Trypanosoma cruzi in Nonischemic Cardiomyopathy Patients, Houston, Texas, USA.

To investigate possible cardiac manifestations of Chagas disease, we tested 97 Latinx patients with nonischemic cardiomyopathy in Houston, Texas, USA, for Trypanosoma cruzi infection. We noted a high prevalence of underdiagnosed infection and discrepant results in clinical diagnostic assays. Latinx cardiac patients in the United States would benefit from laboratory screening for T. cruzi infection.

Phenotypic and Genotypic Characterization of West Nile Virus Isolate 2004Hou3.

West Nile virus (WNV) is an arbovirus with important public health implications globally. This study characterizes a viral isolate, 2004Hou3, in comparison with the NY99 strain from the original WNV outbreak in New York, USA. NextGen sequencing was used to compare the viral isolates genetically, while wild-type C57/BL6 mice were used to compare pathogenicity and viral persistence. Significant differences in survival and clinical presentations were noted, with minor genetic variations between the two strains potentially offering an explanation. One notable difference is that 5 of 35 mice infected with the 2004Hou3 strain developed hind limb flaccid paralysis, suggesting its possible use as a small animal pathogenesis model for this clinical characteristic often observed in human WN neuroinvasive disease patients but not reported in other animal models of infection. Overall, this study suggests that 2004Hou3 is a less pathogenic strain with potential for use in long-term outcome studies using small animal models.

Optimizing PCR Detection of West Nile Virus from Body Fluid Specimens to Delineate Natural History in an Infected Human Cohort.

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.

Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge.

The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans.IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

Timing of gestational exposure to Zika virus is associated with postnatal growth restriction in a murine model.

BACKGROUND: Vertical transmission of Zika virus leads to infection of neuroprogenitor cells and destruction of brain parenchyma. Recent evidence suggests that the timing of infection as well as host factors may affect vertical transmission. As a result, congenital Zika virus infection may only become clinically apparent in the postnatal period. OBJECTIVE: We sought to develop an outbred mouse model of Zika virus vertical transmission to determine if the timing of gestational Zika virus exposure yields phenotypic differences at birth and through adolescence. We hypothesized that later gestational inoculations would only become apparent in adolescence. STUDY DESIGN: To better recapitulate human exposures, timed pregnant Swiss-Webster dams (n = 15) were subcutaneously inoculated with 1 × 104 plaque-forming units of first passage contemporary Zika virus HN16 strain or a mock injection on embryonic day 4, 8, or 12 with bioactive antiinterferon alpha receptor antibody administered in days preceding and proceeding inoculation. The antibody was given to prevent the robust type I interferon signaling cascade that make mice inherently resistant to Zika virus infection. At birth and adolescence (6 weeks of age) offspring were assessed for growth, brain weight, and biparietal head diameters, and Zika virus viral levels by reverse transcription-polymerase chain reaction or in situ hybridization. RESULTS: Pups of Zika virus-infected dams infected at embryonic days 4 and 8 but not 12 were growth restricted (P < .003). Brain weights were significantly smaller at birth (P = .01) for embryonic day 8 Zika virus-exposed offspring. At 6 weeks of age, biparietal diameters were smaller for all Zika virus-exposed males and females (P < .05), with embryonic day 8-exposed males smallest by biparietal diameter and growth-restriction measurements (weight >2 SD, P = .0007). All pups and adolescent mice were assessed for Zika virus infection by reverse transcription-polymerase chain reaction. Analysis of all underweight pups reveled 1 to be positive for neuronal Zika virus infection by in situ hybridization, while a second moribund animal was diffusely positive at 8 days of age by Zika virus infectivity throughout the brain, kidneys, and intestine. CONCLUSION: These findings demonstrate that postnatal effects of infection occurring at single time points continue to be detrimental to offspring in the postnatal period in a subset of littermates and subject to a window of gestational susceptibility coinciding with placentation. This model recapitulates frequently encountered clinical scenarios in nonendemic regions, including the majority of the United States, where travel-related exposure occurs in short and well-defined windows of gestation. Our low rate of infection and relatively rare evidence of congenital Zika syndrome parallels human population-based data.

Prolonged Detection of Zika Virus in Vaginal Secretions and Whole Blood.

Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies.

Likely Autochthonous Transmission of Trypanosoma cruzi to Humans, South Central Texas, USA.

Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease affecting the Americas. The epidemiology of this disease in the United States is incomplete. We report evidence of likely autochthonous vectorborne transmission of T. cruzi and health outcomes in T. cruzi-seropositive blood donors in south central Texas, USA.

Eilat virus host range restriction is present at multiple levels of the virus life cycle.

UNLABELLED: Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described, and in contrast to all other mosquito-borne viruses, it is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis virus (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3' untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV nonstructural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both nonstructural protein (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses. IMPORTANCE: Our work explores the nature of host range restriction of the first "mosquito-only alphavirus," EILV. EILV is related to pathogenic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezuelan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission.

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.

Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication.

Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.

Chikungunya vaccine candidate is highly attenuated and protects nonhuman primates against telemetrically monitored disease following a single dose.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes major epidemics of rash, fever, and debilitating arthritis. Currently, there are no vaccines or antivirals available for prevention or treatment. We therefore generated 2 live-attenuated vaccine candidates based on the insertion of a picornavirus internal ribosome entry site (IRES) sequence into the genome of CHIKV. Vaccination of cynomolgus macaques with a single dose of either vaccine produced no signs of disease but was highly immunogenic. After challenge with a subcutaneous inoculation of wild-type CHIKV, both vaccine candidates prevented the development of detectable viremia. Protected animals also exhibited no significant changes in core body temperature or cardiovascular rhythm, whereas sham-vaccinated animals showed hyperthermia, followed by sustained hypothermia, as well as significant changes in heart rate. These CHIKV/IRES vaccine candidates appear to be safe and efficacious, supporting their strong potential as human vaccines to protect against CHIKV infection and reduce transmission and further spread.

Generation of an infectious Negev virus cDNA clone.

The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus.

Negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution.

Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ∼45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 10(10) PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.

Attenuation of Chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein.

Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism.

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.

Prevalence of Trypanosoma cruzi, the Etiologic Agent of Chagas Disease, Infection in Texas Skunks (Mammalia: Mephitidae).

Background: Chagas disease is one of the world's most neglected tropical diseases, infecting over six million people across the Americas. The hemoparasite Trypanosoma cruzi is the etiological agent for the disease, circulating in domestic, peridomestic, and sylvatic transmission cycles that are maintained by triatomine vectors and a diversity of wild and synanthropic hosts. Public health and wildlife management interventions targeting the interruption of T. cruzi transmission rely on an understanding of the dynamics driving the ecology of this zoonotic pathogen. One wildlife host that purportedly plays a role in the transmission of Chagas disease within the southern United States is the striped skunk (Mephitis mephitis), although infection prevalence in this species is poorly understood. Materials and Methods: To this end, we conducted a PCR-based surveillance of T. cruzi in 235 wild skunks, representing 4 species, across 76 counties and 10 ecoregions in Texas, United States, along with an evaluation of risk factors associated with the infection. Results: We recovered an overall T. cruzi prevalence of 17.9% for all mephitid taxa aggregated, ranging between 6.7% for plains spotted skunks (Spilogale putorius interrupta) and 42.9% for western spotted skunks (Spilogale gracilis). We report the first cases of T. cruzi infection in plains spotted and American hog-nosed skunks (Conepatus leuconotus), of important note for conservation medicine since populations of both species are declining within Texas. Although not statistically significant, we also detected trends for juveniles to exhibit greater infection risk than adults and for differential sex biases in T. cruzi prevalence between taxa, which align with variations in species-specific seasonal activity patterns. No geographic or taxonomic risk factors were identified. Conclusion: Our study contributed key data for population viability analyses and epidemiologic models in addition to providing a baseline for future T. cruzi surveillance among skunks and other wildlife species.

An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing.

The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.

Functional Sindbis virus replicative complexes are formed at the plasma membrane.

Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However, our recent studies have demonstrated that Sindbis virus (SINV)-specific, double-stranded RNA (dsRNA)- and nonstructural protein (nsP)-containing RCs are initially formed at the plasma membrane. In this new study, we present extensive evidence that (i) in cells of vertebrate origin, at early times postinfection, viral nsPs colocalize with spherules at the plasma membrane; (ii) viral dsRNA intermediates are packed into membrane spherules and are located in their cavities on the external surface of the plasma membrane; (iii) formation of the membrane spherules is induced by the partially processed nonstructural polyprotein P123 and nsP4, but synthesis of dsRNA is an essential prerequisite of their formation; (iv) plasma membrane-associated dsRNA and protein structures are the active sites of single-stranded RNA (ssRNA) synthesis; (v) at late times postinfection, only a small fraction of SINV nsP-containing complexes are relocalized into the cytoplasm on the endosome membrane. (vi) pharmacological drugs inhibiting different endocytotic pathways have either only minor or no negative effects on SINV RNA replication; and (vii) in mosquito cells, at any times postinfection, dsRNA/nsP complexes and spherules are associated with both endosomal/lysosomal and plasma membranes, suggesting that mechanisms of RC formation may differ in cells of insect and vertebrate origins.

Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection.

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen's DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of ß-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

Unique Cytokine Response in West Nile Virus Patients Who Developed Chronic Kidney Disease: A Prospective Cohort Study.

West Nile virus (WNV) is a widespread and devastating disease, especially in those who develop neuroinvasive disease. A growing body of evidence describes sequelae years after infection, including neurological complications and chronic kidney disease (CKD). Eighty-nine out of 373 WNV-positive cases were followed for approximately two years and compared to 127 WNV-negative controls with and without CKD. Adjusted risk ratios (aRRs) were calculated via a log binomial regression to determine the impact of WNV exposure and other possible confounders on the likelihood of developing CKD. Cytokine profiles of WNV patients and controls were evaluated to characterize differences and describe potential underlying pathophysiological mechanisms. The associated risk for developing CKD was significantly associated with history of WNV infection (aRR = 1.91, 95% CI 1.13-3.25). Additionally, five distinct cytokines were found to be significantly associated with WNV infection (eotaxin, IL-8, IL-12p70, IP-10, and TNFα) after the p-value was adjusted to <0.0019 due to the Bonferroni correction. These data support that WNV infection is an independent risk factor for CKD, even after accounting for confounding comorbidities. WNV participants who developed CKD had high activity of proinflammatory markers, indicating underlying inflammatory disease. This study provides new insights into CKD resultant of WNV infection.