications, and higher morbidity and mortality. Beyond severity of pulmonic infection, highly pathogenic strains can have the additional capacity to spread systemically. Systemic spread is in part associated with enzymatic stability of the HA molecule and escape from host immunity. HA of highly pathogenic strains can be cleaved by ubiquitous host proteases and thus these strains are capable of maturing in any body compartment rather than being limited to the respiratory and gastrointestinal system. In ferret and murine models, neurons are the predominant infected cell type in the CNS. Whether this is true in humans remains to be proven (36, 37). There are reports suggesting microglia are infected, but this has not been universally accepted. Four days after intranasal IAV infection of mice, virus can be detected in the brainstem and olfactory cortex. This infection quickly disseminates throughout the cortex, occasionally appearing in small foci reminiscent of hematogenous dissemination. A somewhat analogous pattern of infection is seen in ferrets (33). In ferrets, pulmonary infection is more variable and quickly spreads to the liver, spleen, gut and brain. The brain shows a panencephalitis with neurons the predominant infected cell (Fig. 1). Infection of ependymal cells lining the ventricles or cells within the leptomeninges, are consistent with CSF dissemination. FIGURE 1. Open in new tabDownload slide H5N1 virus infection of the ferret. Whole mount of a midsagittal section of a ferret brain 7 days after H5N1 infection. In situ hybridization to detect H5N1 nucleic acid appears as black grains. Section is counterstained with hematoxylin to stain nuclei blue. Multiple loci in the brain show dense staining for the virus (black grains) not only in the olfactory bulb (“O”) but also in the brainstem (“BS”) and other locations. The cerebellum (“CB”) appears mostly spared. This pattern of infection would be consistent with either axonal transport from respiratory epithelium to olfactory bulb and brainstem or hematogenous dissemination of a viremia to multiple foci in cortex. At each site of infection, the virus demonstrates an extreme neurotropism with close to all of the neurons infected. FIGURE 1. Open in new tabDownload slide H5N1 virus infection of the ferret. Whole mount of a midsagittal section of a ferret brain 7 days after H5N1 infection. In situ hybridization to detect H5N1 nucleic acid appears as black grains. Section is counterstained with hematoxylin to stain nuclei blue. Multiple loci in the brain show dense staining for the virus (black grains) not only in the olfactory bulb (“O”) but also in the brainstem (“BS”) and other locations. The cerebellum (“CB”) appears mostly spared. This pattern of infection would be consistent with either axonal transport from respiratory epithelium to olfactory bulb and brainstem or hematogenous dissemination of a viremia to multiple foci in cortex. At each site of infection, the virus demonstrates an extreme neurotropism with close to all of the neurons infected. IAV Treatment Knowing IAV replication strategy and pathogenesis has permitted the design of drug and immune therapy. Drug therapies have focused on blocking the viral ion channel essential for acidification of the endocytosed viral compartment or inhibiting viral NA activity, essential for viral particle release. New drugs targeting different points of viral replication (e.g. inhibition of target membrane fusion and inhibition of the HA cleavage) are undergoing clinical trials. Vaccines targeting IAV HA and NA have been highly successful in generating protective antibodies, abrogating illness and blocking epidemics (38). The major problem with creating vaccines is that it is not possible to predict what new strain will emerge. Our current strategy is to use the seasonal cycle of the earth to observe what strain emerges in the Southern hemisphere and within 6 months produce a vaccine for the Northern hemisphere. The pressure of that time frame is not very forgiving if there are production problems (e.g. inadequate replication of virus in eggs to generate inoculum). Given the historical importance of IAV, development of better vaccines has been given high priority. Conclusions: IAV Emergence as a Neuropathogen Abundance of IAV in aquatic fowl and its segmented genome are ideal breeding grounds for emergence of novel pathogens. History has shown that a series of mutations and selections in mammalian species are necessary before a pandemic strain capable of human to human transmission develops. Aerosol transmission between humans requires high titer replication in upper respiratory system. Because neurotropic strains do not replicate well in mammalian upper respiratory system, the lethal neurological IAV disease has been linked to close contact with infected birds and lacks the capacity for aerosol transmission between humans. That is a great barrier but not one we should rely on in perpetuity. Since eliminating the virus is impossible, we need to focus on vaccines and antiviral therapy. WEST NILE VIRUS West Nile Virus Virology Sequence analysis suggests that West Nile virus (WNV) emerged in Africa ∼2000 years ago (39). In the wild, WNV participates in an enzootic cycle with birds serving as the main animal reservoir and many species of mosquitoes serving as vectors. Because infection of mammals is not associated with significant prolonged viremia, they are considered incidental hosts. The first isolation of WNV from a human was in the West Nile province of Uganda in 1937, thus its name. After its discovery, this mosquito-borne flavivirus was linked to periodic epidemics of febrile illness and more rarely sporadic encephalitis in Africa, the Mediterranean, eastern Europe, southwest Asia, and Australia (40, 41). In 1999, the virus migrated to New York City from Israel (42). While spread of WNV from Africa to the old world was the result of bird migrations, WNV is presumed to have arrived in NYC aboard an airliner either in the form of an infected passenger or mosquito. Since arriving in North America, it has spread rapidly across the continent and southward into Latin America and the Caribbean (43). As such, WNV offers an excellent contemporary example of the degree and speed with which emergent infections can penetrate an ecosystem. WNV Epidemiology In the first 20 years since its arrival in the United States, there have been over 50 000 cases of human WNV disease reported to the CDC, with approximately half of those reports being neuroinvasive disease (http://www.cdc.gov/ncidod/dvbid/westnile/index.htm). Annual incidence waxes and wanes in accordance with variations in the ecosystem (years with increased temperature show increased transmission). Consistent with mosquito born transmission, infections are confined between late spring and early fall commensurate with vector feeding. The majority (up to 80%) of human infections with WNV are clinically silent, with the remaining 20% of infections apparent as self-limited fever (i.e. WNV fever), half with a rash. WNV fever is characterized by the classic viral syndrome of: Acute onset of fever, headache, fatigue, malaise, muscle pain, and weakness; gastrointestinal symptoms and a transient macular rash. Less than 1% of all infected patients develop neuroinvasive disease. The severity of neurological disease ranges from mild disorientation to coma and death, and many manifest movement disorders, including severe tremors and parkinsonism (44, 45). Approximately a third of those with neuroinvasive disease develop a meningitis, half develop encephalitis and 5%–10% develop an asymmetric flaccid paralysis similar to that seen with poliomyelitis (46). Immunosuppressed, and in particular elderly subjects, are at higher risk of neuroinvasive disease (47–49). There is some suggestion that genes related to the innate immune response may be responsible for predisposition to develop neurological disease (50–52). WNV Neuropathology Computed tomography of the head is generally normal and not helpful in the diagnosis of neuroinvasive WNV, except to exclude other etiologies of encephalopathy. Brain magnetic resonance imaging (MRI) may frequently be normal, even in severe encephalitis (44, 53). However, when abnormalities are noted on MRI (particularly T2 and fluid attenuation inversion recovery [FLAIR] sequences), they include prominent signal abnormalities in the deep gray matter, predominantly in the basal ganglia, thalamus, brainstem, and cerebellum of patients with encephalitis, and in the anterior spinal cord in patients with poliomyelitis-like syndrome (44). Histopathologic examination of CNS tissues from patients with WNV neuroinvasive disease shows perivascular inflammation, microglial nodules, neuronophagia, and variable necrosis and neuronal loss, with pathologic changes concentrated in the brainstem, deep gray nuclei, and anterior horns of the spinal cells (see [54, 55] for review). Foci of demyelination, gliosis, and occasional perivascular infiltrates may be found in persons who survive acute disease and suffer chronic sequelae (56). Generally, the presence of WNV has been documented in persons who died early during the course of WNV infection (56–58); however, persistence of WNV in human brain tissue samples 4 months after initial diagnosis has been recently documented (58). Using IHC staining, WNV antigen has been demonstrated variably in brain, kidney, spleen, liver, lung, skin, bone marrow, and intravascular mononuclear cells (see [54, 55] for review) (Fig. 2). IHC staining is more often positive than viral culture, showing WNV antigens in ∼50% of fatal WNV neuroinvasive disease cases. IHC staining is more commonly positive in patients who died during the first week of illness when viral antigen concentrations in CNS tissues are high (56), although these studies have been conducted primarily in immunocompetent persons. However, persistence of viral antigen for considerably longer time periods has been demonstrated by IHC in patients with severe immunocompromising conditions, such as solid organ or bone marrow transplant recipients (56, 58). Viral antigens are found principally within neurons and neuronal processes, prominently in the brainstem, basal ganglia, thalami, and spinal cord anterior horn cells, consistent with WNV’s neurotropic reputation. In general, viral antigens are focal and sparse, except in severely immunosuppressed patients where they are detected extensively throughout the CNS. FIGURE 2. Open in new tabDownload slide West Nile virus (WNV) infection of mice and humans. (A) Low-power view of the mouse hippocampus 5 days after infection with WNV. In situ hybridization labels viral nucleic acids black. Essentially all neurons of the hippocampus are infected by WNV (black dots). (B) Immunohistochemical staining for WNV (green) and macrophages (red) in the brain of an immunosuppressed patient. A large neuron cell body is filled with WNV antigens (green) and surrounded by red macrophages that are phagocytosing infected tissue. An H&E stained section from the testis of an immunosuppressed patient who died of fulminant WNV infection. (C) The testis seminiferous tubules are densely infiltrated by inflammatory cells (blue) responding to WNV infected tissue. FIGURE 2. Open in new tabDownload slide West Nile virus (WNV) infection of mice and humans. (A) Low-power view of the mouse hippocampus 5 days after infection with WNV. In situ hybridization labels viral nucleic acids black. Essentially all neurons of the hippocampus are infected by WNV (black dots). (B) Immunohistochemical staining for WNV (green) and macrophages (red) in the brain of an immunosuppressed patient. A large neuron cell body is filled with WNV antigens (green) and surrounded by red macrophages that are phagocytosing infected tissue. An H&E stained section from the testis of an immunosuppressed patient who died of fulminant WNV infection. (C) The testis seminiferous tubules are densely infiltrated by inflammatory cells (blue) responding to WNV infected tissue. Rare cases of clinically and/or histologically confirmed WNV-associated hepatitis, pancreatitis, myocarditis, cardiac dysrhythmia, myositis, rhabdomyolysis, orchitis, nephritis, chorioretinitis, uveitis, vitritis, and optic neuritis have been reported. There are few systematic surveys of WNV in human postmortem tissues, apart from the CNS, during neuroinvasive WNV infection. WNV has been identified at autopsy in renal and splenic tissues by culture, IHC, and reverse transcriptase polymerase chain reaction (RT-PCR) (56, 59); in testicular tissue by electron microscopy (60, 61); and in liver, lung, bone marrow, intravascular mononuclear cells, and skin by IHC (62). WNV infection of non-CNS tissues has important implications for organ and blood donations. WNV Diagnosis and Treatment By the time patients develop WNV neuroinvasive disease, WNV-specific IgM is detectable in serum and/or CSF (63). Real-time RT-PCR is the most sensitive nucleic acid amplification test for WNV, able to detect as low as 50 viral RNA copies per mL, which is ∼1000-fold more sensitive than culture (64). The sensitivity of RT-PCR among 28 patients with serologically confirmed neuroinvasive WNV disease was 57% in CSF and 14% in serum. Using RT-PCR, WNV RNA has been detected in serum, blood, CSF, brain, spleen, and kidney (65). The pronounced epithelial tropism and polar distribution of WNV suggests a cautionary note regarding the potential for asymptomatic or less severe human carriers. WNV may evade the immune system after primary acute infection by budding toward the apical surface of glandular epithelium. This polar budding may permit individuals to be chronic carriers without immune or inflammatory clearance of virus. While excretion of virus in bile and urine is unlikely to transmit to new hosts, the presence of virus in the testes leaves open the possibility of sexual transmission, while the presence of virus chronically in sweat glands may foster further arthropod dissemination. Persistence of viral antigens in systemic organs and the CNS after the initial onset of symptoms has been observed in transplant recipients (56, 58). Since IHC positivity indicates productive viral infection, this suggests that WNV can persist in certain systemic organs and CNS for almost 5 months after the initial infection in patients with severe immunocompromising conditions (Fig. 2). This may have treatment implications for transplantation patients who contract WNV. WNV Animal Models Numerous animal models of WNV infection have been described (Fig. 2). For economic and experimental design reasons, rodents have been most commonly studied. The various animal models in immunocompetent animals show remarkable similarity to human infection, with most animals remaining asymptomatic, but developing a brief viremia that quickly abates with production of specific neutralizing antibody. Recent studies in hamsters have shown chronic or persistent WNV infection may occur (66). Intriguingly, the persistently infected hamsters have shown WNV infection in organs similar to what was previously describe in humans (e.g. kidney and lung). The mechanism of viral persistence in immunocompetent animals remains to be fully elucidated; however, some selected viral mutations may facilitate chronic infection (67). In contrast, experimental infection of immunosuppressed hamsters with WNV produced a fulminant encephalitis with multiple organ involvement and death, as we have observed in immunosuppressed patients. Viral infection cycle begins with a mosquito bite and introduction of virus below the epidermis. WNV replicates within dendritic cells and instigates a low-level primary viremia. Spread of virus to the lymphoreticular system results in a more robust but short-lived secondary viremia, which in rare instances can be followed by neuroinvasion. Once the new host develops IgM, blood-borne virus has been neutralized and is difficult to detect by culture. While the vast majority of transmissions are the result of mosquito bites, WNV can be transmitted by organ transplant, infected milk and blood products. This quickly became an issue for blood banks. Conclusions: WNV Emergence as a Neuropathogen Sylvatic reservoirs of WNV are well established worldwide. Man lives in close association with the birds that harbor the virus and the vectors that deliver it. While theoretically possible to create a vaccine, in the developed world it is currently safer and more economically viable to control exposure to the mosquito vector. To prevent neurological disease, these measures can be focused on immunosuppressed individuals. ZIKA VIRUS Zika Epidemiology Less than a decade after the emergence of pandemic WNV, the first major human Zika outbreak occurred in the island archipelago of Micronesia, on the opposite side of the globe from its origin in Uganda. First isolated in 1947 from blood of a febrile primate in Zika forest, the virus was first recovered from a human in 1952. The insect vector is predominantly Aedes mosquitoes (68, 69). Since then, 2 genotypes (Asian and African) of Zika have been endemic in mankind throughout the old world where it is notable for an exanthematic fever. In 2007, there was an outbreak of 5000 infections (3 quarters of the population) on the island of Yap. This was followed 6 years later by an outbreak in French Polynesia with a similar infection rate in the total 280 000 population. From Asia the infection crossed the Pacific (possibly associated with travel of competitive water sports teams) to reach the Americas in 2015 where up to 1 million infections were reported in Brazil. While there have been rare reports of meningoencephalitis (70), fatal encephalitis (71), and myelitis (72), most adult Zika infections were clinically mild. Nevertheless, 62% of the Brazilian Guillain-Barre syndrome cases report prior Zika infection (73). The highly variable disease manifestations could be explained by different host genetics determining unique immune responses or prior exposure to different flaviviruses. Zika Neuropathology Zika transmission mirrors that of WNV and other arboviral infections with the important exception of transplacental infection. In Brazil a 20-fold spike in microcephalic births was noted coincident with the 2015 Zika epidemic. Detailed pathological evaluation of these cases confirmed the presence of ZIKA specifically when maternal infection occurred in the first trimester. Two general forms of CNS Zika microcephalic disease were described: An early neurotropic infection where immature neuroglial elements were infected (Fig. 3) and a later meningoencephalitis linked to tissue destruction (74). The scale of the Zika microcephaly outbreak is difficult to discern. Estimates ranged from 1% to 13% of births of infected individuals (75); however, further follow up could not always confirm the association with Zika. A 2016 assessment of the epidemic from the Brazilian Ministry of Health indicates 7830 reported cases of microcephaly. Of these 1501 live births could be investigated, of which only 76 could be definitely associated with Zika infection (76). FIGURE 3. Open in new tabDownload slide ZIKA infection of humans. Tissue section from the brain of an infant infected during the first trimester with ZIKA virus. In situ hybridization for ZIKA nucleic acids (red) show infection of subventricular embryonic neurons. Infection of these cells leads to death of the neuronal tissue and microencephaly. “V,” cerebral ventricle. FIGURE 3. Open in new tabDownload slide ZIKA infection of humans. Tissue section from the brain of an infant infected during the first trimester with ZIKA virus. In situ hybridization for ZIKA nucleic acids (red) show infection of subventricular embryonic neurons. Infection of these cells leads to death of the neuronal tissue and microencephaly. “V,” cerebral ventricle. These findings suggest that while potentially devastating, neurological complications from Zika are relatively uncommon. One possible explanation for this is the concept of heterotypic adaptive immunity (77). This theory suggests that exposure to other flaviviruses, which are endemic in these regions, confers some level of adaptive immunity protection when the host encounters Zika. While this is more than theoretically possible, the exact opposite has been observed with Dengue virus where infection with different viral strains can be associated with a worse clinical outcome through antibody-dependent enhancement. Zika Treatment In the case of transplacental infections, it could be hypothesized that once Zika is endemic, the chance of a female developing a primary infection during pregnancy will be vanishingly small. As observed with other congenital infections (e.g. rubella) the maternal host develops an adaptive immunity after primary infection that protects from future maternal-fetal transmission. However, for tourists living outside of endemic regions, vaccination or avoidance of travel would be imperative. The discovery of a potential sexual transmission of Zika (78) raised the possibility of transmission modes beyond the usual mosquito vector (e.g. transplantation, transfusion, breast feeding, and sex). Conclusions: Zika Emergence as a Neuropathogen The lessons and conclusions for WNV apply also to Zika with some modest twists. For unclear reasons, Zika has proven capable of crossing the first trimester placenta to infect the immune incompetent host fetus. So, in addition to focusing on protecting immune incompetent adults from Zika exposure, we need to focus on protecting naive pregnant females from exposure. Women living within the Zika ecosystem likely develop immunity to Zika in their prepubescents. But women living outside endemic Zika regions should be discouraged from traveling to Zika regions while they are pregnant. The rare capacity for Zika to be transmitted sexually should discourage sex between exposed males and potentially pregnant naïve females. RIFT VALLEY FEVER VIRUS Rift Valley Fever Virus Virology Rift Valley Fever Virus (RVFV) is a member of the Bunyaviridae family. These single-stranded RNA viruses have 3 segments defined by size (small, medium, and large). Several genera of Bunyaviridae infect both arthropods and mammalian hosts. In arthropods, the viruses infect salivary glands and gonads permitting horizontal, vertical and venereal transmission. Viral replication strategy is similar to IAV except viral surface envelope proteins (Gn and Gc) bind to unknown host receptors; viral assembly occurs in Golgi and viral release from mammalian cells involves cytolysis rather than viral budding as in arthropods. RVFV Epidemiology RVFV was first isolated in Kenya in the 1930s, but clinical outbreaks presumably occurred decades earlier. Transmitted by Aedes and Culex mosquitoes, the first recorded outbreaks occurred in Egypt after completion of dam projects along the Nile (79, 80). The viral emergence appears to be the result of introduction of cattle following completion of the Nile river dams. Mosquitoes that bite both cattle and humans can transmit the virus. Epidemic cattle infections are notable for increased incidence of abortions. Transmission to humans is not associated with abortions; however, 1% of humans infected by mosquitoes develop severe disease including fulminant hepatitis, retinitis, encephalitis, and hemorrhagic syndromes (81, 82). When inhaled, the virus has a completely different pathogenesis. The location of systemic replication is unknown; however, after several days, the virus enters the brain and mediates a lethal encephalitis (Fig. 4). FIGURE 4. Open in new tabDownload slide Aerosol infection of mice with pathogenic Rift Valley Fever Virus (RVFV). In situ hybridization (black grains) for RVFV nucleic acids, in a mouse 6 days after inhalation of aerosol RVFV. Essentially all of the neurons have been infected with RVFV leading to a lethal disease. FIGURE 4. Open in new tabDownload slide Aerosol infection of mice with pathogenic Rift Valley Fever Virus (RVFV). In situ hybridization (black grains) for RVFV nucleic acids, in a mouse 6 days after inhalation of aerosol RVFV. Essentially all of the neurons have been infected with RVFV leading to a lethal disease. RVFV emerged as an important pathogen in humans who participated in shearing and butchering of infected livestock. In these instances, inhalation appears to be route of entry. The exact route of entry of RVFV into the CNS after aerosol exposure is unknown. The capacity to grow RVFV in concentrated stocks raised the prospects that it could function as a bioterrorism agent. RVFV Treatment Immunization for RVFV is available for livestock and indirectly protects humans. Unfortunately, to date, protection conferred by vaccines is limited to conventional exposure (i.e. mosquito bite) and provides no protection from inhalation exposure. While there are currently no vaccines for humans, the capacity to maliciously prepare stocks of this virus for artificial aerosol delivery has spurred research in developing vaccines. Conclusions: RVFV Emergence as a Neuropathogen From a neurologic perspective it is not critical to protect humans from exposure to mosquito born RVFV. However, aerosol exposure must be controlled through either more cautious animal husbandry or more widespread use of animal vaccines. The capacity to weaponize RVFV through aerosol dispersion may require development of a vaccine and assume all associated costs and risks. HENIPAVIRUSES Henipavirology Henipavirus genus is a part of the Paramyxoviridae family, which also includes measles virus (4). In the 1990s, outbreaks of Hendra and Nipah virus heralded the emergence of these pathogens. These are antisense, nonsegmented, single-stranded RNA viruses with genomes approximately twice the size of flaviviruses. They have 2 envelope proteins that facilitate attachment and fusion after binding to the cell surface receptor ephrin. Their reservoir host is bats and spread to humans and other species requires contamination with infected secretions. Henipavirus Neuropathology Only a handful of human Hendra encephalitis cases have been reported, while several hundred human Nipah cases have been reported in different outbreaks (for review [19]). The human Hendra cases have been associated with outbreaks in, and close contact with, horses, while the Nipah cases were associated with slaughter of infected pigs. Both viruses are associated with an acute encephalitis syndrome and less commonly respiratory infection. Fatality rate with Nipah encephalitis approaches 70% (83). Survivors can experience a relapsing encephalitis during which virus cannot be recovered from the host. Human autopsy cases have shown encephalitis with neuronal and endothelial cell infection. A comparable vasculopathy has been noted in experimentally infected pigs (84). While endothelial cell infection suggests hematogenous dissemination, the early presence of virus in the nasal mucosa and olfactory bulb support the possibility of intra-axonal transport of this virus. There is no available vaccine and control of infection is mostly obtained through quarantined culling of livestock. Conclusions: Henipavirus Emergence as a Neuropathogen To control human infection from Henipaviruses requires conscious control of exposure through better animal husbandry. Neurologic disease is the result of rare exposure to high titers of virus. However, this may not be true in the future. Measles is a member of the paramyxovirus family that is phylogenetically related to rinderpest virus of livestock. A few thousand years ago, measles made the cross species jump to humans. Other rinderpest viruses may have made the cross species jump previously, but it was not until human populations achieved a size between 100 000 and 500 000 that sustained infection could be propagated by spreading from infected human to uninfected naïve human without depleting the population leading to extinction of the virus. Obviously, the earth’s current human population has no such barrier to new infections. ENTEROVIRUS A71 AND D68 Enterovirus Epidemiology Picornaviruses are a large family of small single-stranded, positive-sense RNA viruses (4). The genus enterovirus consists of a plethora of agents that have prominent replication within the gastrointestinal system. One important feature that distinguishes them from all of the previously described agents is there is no zoonotic reservoir for the agents that infect man. In an important sense, this should make them more tractable to our intervention. However, there are 7 billion humans on this planet which provides ample reservoir for future infections. Several viruses have emerged with notorious neurovirulence (e.g. polio). Through intensive efforts with vaccination, mankind has come very close to eliminating polio from the face of the earth (85). Unfortunately, given the diversity of enteroviruses and their capacity to recombine, new neurovirulent strains of enteroviruses continue to emerge. Five to 6 days after EV-A71 infection, patients develop a self-limiting acute febrile illness with oral ulcers and vesicular rash on hands and feet (hand-foot-mouth disease) with rare neurologic disease. Viral transmission between humans occurs through fecal-oral or oral-oral routes. Virus replicates to high levels in the oropharynx and is readily recovered from oral swabs or fecal samples. It is not clear why, on rare occasions, EV-A71 spreads to the nervous system. While virus can disseminate hematogenously, most clinical and experimental data suggest that ascent to the CNS occurs most commonly via retrograde transport in peripheral and central nerves. Enterovirus Neuropathology Within the CNS EV-A71 manifests as an acute self-limiting meningitis or a fatal encephalomyelitis. The virus is neurotropic binding to a spectrum of host neuronal receptors. Like its predecessor polio, EV-A71 preferentially replicates within spinal cord anterior horn neurons. Death of these cells accounts for the clinically irreversible paralysis. Currently there are no approved antiviral drug regimens for treating EV-A71; however, numerous therapies have been attempted to block viral binding (suramin), neutralize extracellular virus (intravenous immunoglobulin) or antagonize viral metabolism (e.g. type 1 interferon). Given the success of vaccines in combatting polio, there has been much interest in creating an EV-A71 vaccine (86). Several clinical trials have demonstrated efficacy in protecting children; however, the immunity generated is EV-A71 specific and does not confer protection from other known neuropathogenic enteroviruses (e.g. Coxsackie virus A16). This would suggest that man’s battle to prevent enteroviral neurological disease will require continued vigilance creating new vaccines to counter newly emergent enteroviruses (e.g. EV-D68). Conclusions: Enterovirus Emergence as a Neuropathogen Without a sylvatic reservoir, humans have an abundance of enteroviruses from which neurotropic strains periodically emerge. We have successfully controlled (for the most part) the worst of these, Polio, but EV-A71 and EV-D68 show us that new strains will emerge for which we will need to mount future vaccines. If we are prepared, we can rapidly respond and with appropriate commitment of resources, create new vaccines. But previously with Polio, we were incredibly lucky that there were only 3 neurotropic polio strains and thus readily targetable for vaccine development. When it comes to emergent enteroviruses, the genetic pool is so deep it could catch us by surprise. HUMAN PARECHOVIRUS Human Parechovirus Virology Originally classified within the Enterovirus genus, Parechoviruses were reclassified in 1999 into their own genus on the basis of nucleotide sequence. Human parechovirus (HPeVs) are common and cause a range of illnesses (respiratory to gastrointestinal to neurological); however, most infections are asymptomatic. Exposure to different strains of HPeVs leads to near universal seroconversion within the first 2 years of life. Primary infection is associated with diarrhea and respiratory disease with rare myocarditis or encephalopathy. Genomic sequence analysis has shown that different strains of HPeV possess different degrees of virulence and different organ tropism. As HPeV cannot be cultured, its detection requires targeted PCR, which, if not specifically requested, would be missed. Nevertheless, HPeV accounts for many neonatal admissions for encephalitis (87), with HPeV3 being the most common pathogen. Genetic analysis of HPeV3 suggests that this agent emerged recently within the last 400 years (88). HPeV Neuropathology Our understanding of the clinical significance of neonatal HPeV infection is in, well, its infancy. The largest study to date included 10 infants (89). Head MRI and ultrasound showed abnormal white matter in all of these infants compatible with clinical signs. None of these infants died and long term follow up showed no to variable neurological sequelae. Since the initial study several follow up studies have shown similar results (90). Our understanding of the pathogenesis of HPeV infection is limited to the very rare autopsies (91). These autopsies have confirmed the white matter pathology seen on radiologic evaluation (Fig. 5). However, there was no evidence of significant neuroglial infection. Rather HPeV was detected specifically within the smooth muscle of arterial walls. This suggests that the neurologic damage in HPeV-infected infants results from infection of the vasculature, leading to ischemic white matter pathology. The pattern of this damage is very similar to periventricular leukoencephalopathy which is one of the more common afflictions of premature children. FIGURE 5. Open in new tabDownload slide HPeV3 infection of neonates. (A) T1-weighted noncontrast MRI from an HPeV3-infected infant. Healthy at birth, the child developed “neonatal sepsis” after exposure to an ill adult 30 days after delivery. Initially radiologic studies were normal; but the infant developed seizures, and subsequent MRIs demonstrated cavitary deep white mater lesions. (C) The infant died the following day and gross coronal section of its brain shows deep-seated periventricular cavitary lesions. (B) Paraffin-embedded brain (“BR”) and overlying leptomeninges (“M”) probed for HPeV3 RNA using in situ hybridization (red) (counterstained with hematoxylin). Abundant HPeV3 viral RNA (red) is seen in the modestly hypercellular leptomeninges with no evidence of infection of the brain parenchyma. (D) Higher-power image confirms presence of HPeV3 RNA (red) in leptomeningeal cells and particularly in smooth muscle cells of blood vessel (“BV”) walls. These findings suggest that damage noted in severe periventricular leukoencephalopathy damage is an indirect effect of vascular compromise. FIGURE 5. Open in new tabDownload slide HPeV3 infection of neonates. (A) T1-weighted noncontrast MRI from an HPeV3-infected infant. Healthy at birth, the child developed “neonatal sepsis” after exposure to an ill adult 30 days after delivery. Initially radiologic studies were normal; but the infant developed seizures, and subsequent MRIs demonstrated cavitary deep white mater lesions. (C) The infant died the following day and gross coronal section of its brain shows deep-seated periventricular cavitary lesions. (B) Paraffin-embedded brain (“BR”) and overlying leptomeninges (“M”) probed for HPeV3 RNA using in situ hybridization (red) (counterstained with hematoxylin). Abundant HPeV3 viral RNA (red) is seen in the modestly hypercellular leptomeninges with no evidence of infection of the brain parenchyma. (D) Higher-power image confirms presence of HPeV3 RNA (red) in leptomeningeal cells and particularly in smooth muscle cells of blood vessel (“BV”) walls. These findings suggest that damage noted in severe periventricular leukoencephalopathy damage is an indirect effect of vascular compromise. HPeV Treatment There are no approved widely used antiviral therapies for enteroviral diseases. As with other enteroviral infections, vaccination is a possible preventative approach; however, in the case of HPEV, it would have an unusual twist. Vaccinations have worked well in preventing childhood polio. But HPeV infection in the first few weeks after birth occurs prior to most pediatric immunizations. Therefore, to achieve adequate protection from HPeV neonatal infection, it would be necessary to immunize the mother and expect enough antibodies to pass through the placenta to protect the newborn from early exposure. Conclusions: HPeV Emergence as a Neuropathogen Neurologic disease associated with HPeV is rare but represents a novel form of pathogenesis and thus a novel approach to therapy. It is well recognized that leaving the protected womb environment is our first big mistake and met with a barrage of infectious agents. We accept that many unknown agents contribute to a plethora of neonatal and childhood disease, from most of which, we quickly recover. That HPeV infection occurs within weeks of birth and can mediate significant neurologic disease accents the importance of discovering unknown unknowns. In the absence of effective antivirals for HPeV, we will have to focus on vaccines, but the newborn does not have time to develop adaptive immunity before exposure. This scenario forces us to explore developing vaccines that can work transplacentally by passing high titers of antibody from mom to fetus providing passive immunity. HUMAN IMMUNODEFICIENCY VIRUS HIV-1 Virology Human immunodeficiency virus (HIV-1), the etiologic agent of the AIDS epidemic, was discovered in 1983. Molecular analysis has shown that the retrovirus was derived from an endogenous chimpanzee agent that crossed species ∼100 years ago (92). HIV-1 rapidly adapted to its new human host. Because of an error-prone RNA polymerase that creates mutant viral swarms capable of evading the host immune system, and a lengthy period of high viremia during which the host is capable of spreading HIV to new hosts through close contact and exchange of blood or bodily secretions, HIV-1 rapidly spread, eventually infecting over 70 million humans worldwide. A second virus, HIV-2, leapt from sooty mangabey monkeys into humans around the same time, but proved much less capable of mediating a global infection (93). That only 2 lentiviruses have crossed into humans suggests there are substantial, but not insurmountable, ecological, physiological and molecular barriers preventing cross species infections. Retroviruses are named for their ability to create stable DNA copies of their RNA genome (4). The lentivirus subfamily spends little of its life cycle in DNA form and instead replicates to high levels as cell associated RNA and cell free virion. HIV-1 has an ∼10-kb genome packaged as a dimer in an enveloped virion ∼110 nm in diameter. The viral envelope glycoprotein complex binds the host receptor molecule CD4 in addition to a chemokine coreceptor. As a genus, lentiviruses are notable for their infection of macrophages. However, initially HIV-1 was notable for strains that infect and destroy lymphocytes. HIV-1 infection of CD4 T-lymphocytes results in their depletion and eventual immune exhaustion with host immunosuppression. HIV Neuropathology Approximately 25% of the severely immunosuppressed patients developed HIV encephalitis in which macrophages in the CNS produced the highest concentration of virus anywhere in the body (1). It has never been clear how in the absence of neuro-glial infection, macrophage infection led to neurodegeneration. Hypotheses spanned the spectrum of neurotoxic viral proteins to neurotoxic inflammatory mediators. The advent of highly effective combined anti-retroviral therapy (CART) permitted CD4 T-cell recovery, immune reconstitution, suppression of CNS infection and the disappearance of HIV encephalitis/dementia. While CART appears to have cured HIV encephalitis, infected patients frequently experience less well-defined neurological symptoms of unknown cause (HIV-associated neurological disease [HAND]) (94). There are several excellent animal models of HIV infection of humans (95). Infecting specific species of old-world macaques with retroviruses (simian immunodeficiency virus [SIV]) from other primates causes disease with similarities to HIV in humans. SIV infection of vervets (Chlorocebus pygerythrus) mediates chronic host infection without significant immunosuppression. Infection of pigtailed or rhesus macaques of Indian subcontinent extraction mediates a rapid CDT-cell depletion and immunosuppression with high incidence of SIV encephalitis. Each virus/host pair generates a unique outcome in which the pathogenesis of different lentiviral infections can be studied. Conclusions: HIV Emergence as a Neuropathogen Lentiviruses are notorious for infecting host macrophages. With microglia representing up to 5% of CNS cells, the human brain provides an abundance of permissive targets for HIV infection. After destruction of systemic CD4 T-cells, infected microglia escape immune surveillance. A variety of mechanisms have been proposed to explain how active, uncontrolled HIV-infection of microglia leads to neurodegeneration and dementia. For now, we are fortunate to have discovered pharmaceutical means of blocking systemic HIV replication, preventing immunosuppression and preserving brain integrity. If we are lucky and persistent, eventually we will create a vaccine and be spared the enormous expense of pharmaceutical therapy. If we are cautious and avoid extraordinary contact with our primate cousins, we can maintain the barrier to cross species transmission of lentiviral pathogens. CORONAVIRUSES Coronavirus Virology The recent COVID-19 pandemic raises the question of whether this emergent virus infects or otherwise perturbs the human CNS. Coronaviruses (CoVs) are large enveloped positive-stranded RNA viruses. The CoVs surface spike glycoprotein (S) binds to specific cellular receptors and mediates fusion. In addition to the spike glycoprotein, viral surface HA-esterase glycoprotein also binds specific host sialic acid conjugate configurations to achieve cell entry. With the possible exception of SARS (see below), CoV are highly species specific. There are numerous well-studied animal models of endogenous CoV infection, some of which show CNS infection (96). As with all infectious models, the complex nature of the virus-host interaction is highly specific for the strain of virus and strain of host. Select strains of the CoV mouse hepatitis virus can cause either an acute T-cell-mediated encephalitis or a chronic demyelinating disease depending upon the precise strain of virus and age and immune status of the host (97). To circumvent one of the species specificity restrictions, transgenic mice have been constructed in which human CoV receptors are expressed. While these transgenic animals are highly infectable, the artificial expression of a transgene and introduction of a novel pathogen complicates their contribution to our understanding of human pathogenesis. Nevertheless, transgenic mice expressing the human Middle East respiratory syndrome CoV (MERS) receptor DPP4 (98) show lethal lung and brain infections and transgenic mice expressing the human SARS receptor angiotensin-converting enzyme 2 (ACE2) (99) show limited lung infection and lethal encephalitis. CoV Epidemiology There are 4 known endemic human CoVs that mediate mild respiratory infections. To date there has been a single nonreplicated report of these viruses in human CNS tissue (100–105). However, there are an unknown number of zoonotic CoVs of which 3 have made spectacular recent jumps into humans; severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, and currently SARS-CoV-2 (also known as COVID-19). These 3 emergent CoVs are suspected to be derived from bat viral species with different intermediate hosts (palm civet cats for SARS-CoV, camels for MERS-CoV, and pangolins for SARS-CoV-2). All 3 of these CoVs mediate severe pneumonia and their appearance in humans was unanticipated. The first 2 CoVs had limited spread (∼800 fatalities in the world each) but the third caused catastrophic disruption of the world economy the likes of which have not been seen since the influenza A pandemic of 1918. There are 3 human autopsy studies of SARS-CoV patients suggesting CNS infection (106–108). Unfortunately, the reported cases are highly idiosyncratic and it is difficult to discern how much the reports confirm each other. So, whether or not SARS-CoV infects the human CNS remains to be elucidated. CoV Neuropathology This early in the SARS-CoV-2 pandemic, it is distressingly difficult to weed through the myriad of reports and discern the scientific facts related to infection. Nevertheless, there have been reports of nervous system involvement by SARS-CoV-2, so it is important to review the potential pathogenesis of this emergent viral infection. While notorious as a respiratory pathogen, like influenza, SARS-CoV-2 could mediate neurological disease through a variety of mechanisms. One is direct infection of the CNS causing a meningoencephalitis. Like influenza A, CoV bind to host receptors with specific protein terminal sialic acid conjugates. Only a limited number of influenza A strains, and none of the pandemic strains, mediate encephalitis. This limitation in part is due to tissue specific distribution of viral receptors. Avian flu binds to terminal alpha2-3 sialic acid conjugated host proteins (present in lower respiratory tract and brain) while pandemic influenza binds to terminal alpha2-6 sialic acid linkages (present in upper respiratory tract). This restriction, along with maturation of the viral hemagglutinin, limits pandemic flu to severe respiratory infection while avian flu can invade the CNS by binding to cells with abundant alpha2-3 sialic acid conjugated surface proteins. The receptor for SARS-CoV-2 has yet to be definitively identified but probably includes ACE2 and is further facilitated by other glycosylated host cell surface proteins. As with influenza, utilizing terminal alpha2-6 sialic acid linked host proteins is an effective strategy to secure human-human transmission through high titer replication in the upper respiratory tract. However, SARSCoV2 also appears capable of utilizing terminal alpha2-3 conjugated receptors, which is one of the reasons it causes a severe infection of the lower respiratory tract. Theoretically, this binding property could also facilitate its infection of the CNS. Whether such an infection occurs in humans (101) awaits confirmation (102). Besides direct infection of the CNS, SARS-CoV-2 could cause CNS dysfunction by other mechanisms. Severe pneumonia could lead to hypoxic-ischemic CNS injury. Metabolic derangements, such as syndrome of inappropriate antidiuretic hormone, could disrupt basic electrolyte balance and brain function. Given the severity of the infection, a robust immune response could result in cytokine storm syndrome, physiologically affecting CNS function or hematologic disorders like platelet reduction leading to strokes. As with other viral infections, overstimulation of the immune system predisposes to autoimmune disease and there have been reports of acute hemorrhagic encephalitis and Guillain-Barre syndrome. Whether or not SARS-CoV-2 proves capable of infecting/affecting the human CNS, its appearance in 2019 proves how poorly prepared we are for emergent viral infections and how devastating an impact they can have. Conclusions: SARS-CoV Emergence as a Neuropathogen Human SARS-CoV infections are under active investigation. While there are only rare reports of human CNS infections, there are abundant examples of CoVs infecting the CNS of animals. Clearly the brain is susceptible to infection by CoVs and there are now notorious recent examples of the sylvatic viruses successfully adapting to human receptors. CONCLUSIONS Our innate and adaptive immune systems have evolved to protect us from an ecosystem replete with deadly pathogens. Throughout our past, new pathogens emerged from this pool to threaten the survival of our species. Our immune system successfully protected us from extinction but, as the history of past plagues has taught us, at great cost. Fortunately, man has evolved a brain capable of supplementing our immune system to devise new ways to evade the carnage of infectious disease. As our understanding of infectious disease improved, we learned how to escape lethal infection. Beginning with simple quarantine, we discovered vaccination, a means of giving our immune system the critical time-advantage to develop adaptive immunity prior to pathogen exposure. Intelligently designed vaccines have gone a long way in protecting us from known infectious agents. But vaccine development is a lengthy process, and emergent infectious diseases frequently catch us unawares. In those instances, we must rely upon our understanding of the viral pathogenesis to develop an armamentarium of small molecule drugs to disrupt the pathogen’s life-cycle. The best contemporary example of this is HIV, where we are still struggling to create a vaccine but have created an effective treatment regimen, completely preventing HIV encephalitis. We face many old and new challenges to successfully stay ahead of emergent threats. The first new challenge is that our population has grown and spread such that we have invaded every ecosystem on earth. Each time we disrupt an existing host/pathogen cycle, we risk becoming new infection targets. Viruses don’t have legs, but nature has given them wings in the form of infected mosquitoes and birds. But even with wings, many parts of the globe were isolated with limited genetic exchange. Man has given viruses jet planes capable of moving them around the world in a day (WNV, Zika, and SARS). Because we are one of the few organisms capable of circling the globe, we have erased all-natural geographical barriers and facilitated global distribution of these new pathogens. Through our animal husbandry and massive overpopulation, we have greatly expanded the host target further flattening the earth from the pathogen’s perspective. Of the viruses we know, perhaps our greatest challenge is influenza. The enormous sylvatic reservoir and diverse IAV genetic pool forms a trove of virulent potential emergent pathogens. IAV has a protein coat that can rapidly mutate or recombine to elude our immune system. History has shown us that emergent flu is capable of decimating a significant portion of our population causing untold misery and economic collapse. While considered a pulmonary pathogen, certain strains of flu have the proven capacity to infect the CNS. The perfect storm would entail emergence of an IAV strain that supports human to human aerosol transmission and the capacity to infect the brain. Contagious IAV encephalitis could be the plot of horror stories. Beyond IAV, the brain appears to be uniquely vulnerable to aerosol transmission. Traditionally transmitted agents notorious for causing systemic disease (e.g. arboviruses) for unknown reasons become highly neurotropic when delivered by aerosol. Perhaps this relates to some selective hole in our natural immune response to inhaled agents that for whatever reason, such exposure is not associated with effective immunity for the brain. Such a hole can be readily targeted, if not by nature, then by malignant members of our own species. Arboviruses can be grown to high titers and then packaged as biological weapons for aerosol transmission. This brief survey of emergent viral infections begs the question: Why are RNA viruses such devastating infections of the CNS? As discussed above, the brain generally does not mount the full armamentarium of viral clearance strategies as seen with other organs. This limited immune response has been thought to curtail death of a cell population with limited regenerative capacity. However, recent studies have revealed that the brain is unique not only in its massive expression of numerous genes, but also in its overall processing of RNA (109). Critical to the brain’s function is the capacity to modulate RNA metabolism in an intricate and spatially refined way. While systemic organs like the gut and lung can utilize generic innate immune responses that entail shutting down RNA processing, such a defense might not be available to the CNS. The inability of the brain to defend itself from viral infection may reside in the importance of meticulous control of RNA processing necessary for brain functionality. 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TI - Emergent Viral Infections of the CNS
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nlaa054
DA - 2020-05-11
UR - https://www.deepdyve.com/lp/oxford-university-press/emergent-viral-infections-of-the-cns-yAQwfkI7Ng
DP - DeepDyve
ER -