The tissue was cultured in a Vero cell for a 3-day incubation period. The Vero cells were fixed in glutaraldehyde, dehyrated, placed in an Epon resin, thin sectioned, placed on a copper grid, and stained with uranyl acetate and lead citrate. The grids were then placed in the electron microscope and viewed. Total magnifications, image 65,x.
The most current phylogenetic studies based upon sequences of entire or partial genome sequences indicate five lineages of WNV [ 28 ]. The virus that entered North America belongs to lineage I clade Ia. This lineage also contains viruses found in Europe, the Middle East and Africa. Although there are exceptions, in general, lineage I clade Ia viruses can cause severe human neurologic disease whereas lineage I clade Ib , and lineage II viruses generally cause a mild, self-limiting disease.
WNV is maintained in nature in a cycle between birds and mosquitoes Figure 3. Although many different species of mosquito are capable of maintaining this cycle, the Culex species play the largest role in natural transmission Figure 4. Not all infected mosquitoes preferentially feed upon birds, which can lead to other animals including humans becoming infected.
Other natural modes of WNV transmission have been documented, but occur rarely. WNV transmission can occur between infected mother and newborn via the intrauterine route [ 29 - 31 ]or possibly by breast-feeding [ 32 ].
The maintenance of WNV in nature depends upon many avian and mosquito species. Humans and other incidental hosts like horses become infected when WNV-infected mosquito takes a bloodmeal from them. The Culex species of mosquito is the most common vector of WNV.
Photograph of Culex species mosquito feeding. Courtesy of USGS. Within the human population, the virus can spread between individuals by more artificial means. These events highlighted the need to safeguard blood and organ donations from potentially viremic yet healthy donors, and relatively few infections via this route of transmission were reported since The epidemiology of WNV is continuously changing. The virus was initially isolated from a febrile woman in Uganda in [ 38 ].
Since that time, few outbreaks of WNV in human or horse populations were recorded until the beginning of the s. Noteworthy exceptions during this time were outbreaks in Israel in the early s and France in the s, which were characterized by encephalitis in humans and horses.
A series of outbreaks in the s brought WNV into the spotlight; epidemics in Algeria, Morocco, Tunisia, Italy, France, Romania, Israel, and Russia were associated with uncharacteristically severe human disease, including neurologic complications and death [ 39 , 41 - 43 ].
The sequence of the New York strain of WNV is closest in identity to a viral isolate from Israel [ 44 ], but it is still a mystery how the virus traversed the Atlantic Ocean. In the past decade, there have been thousands of reported human cases of WNV disease WN fever and WN encephalitis accompanied by over a thousand deaths Table 1.
Countries with historic or recent present WNV activity isolations from mosquitoes, birds, horses or humans are highlighted in red and blue, respectively. Courtesy of CDC. Summary of confirmed human cases of WN disease in the United States, a. Symptomatic infections are primarily a mild, self-limiting febrile illness. Most symptomatic patients exhibit mild illness with fever, sometimes associated with headache, myalgias, nausea and vomiting, and chills [ 34 , 46 - 49 ].
Further, some patients briefly present papular rash on the arms, legs, or trunk. These symptoms follow relatively predictable pattern with illness generally lasting less than seven days. However, a number of patients experience severe fatigue and malaise during convalescence. Approximately 5 percent of patients with symptomatic WNV infection develop neurologic disease.
WNV neurologic symptoms include meningitis, encephalitis, and poliomyelitis-like disease, presented as acute flaccid paralysis [ 50 ]. WNV poliomyelitis-like syndrome is characterized by acute onset of asymmetric weakness and absent reflexes without pain. Patients presenting with flaccid paralysis require further testing to determine nature and degree of disease. Diagnostic tests including cerebrospinal fluid CSF examination should be performed in order to differentiate WNV infection from stroke, myopathy, and Guillain-Barre Syndrome.
Other clinical symptoms may include tremor, myoclonus, postural instability, bradykinesia, and signs of parkinsonism. Understanding the full range of WNV pathogenesis in humans has been difficult, mainly due to the difference in virulence between WNV strains, the high prevalence of asymptomatic or sub-clinical infections, and the relative infrequency of laboratory-confirmed human infections.
Little has been published about human infections with WNV of limited virulence. The vast majority of our current knowledge regarding WNV pathogenesis resulted from animal models mostly rodent infected under controlled conditions with a known amount of needle-inoculated virus, which may not accurately reflect the course of a natural infection in humans.
Nevertheless, many descriptive accounts have been documented following the course of infection in humans suffering from WN fever and WN encephalitis resulting from a virulent lineage I WNV infection.
WNV-infected mosquitoes transmit the virus to humans following a bloodmeal from the host. During this process, mosquito saliva contaminated with WNV is deposited in the blood and skin tissue. Shortly thereafter, virus amplifies in the tissues and results in a transient, low-level viremia, lasting a few days, and typically wanes with the production of anti-WNV IgM antibodies [ 53 ].
Following viremia the virus infects multiple organs in the body of the host, including the spleen, liver, and kidneys. Interestingly, 8 days after onset of symptoms, WNV was detected in the urine viruria of a patient with encephalitis [ 54 ], which is consistent with animal hamster experiments demonstrating viruria [ 54 ] and the presence of viral infection in the kidneys [ 55 , 56 ].
High viremia may easily lead to an infection of the brain if the BBB is disrupted and high viremia is correlated with severity of infection in experimentally infected mice [ 57 ].
Viremia and high viral titers in the periphery alone do not predict neuroinvasion. However, an increase in BBB leakage does not accurately predict WNV-induced mortality in hamsters, nor does lethal infection increase BBB permeability in all strains of mice [ 66 ].
WNV may enter the brain by directly infecting and retrograde spreading along neurons in the periphery [ 67 ]. Entering the brain via infected peripheral neurons is a likely entry route since the level of viremia is low and leakage into the CNS by a breakdown of the BBB is less likely compared to animals with a high titer of circulating WNV in the blood. Diagnosis of WNV infection depends on a number of factors, including environmental conditions, behaviors, and clinical symptoms.
Patient history will give crucial clues to diagnosis. For example, if a patient presents with clinical symptoms, including fever and headache, one must consider the distribution of WNV and its mosquito vector.
Endemic areas must consider WNV infection, especially during the summer months. Further, the patient history should suggest exposure to mosquitoes through outdoor activities. An initial physical examination will confirm clinical symptoms of fever, headache, myalgia, or the more severe meningitis and flaccid paralysis. Also, the presence of mosquito bites on the skin will assist in diagnosis. To confirm the initial diagnosis, specific laboratory tests must be ordered Table 2.
To date, the most consistent manner to verify WNV infection is serology [ 47 , 49 ]. This test is commercially available and relatively inexpensive [ 34 ]. Also, serology can be performed to analyze immune responses.
More dramatically, a massive influx of polymorphonuclear cells occurs. Plaque reduction and neutralization tests PRNT allow for identification of virus specificity. Virology tests can directly confirm the presence of virus. Serum or CSF is collected and virus is amplified within permissive cells and sequenced. This test is time-consuming and expensive. Finally, molecular biology tools can be employed to confirm the presence of virus. Serum or CSF is collected during the initial phases of virus infection can be directly amplified, or used to detect viral RNA by quantitative reverse transcription polymerase chain reaction Q-RT-PCR with virus-specific primers.
The regions of the CNS most commonly affected are basal gangli, thalami, brain stem, ventral horns, and spinal cord. However, the majority of these studies were performed retrospectively.
Thus, the results do not provide predictive capabilities to WNV infection. A Coronal fluid-attenuated inversion recovery FLAIR magnetic resonance image shows an area of abnormally increased signal in the thalami, substantia nigra extending superiorly toward the subthalamic nuclei and white matter. B Corresponding tissue section from the same patient at autopsy 15 days later, stained with Luxol fast blue—periodic acid Schiff for myelin, shows numerous ovoid foci of necrosis and pallor throughout the thalamus and subthalamic nucleus arrows.
C Axial proton density image at the level of the midbrain shows a bilaterally increased signal in the substantia nigra arrows. E Axial FLAIR image at the level of the lateral ventricle bodies shows a bilaterally increased signal within the white matter.
A scan performed approximately 5 months earlier demonstrated an abnormal signal in the left periventricular white matter. This signal increased once West Nile virus encephalitis developed, and the lesions in the right cerebral white matter left side of photograph were new. F Photomicrograph taken from the right periventricular white matter, immunostained with the HAM56 antibody, shows numerous macrophages, both in perivascular areas lower right and diffusely throughout the white matter center. Arch Neurology A number of diseases manifest as symptoms similar to West Nile virus, including the encephalitides viruses such as JEV and Murray Valley encephalitis virus and bacterial meningitis.
Therefore, differential diagnosis is crucial to determining WNV infection. A differential diagnosis is required when a patient presents with unexplained febrile illness, encephalitis or extreme headache, or meningitis. Currently, patients infected with WNV have limited treatment options. The primary course of action is supportive care.
There is no FDA-licensed vaccine to combat WN disease in humans, despite the research of many laboratories and institutions and vaccines available for use in horses. Furthermore, there are no effective antiviral to combat WNV infection.
Two classical antiviral compounds, interferon and ribavirin, showed promising results in vitro [ 71 , 72 ] but it is unclear if these compounds are effective in patients [ 73 - 77 ]. Passively transferring anti-WNV immunoglobulin has been shown to be effective in mouse and hamster models [ 78 ] and may be helpful in patients [ 79 , 80 ].
Long-term complications 1 year or greater after infection are common in patients recovering from WNV infection. The most common self-reported symptom is fatigue and weakness, although myalgia, arthralgia, headaches, and neurologic complications, such as altered mental depression, tremors and loss of memory and concentration are not uncommon [ 81 ].
There is also evidence from animal models [ 55 , 82 , 83 ] and human autopsies [ 84 , 85 ] that the virus may persist in some individuals, as measured by isolation of virus or viral genomes or antigen months after infection or symptom presentation.
Experimentally infected hamsters show long-term neurological sequela, which appears to coincide with the presence of both viral antigen and genome within areas of the brain showing neuropathology [ 83 ].
Although the direct evidence of persistence in humans is limited at this time, many patients have long-lasting WNV-specific IgM titers in the serum and CNS, suggesting that persistent infections may be more common than previously indicated [ 86 - 88 ].
Both the innate and adaptive immune responses mounted against WNV are critically important for controlling infection. Type I interferons alpha and beta are important for limiting virus levels, reducing neuronal death, and increasing survival [ 57 ]. The adaptive immune response also plays a role in controlling infection. Studies using WNV-infected genetically engineered knockout mice indicate that both T- [ 91 - 96 ]and B- [ 97 ]cells are critical for controlling infection.
IgG is the predominant antibody most likely confering long-term immunity against WNV re-infection. Diagnostic procedures for viral, rickettsial, and chlamydial infections; 7th ed.
Washington: American Public Health Association; Investigation of Eastern equine encephalomyelitis IV: susceptibility and transmission studies with virus of pheasant origin. Am J Hyg. Polymerase chain reaction for detection of avian leucosis virus subgroup J in feather pulp.
Avian Dis. High virus titer in feather pulp of chickens infected with subgroup J avian leucosis virus. Direct non-vector transmission of West Nile virus in geese. Avian Pathol. Pyle P. Identification guide to North American birds. Articles by Country Search — Search articles by the topic country. Article Type Search — Search articles by article type and issue. Please use the form below to submit correspondence to the authors or contact them at the following address: Douglas Docherty, National Wildlife Health Center, Schroeder Rd.
Comments character s remaining. Comment submitted successfully, thank you for your feedback. There was an unexpected error. Message not sent. Page created: February 22, The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U. Use of trade names is for identification only and does not imply endorsement by any of the groups named above. Links with this icon indicate that you are leaving the CDC website.
Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website. You will be subject to the destination website's privacy policy when you follow the link. We wish to thank our collaborators in the Systems Immunogenetics Group for helpful discussions and generation of mice. Software: MM SM. Abstract Infection with West Nile virus WNV leads to a range of disease outcomes, including chronic infection, though lack of a robust mouse model of chronic WNV infection has precluded identification of the immune events contributing to persistent infection.
Author Summary Much experimental work has been directed at understanding the host immune response to flavivirus infections such as West Nile virus WNV using mouse models of infection. Introduction West Nile virus WNV is an emerging flavivirus, and a potential model pathogen for other viruses in its genus such as Zika, dengue, and yellow fever viruses. Download: PPT. Fig 1. Fig 2. Comparison of WNV disease models by clinical presentation and neuropathology.
Altered innate immune responses in lymphoid tissues and the CNS distinguishes CC x F1 from RIX lines with no disease and those with disease Because CC x F1 mice clearly exhibited a unique disease course following WNV infection as compared to the conventional inbred line B6 as well as the three RIX lines that showed no signs of disease and three RIX lines that did, we further examined the immune responses mounted upon infection in two target organs of WNV infection: the spleen, which notably is an immune inductive site as well as a target for virus replication, and the brain.
Fig 3. Differential innate immune signatures and viral load in the tissues. A strong immunoregulatory signature distinguishes CC x F1 from CC-RIX lines with no disease Due to the unique differences in innate immune responses observed in the spleen and brain of the chronic RIX line, we next examined the later stages of acquired immunity to WNV infection, with a focus on T cell responses using systematic multi-parameter flow cytometry.
Fig 4. Flow cytometry heatmaps allow visualization of intra- and inter-strain comparisons of the immune response. An early increase in regulatory T cell numbers in the brain allows for establishment of WNV chronic infection over traditional disease course When comparing the chronic RIX to RIX lines with signs of disease, a distinct immunoregulatory signature in the spleen again marks the chronic RIX as unique compared to three RIX lines with disease Fig 5A. Fig 5. Chronic infection model mice show increased splenic Tregs over the time course of WNV infection compared to mice with disease.
Fig 6. Flow cytometry visualization of the kinetics of the T cell response to WNV infection. Mice chronically infected with WNV display a unique gene expression profile including reduced cytolytic ability Finally, we examined gene expression changes within the spleen following WNV infection in the chronic RIX versus three RIX lines with no signs of disease and three with signs of disease, via microarray analysis. Fig 7. Mouse model of chronic WNV infection reveals that maintenance of cytolytic ability, associated with reduced activation of regulatory T cells, is critical for preventing viral persistence.
Discussion We report here for the first time a mouse model of chronic WNV infection that can be used to elucidate the immunological mechanisms underlying chronic flaviviral infection and disease in humans.
RNA extraction and analysis procedures Spleen, kidney and brain were removed from mock infected or WNV infected mice after perfusion as described above. Transcriptomic analysis Samples were screened for QC and outlier detection using the Affymetrix expression console using boxplots, as well as multi-dimensional scaling MDS analysis and inter-array correlation IAC plots using the R statistical programming language version 3.
CC-probe masking To ensure that all Affymetrix probes work properly across all CC lines, previously described masking techniques were applied based off the CC founder data. Cell preparation for flow cytometry assays Following euthanasia, mice were perfused with 10 ml PBS to remove any residual intravascular leukocytes. Supporting Information.
S1 Fig. S2 Fig. S1 Table. Histology Scoring Methods. S2 Table. Acknowledgments We wish to thank our collaborators in the Systems Immunogenetics Group for helpful discussions and generation of mice. References 1. Clinical microbiology reviews — Clinical infectious diseases: an official publication of the Infectious Diseases Society of America — View Article Google Scholar 3.
J Infect Dis 2—4. PLoS Pathog 3: e Journal of virology — Nat Rev Microbiol — PLoS Pathog 6: e Nature genetics — Collaborative Cross C The genome architecture of the Collaborative Cross mouse genetic reference population. Genetics — View Article Google Scholar Mammalian genome: official journal of the International Mammalian Genome Society — Nature — MBio 6: e— PLoS One 5: e J Virol — Virology — Eur J Immunol — Clin Exp Immunol — PLoS One 9: e Clin Immunol 50— Inflamm Allergy Drug Targets — Hepatology — Plaque-reduction neutralization assay PRNT Traditional test used for flavivirus antibody detection where cross-reactivity with other viruses may occur, e.
St Louis encephalitis virus. Fresh tissues: brain, spinal cord, CSF. Can be done with plasma, but starting dilution of neutralization assays will be higher in order to dilute out anti-coagulant. Test is used to detect an early immune response due to infection. Test interpretation must take into account vaccination status of the animal. Test is used to define the infection or vaccination status of the horse.
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