A Comparative Analysis of Models for West Nile Virus

West Nile virus bites back

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Until recently, West Nile (WN) virus and Kunjin (KUN) virus were classified as distinct virus types within the Flavivirus genus. However genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To properly define the relationship between KUN and WN viruses, sequence analyses were performed on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN virus isolate from the recent outbreak in New York. Two independent 500 - 550 nucleotide regions of the genome were sequenced: (i) a region coding for a portion of the envelope protein surrounding the potential glycosylation site at amino acid 154; and (ii) a region encompassing the 3' end of the non-structural (NS) 5 gene and 5' end of the 3' untranslated region (UTR). Sequence comparisons from both regions revealed that the KUN virus isolates from Australia were tightly grouped whereas the WN virus isolates exhibited significant divergence and could be differentiated into three distinct groups. Some WN virus isolates shared greater sequence identity with Australian KUN virus isolates than with other WN virus isolates, while a KUN virus isolate from Sarawak, Malaysia, showed greatest sequence identity with WN virus strains. Virus isolates were also tested for antigenic variation using a panel of seven monoclonal antibodies (Mab) produced to either KUN or WN viruses. The binding patterns of these antibodies in ELISA demonstrated that KUN virus isolates from Australia were antigenically homologous and distinct from the WN virus isolates and the Malaysian KUN virus isolate. Similarly, all WN virus isolates except one displayed a distinct Mab binding profile. The remaining WN virus isolate (Sarafend), the Malaysian KUN virus isolate and Koutango virus also displayed distinct Mab binding patterns. The results in this thesis suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into a number of subgroups on the basis of genetic and antigenic analyses.During the course of this study, viral cultures were identified that contained both a KUN-like virus and a WN-like virus. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN virus population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. A novel strategy was then designed to separate the two virus populations. Limit dilution of the enriched virus preparations was then performed in the appropriate cell line by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by removing and retaining the culture fluid from each well and immunostaining fixed cell monolayers with virus-specific Mabs. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific Mab staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogenous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.

In October 1999, the Centers for Disease Control and Prevention (CDC) reported on an outbreak of human arboviral encephalitis in New York City, beginning in late August (1). The encephalitis was initially thought to be due to St Louis encephalitis (SLE) virus because of positive serological results from the cerebrospinal fluid (CSF) and serum of affected patients. At the same time, there was an increase in avian mortality including wild crows and exotic birds at the Bronx Zoo (2). Because avian mortality is not common with SLE, other pathogenic arboviruses were investigated as the cause of this unusual phenomenon. Subsequent DNA sequencing of human and avian viral isolates indicated that they were closely related to West Nile (WN) virus, not previously isolated in the Western Hemisphere (1). Serological testing of CSF and serum, including those specimens positive for SLE virus, from a number of patients was positive for antibody to WN virus (3). A new infectious disease had emerged in the Americas. WN virus is an arthropod-borne virus belonging to the Japanese encephalitis complex of the Flavivirus genus (4). Flaviviruses are lipid-enveloped, single stranded RNA viruses, with a genome of approximately 11,000 nucleotides. Flaviviruses belong to the family Flaviviridae with Pestivirus (of veterinary importance) being the other genus in this family. There are over 68 viruses in this genus, of which 30 are known to cause human disease (5). Within the genus, the flaviviruses are classified into distinct species or serotypes by antigenic distinctions. There are at least eight antigenic complexes, six of which contain human pathogens. Japanese encephalitis, SLE, Murray Valley encephalitis, Kunjin, Kokobera, Koutango, Usutu and WN viruses all belong to the Japanese encephalitis complex (4). Viruses within the Japanese encephalitis complex share up to 77% of their amino acid sequences, resulting in cross-reactive serological tests and providing an explanation for the original identification of the outbreak as due to SLE virus. Although the Flaviviruses are closely related antigenically and cross-react in serological tests with polyclonal antisera, most have a distinctive geographical distribution (6). WN virus was first isolated in the WN province of Uganda in 1937 (7). The first recorded epidemics were reported in Israel in the 1950s (8,9) and in Europe in 1962 (1). Sporadic cases and outbreaks have been reported from Africa (10,11), India (12) and Romania (13). The virus is the most widely distributed of the arboviruses, causing infections in Africa, the Middle East and South Asia, where it is endemic, and in Europe more sporadically (13-15). However, it had never been identified in the Americas before 1999. A closely related serotype, Kunjin, has been found in Australia and Southeast Asia (15). Subtypes of WN virus are distinguished by antigenic variations in the envelope (E) protein and the presence of an N-glycosylation site at amino acids 154 to 156 (16). Two lineages have been proposed: lineage I includes Kunjin and WN virus from Europe, the Middle East, and North, Central and West Africa; and lineage II includes WN virus from West, Central and East Africa, and Madagascar. The complete nucleotide sequence of one of the viral isolates (from the dead Chilean flamingo at the Bronx zoo) was determined (6). Analysis showed it to be a lineage I WN virus (16), and most closely related to WN virus isolated recently from North Africa, Romania, Kenya, Italy and the Middle East (6). Lanciotti et al (6) demonstrated a high degree of sequence similarity among the various strains circulating throughout New York City and surrounding counties and states. The ecology of WN virus has recently been described by Hubalek and Halouzka (15). Similar to other Flaviviruses, WN virus has an arthropod vector, which serves as a true biological vector. Mosquitoes, mainly bird-feeding species, are the principal vectors. WN virus has been isolated from 43 mosquito species, predominantly Culex but also Aedes and Anopheles species. Experimental and field evidence demonstrates vertical transmission from parent to offspring mosquitoes (17-19). While the exact role of vertical transmission is

West Nile (WN) virus has been spreading geographically to non-endemic areas in various parts of the world. However, little is known about the extent of WN virus infection in Russia. Japanese encephalitis (JE) virus, which is closely related to WN virus, is prevalent throughout East Asia. We evaluated the effectiveness of a focus reduction neutralization test in young chicks inoculated with JE and WN viruses, and conducted a survey of WN infection among wild birds in Far Eastern Russia. Following single virus infection, only neutralizing antibodies specific to the homologous virus were detected in chicks. The neutralization test was then applied to serum samples from 145 wild birds for WN and JE virus. Twenty-one samples were positive for neutralizing antibodies to WN. These results suggest that WN virus is prevalent among wild birds in the Far Eastern region of Russia.

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: Effects of WN virus NS1 antibodies induced by inactivated WN vaccine

To the Editor: West Nile virus (WNV) is an arthropod-borne virus that is transmitted to humans by mosquitos, primarily of the genus Culex. Most human infections are asymptomatic. Clinical symptoms occur in ≈20% of case-patients and include fever, headache, and myalgia; <1% of WNV infections develop into severe neuroinvasive disease (1). The virus was discovered in 1937 in the West Nile district of Uganda. WNV is endemic to parts of Africa, Europe, Asia, and the Middle East, and since its introduction in New York in 1999, in North America. In Eurasia, human WNV infections were first reported in Israel and France during the 1950s–1960s, and the first major outbreak in Romania occurred in 1996 (1). The disease emerged recently in Greece; a large outbreak in 2010 caused neuroinvasive disease in 197 patients, of whom 33 died (2). Since 2010, occasional and local epidemics have been ongoing in Greece, Italy, Romania, Hungary, Spain, and the Balkans (3,4). Clinical diagnosis may be difficult because WNV infections resemble other (arbo)viral diseases. Laboratory diagnosis relies primarily on serologic testing. Reverse transcription PCR (RT-PCR) can be used to detect viral RNA during the acute phase of the disease, but its use is hampered by the patient’s low-level and transient viremia (1). We here describe a confirmed case of WNV encephalitis imported by a traveler returning from Greece. A 73-year-old Belgian woman, who had a medical history of lymphoma, traveled to Kavala city (Macedonia, Greece). On August 14, 2012, she sought treatment at the Kavala General Hospital with a 6-day history of fever, headache, malaise, nausea, confusion, decline of consciousness, and neck stiffness. Results of laboratory testing on admission demonstrated an increased leukocyte count (9,670/µL; 80% neutrophils) and lactate dehydrogenase level (522 IU/L), a low C-reactive protein level (0.7 mg/dL), and hyponatremia (131 mEq/L). Cerebrospinal fluid (CSF) testing showed 90 cells/µL (79% lymphocytes) and glucose and protein levels of 72 and 100.9 mg/dL, respectively. Serum obtained on August 15 was sent to the national reference laboratory at Aristotle University (Thessaloniki, Greece), and IgM against WNV was detected by ELISA (WNV IgM Capture DxSelect and IgG DxSelect; Focus Diagnostics, Cypress, CA, USA). IgG was absent. On the second day of hospitalization, the patient exhibited seizures (speech arrest); she was given phenytoin (1/2 amp 3×/day intravenously). On August 18, the patient was transferred to a private hospital. Further treatment included intravenous fluid, antipyretics, antimicrobial drugs, mannitol, and oxygen. On August 30, she was returned by plane to Belgium. CSF obtained 26 days after symptom onset and serum obtained 29 days after symptom onset were sent to the Institute of Tropical Medicine (Antwerp, Belgium) because of its function as a national reference center for Belgium. IgM and IgG against WNV were detected in both samples by ELISA (Focus Diagnostics) (Table). Immunofluorescence assays on serum revealed IgM against WNV only and IgG against West Nile, dengue, yellow fever, and Japanese encephalitis viruses, with the strongest reaction against WNV (Flavivirus Mosaic 1; Euroimmun, Lubeck, Germany). Real-time RT-PCR (adapted from [5]) on the serum demonstrated a weak positive signal. Repeated RNA extraction and RT-PCR were confirmative (Table). Sequencing of the RT-PCR product confirmed the detection of WNV. Although the product was short (116 bp), it was highly suggestive of WNV, lineage 2. Flemish regional authority in Belgium, national authorities (both in Belgium and Greece), and European health authorities were notified of the imported case of WNV encephalitis. According to the case definition of the European Center for Disease Prevention and Control, Stockholm, Sweden, the patient met the laboratory criteria of having a confirmed case. Table Laboratory results confirming WNV infection of 73-year-old woman, Greece, 2012*† To date, autochthonous WNV infections have not been reported in Belgium, although the presence of the mosquito vector provides a potential risk for transmission (6). This WNV infection was acquired in Greece (a leading travel destination for tourists from Belgium), specifically in the Kavala region, which was highly affected by WNV in 2012. The lineage responsible for the WNV encephalitis was identified as lineage 2, the currently circulating strain in Greece (7). Our report highlights the need for physicians and laboratory staff to be aware of imported WNV infections originating from southeastern Europe, especially Greece and its neighboring countries, where recent and recurrent outbreaks have occurred (3,4). Special attention should be given to immunosuppressed and elderly patients who are at higher risk of acquiring neuroinvasive disease. The 73-year-old patient described here was unconscious when she arrived in Belgium. After a short period of relative improvement (more reactive and cooperative), her condition deteriorated, and she died on November 23, 2012. The detection of viral RNA 29 days after symptom onset was surprising but might be explained by the immunocompromised status of the patient. Several studies have reported persistent WNV RNA for 30 days, 77 days, and even years after the symptom onset in serum, CSF, and urine, respectively (8–10), and a prolonged period of viremia in immunocompromised patients (9).

Rôle des oiseaux migrateurs dans l'épidémiologie du virus West Nile

1. Kendra Williams, MS student 1. Medical and Research Technology Department University of Maryland, Baltimore MD The Winter 2003 Clinical Laboratory Science published article on West Nile Virus (WNV): An Emerging Virus in North America , was very informative, organized, and well written. Although the article clearly documents that the transmission of WNV to human occurs most commonly through the bite of an infected mosquito, I would like to comment that there have been a few reported cases whereby other modes of transmission have occurred. There have been reports of WNV transmission to other patients due to blood product transfusions such as red blood cell, plasma, and platelet transfusion. This poses some complications due to the fact that the majority of individuals infected with WNV will show no symptoms.1 It has been estimated that only about 1% of those infected with the virus will develop a severe form of the disease such as West Nile (WN) meningitis, WN encephalitis, or meningoencephalitis.2 Thus it was recommended that all potential blood donors should be screen for the virus.1 WNV meningoencephalitis was also diagnosed in a woman after receiving post-partum blood transfusion from a donor infected with WNV. Breast milk sample taken from her also showed WNV specific IgG and IgM antibodies. The newborn that was reported to have little outdoor exposure was also positive for WNV specific IgM antibodies. Additionally, a previously pregnant woman was admitted to the hospital with symptoms common to WNV meningoencephalitis. Serum and cerebral spinal fluid samples taken from her showed evidence of WNV specific IgM antibodies. The infant who was later delivered also showed…

Since the introduction into North America in 1999 of West Nile virus (WNV) and its subsequent dramatic spread across the United States and Canada, human cases of WNV infection declined from their 2003 peak of 9,862 to only 712 cases and 43 fatalities confirmed in 2011 (http://www.cdc .gov/ncidod/dvbid/westnile/index.htm). The reasons for this decline remain unclear because avian amplification host and human herd immunity remains low in most regions and WNV continues to circulate in most areas of the United States. However, last year an unexpected resurgence in West Nile fever and encephalitis cases occurred; a total of 5,674 human cases was detected in the United States, with 286 deaths, making 2012 the deadliest year yet. The epidemic was focused in the central part of the United States, with Texas reporting 1,739 human infections and 89 deaths, or about 33% of the national total and more than three times the number of any other state. Within Texas, Dallas County was the epicenter, with 398 human infections and 20 deaths. The reasons for this dramatic resurgence in WNV infections are not clear; however, several factors could be involved ranging from increased enzootic bird-mosquito-bird circulation, resulting in more human spillover infections, to increased human virulence. In this issue, Duggal and others report complete genomic sequences of 17 WNV isolates from several regions of Texas and phylogenetic studies to determine if viral genetic changes might have been responsible for the resurgence in human cases. Two distinct WNV genotypes were identified, and both circulated in the Dallas and Houston regions where disease incidence differed markedly (about four times higher in Dallas). Importantly, no signs of adaptive evolution that could have resulted in more efficient infection of mosquitoes or birds were identified in any of the recent WNV strains. These results, along with epidemiological analyses, suggest that the 2012 resurgence in the Dallas area resulted from an increase in transmission by mosquitoes from birds to humans (spillover infections), rather than an increase in WNV neuroinvasiveness. Unusually high summer temperatures in the Dallas area, perhaps combined with ecological changes affecting mosquito or avian populations, probably played important roles in driving the 2012 outbreak. These critical findings presented by Duggal and others 1 and the magnitude of the 2012 West Nile encephalitis epidemic underscore the importance of a better understanding of how WNV, other arboviruses, and vector-borne diseases in general will respond to climate and anthropogenic ecological changes that now appear inevitable. Rising temperatures exert a wide variety of complex effects on mosquito survival, reproduction, and behavior. Because temperature also influences vector infection, dissemination, and transmission of arboviruses, often in unpredictable ways, we remain unable to anticipate the effects of global warming on arboviral disease. Droughts, which are predicted to increase in some regions of the United States and elsewhere, can also have dramatic impacts on mosquito populations, especially on West Nile mosquito vectors because of their affinity for standing, highly polluted water and to irrigated agricultural lands. Drought conditions can also concentrate avian amplifying hosts in wetter locations amenable to mosquito transmission of WNV. Thus, rigorous experimental and modeling studies are needed to improve the ability of our public health systems to anticipate and respond to the effects of climate change and its influences on vector-borne pathogens such as WNV.

Complement Protein C1q Inhibits Antibody-Dependent Enhancement of Flavivirus Infection in an IgG Subclass-Specific Manner

West Nile virus

In this review, we discuss the possibility that the glycosylation of West Nile (WN) virus E-protein may be associated with enhanced pathogenicity and higher replication of WN virus. The results indicate that E-protein glycosylation allows the virus to multiply in a heat-stable manner and therefore, has a critical role in enhanced viremic levels and virulence of WN virus in young-chick infection model. The effect of the glycosylation of the E protein on the pathogenicity of WN virus in young chicks was further investigated. The results indicate that glycosylation of the WN virus E protein is important for viral multiplication in peripheral organs and that it is associated with the strong pathogenicity of WN virus in birds. The micro-focus reduction neutralization test (FRNT) in which a large number of serum samples can be handled at once with a small volume (15 μL) of serum was useful for differential diagnosis between Japanese encephalitis and WN virus infections in infected chicks. Serological investigation was performed among wild birds in the Far Eastern region of Russia using the FRNT. Antibodies specific to WN virus were detected in 21 samples of resident and migratory birds out of 145 wild bird samples in the region.