BAFF Produced by Neutrophils and Dendritic Cells Is Regulated Differently and Has Distinct Roles in Antibody Responses and Protective Immunity against West Nile Virus.

B cell activating factor (BAFF) is essential for B cells to develop and respond to Ags. Dysregulation of BAFF contributes to the development of some autoimmune diseases and malignancies. Little is known about when, where, and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses. To better understand BAFF functions, we created BAFF reporter (BAFF-RFP) mice and Baff floxed (Bafffl/fl ) mice. Splenic and bone marrow neutrophils (Nphs) from BAFF-RFP mice expressed the highest constitutive levels of BAFF; other myeloid subsets, including conventional dendritic cells (cDCs) and monocyte (MO) subsets, expressed lower levels. Treatment of BAFF-RFP mice with polyinosinic:polycytidylic acid increased BAFF expression in splenic Ly6Chi inflammatory MOs, CD11bhi activated NK subset, and in bone marrow myeloid precursors. Postinfection with West Nile virus (WNV), BAFF increased in CD8- cDCs and Nphs, and BAFF+ CD11bhi NK cells expanded in draining lymph nodes. The cell- and tissue-specific increases in BAFF expression were dependent on type I IFN signaling. MAVS also was required or contributed to BAFF expression in dendritic cell and MO subsets, respectively. Mice with deletion of Baff in either cDCs or Nphs had reduced Ab responses after NP-Ficoll immunization; thus, BAFF produced by both cDCs and Nphs contributes to T cell-independent Ab responses. Conversely, mice with a cDC Baff deficiency had increased mortality after WNV infection and decreased WNV-specific IgG and neutralizing Ab responses. BAFF produced by Nphs and cDCs is regulated differently and has key roles in Ab responses and protective immunity.

Modeling the West Nile virus transfusion transmission risk in a nonoutbreak country associated with traveling donors.

BACKGROUND: West Nile virus (WNV) is a mosquito-borne virus and transfusion transmission (TT) has been demonstrated. The European Union and neighboring countries experience an annual transmission season. STUDY DESIGN AND METHODS: We developed a novel probabilistic model to estimate the WNV TT risk in Australia attributable to returned donors who had travelled to the European Union and neighboring countries during the 2018. We estimated weekly WNV TT risks in Australia for each outbreak country and the cumulative risk for all countries. RESULTS: Highest mean weekly TT risk in Australia attributable to donors returning from a specific outbreak country was 1 in 23.3 million (plausible range, 16.8-41.9 million) donations during Week 39 in Croatia. Highest mean weekly cumulative TT risk was 1 in 8.5 million donations (plausible range, 5.1-17.8 million) during Week 35. CONCLUSIONS: The estimated TT risk in Australia attributable to returning donors from the European Union and neighboring countries in 2018 was very small, and additional risk mitigation strategies were not indicated. In the context of such low TT risks, a simpler but effective approach would be to monitor the number of weekly reported West Nile fever cases and implement risk modeling only when the reported cases reached a predefined number or trigger point.

West Nile virus activity in United States blood donors and optimizing detection strategies: 2014-2018.

BACKGROUND: Rare transfusion-transmitted West Nile virus (WNV) cases usually occur due to gaps in testing involving converting to more sensitive nucleic acid testing (NAT) formats (referred to as triggering). Using data from 2014 to 2018, we investigated a strategy used to increase detection early in the triggering period and reviewed its yield as the individual donation (ID)-NAT geographic area was decreased. METHODS: Mini-pool-NAT transitioned to ID-NAT following triggering based on one WNV NAT-reactive donation (having an elevated signal, repeat reactive, or in an area with WNV ongoing activity). ID-NAT-triggered geographic areas included an entire state (2014-2017) or collections within a 50-mile radius of the triggering donor's residential zip code (2018). During the MP- to ID-NAT transition, donation samples were retrieved and tested by ID-NAT for those with results not yet released (referred to as in-process testing). Reactive sample confirmation was performed by repeat NAT of an independent sample or antibody testing. RESULTS: ID-NAT included 3.2 million donations of more than 25 million tested year-round, resulting in 684 confirmed positives; all confirmed-positive donations occurred from June to December (0.64/10,000). Overall, 52% (358/684) required ID-NAT for detection, including 68 (19%) antibody negatives. Ten of 19 (53%) identified in-process were ID-NAT-only detectable, including four antibody negatives, or approximately 1 per year (2.8% of ID-NAT-only detectable). With reduced triggering geography, 12 of 19 (63%) were not identified (including 6/10 ID-NAT-only detectable, and 2/4 ID-NAT-only detectable/antibody negative). CONCLUSION: WNV NAT's utility is between June-December; however, abandoning testing outside of this time may increase risk. While in-process testing identified approximately one ID-NAT-only detectable (antibody-negative) donation per year, reducing the geographic triggered area decreased its effectiveness.

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests.

BACKGROUND: West Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)-reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination. CASE SERIES: Cases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine. STUDY DESIGN AND METHODS: WNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV-reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed. RESULTS: Supplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT-positive, WNV NAT-negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT. CONCLUSIONS: We recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

Serological survey for Saint Louis encephalitis virus and West Nile virus in domestic mammals in Córdoba, Argentina: are our pets potential sentinels?

We evaluated the seroprevalence of Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) in dogs and cats in Córdoba, Argentina. Monotypic and heterotypic serological patterns were differentiated by means of a neutralization test. The SLEV seroprevalence in dogs was 14.6% (44/302; 100% monotypic). Two out of 94 (2.1%, 100% monotypic) cats were positive for WNV only. Four dogs (1.3%) exhibited neutralizing antibody titers against SLEV and WNV. During the study, three dogs seroconverted to SLEV. Our study demonstrates that pets were useful for detecting viral activity and could be considered as sentinels in the local surveillance of SLEV and WNV.

Fatal case of West Nile encephalitis associated with acute anteroseptal ST elevation myocardial infarction (STEMI): a case report.

Cardiac involvement has rarely been reported in West Nile (WNV) infection. We report a fatal case of WNV encephalitis associated with an acute anteroseptal ST elevation myocardial infarction. The patient was hospitalized with a fever, headache, nausea and vomiting. The physical examination revealed positive meningeal signs and an altered level of consciousness. High levels of cardiac enzymes (creatine phosphokinase/MB fraction, lactate dehydrogenase, myoglobin and cardiac troponin I) and ST elevation on electrocardiogram were found. Both CSF and urine samples were positive for WNV RNA. This case highlights the need of awareness of the possibility of a WNV-related myocardial infection, including myocardial infarction.

Evaluation of a new West Nile virus nucleic acid test for screening of blood donations.

BACKGROUND: West Nile virus (WNV) is transmitted to humans through mosquito bites and can be further transmitted to humans through transfusion or transplantation. Because most infected individuals are asymptomatic, blood donor screening is important in areas where WNV is endemic. These studies evaluated the performance of a new test for detection of WNV RNA in blood donations. STUDY DESIGN AND METHODS: Analytical performance evaluation included sensitivity, specificity, inclusivity, and correlation. A clinical specificity study was conducted at four blood donor testing laboratories in parallel with the cobas TaqScreen WNV Test (Roche Molecular Systems, Inc.). RESULTS: The 95% and 50% limit of detection for cobas WNV was 12.9 copies/mL (95% confidence interval [CI], 10.8-16.3) and 2.1 copies/mL (95% CI, 1.9-2.4) for WNV lineage 1, respectively, and 6.2 copies/mL (95% CI, 4.8-8.9) and 1.1 copies/mL (95% CI, 0.8-1.3) for WNV lineage 2, respectively. Clinical specificity was 100% in 10,823 donor samples tested individually (95% CI, 99.966%-100%) and 63,243 tested in pools of 6 (95% CI, 99.994%-100%). Samples of other members of the Japanese encephalitis virus serocomplex, including St Louis encephalitis, Japanese encephalitis, Murray Valley encephalitis, Usutu, and Kunjin viruses were detected by cobas WNV. CONCLUSION: The cobas WNV test for use on the cobas 6800/8800 System, a fully automated test system, demonstrated high sensitivity and specificity and is suitable for the detection of WNV in blood donors.

Usutu virus infection in Dutch blood donors.

BACKGROUND: The screening of Dutch blood donations for West Nile virus (WNV) may be imminent, as WNV emerges in nearby countries and more donors travel to WNV-affected regions. Since 2016 the related, mosquito-borne Usutu virus (USUV) causes seasonal mortality in Dutch birds. To what extent will human USUV infections affect Dutch WNV donor screening? STUDY DESIGN AND METHODS: From April through September 2018, plasma samples from blood donations in blackbird-rich regions were stored. When increased bird mortality was reported in August, samples from July, August, and September were tested for USUV-RNA in pools of eight, using a home-brew combined WNV/USUV-PCR assay. Reactive pools were deconstructed. Original plasma units and samples of previous and follow-up donations of reactive donors were tested for USUV- and WNV-RNA, and for antibody responses. RESULTS: The number of USUV RNA-positive, WNV RNA-negative donations was 0 of 2688 donations in July, 6 of 4416 in August (1:736), and 1 of 4936 in September. The seven infected donors tested negative for USUV-RNA in preceding and follow-up donations. For 6 donors, seroconversion for USUV-antibodies was demonstrated. All index donations tested positive in a commonly used PCR-assay for WNV donor screening. Three exposed recipients did not show signs of infection. Screening a random subset of 1092 donations from September for USUV-IgG antibodies showed that 22 donors tested reactive; for three donors retrospective testing identified an USUV PCR-positive pre-seroconversion donation. CONCLUSION: Seasonal USUV infection in Dutch blood donors is common. Cross-reactivity in molecular assays for WNV-screening occurs, but can be resolved using USUV- and WNV-specific PCR-primers and sequencing of viral RNA.

Who do we gain? Enhancement of blood supplies by additional testing for donors who travel.

AIMS/OBJECTIVES: To describe the impact of additional testing on the England blood supply. BACKGROUND: The blood service for England, NHS Blood and Transplant, applies a system of deferral and testing to donors with potential exposure to Chagas disease, malaria and West Nile virus; however, testing costs must be justified. Here, we describe the donations and donors gained by testing. METHODS: Donation testing results and demographic data on donors in England where additional testing was applied were analysed in 2012-2016. The total number and proportion of donations tested, reactive and confirmed positive were calculated. Proportions of donors requiring additional tests were calculated by ethnic group for first-time and repeat donors. RESULTS: Additional testing for travel was applied to 3·5% of NHSBT blood donations between 2012 and 2016. Over 98% of these tests were non-reactive. Only malaria tests were confirmed positive, in 1·7% of donations tested. In first-time donors, 45 and 40% of Asian and Black donors required an additional test, respectively, mainly for malaria. Testing for West Nile virus increased from 1·5% in 2012 to 2·2% of donations in 2016. CONCLUSION: The majority of additional tests were screened negative, allowing approximately 64 000 donations to be released for issue annually. Donors most affected by malaria testing were more likely to have rare blood groups and be targeted for recruitment, whereas those given West Nile virus testing were mainly regular donors required for continuity of supply. These data show differences in the characteristics of donors by test and can be used to inform decisions about additional testing and deferrals.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus.

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

One Health approach for West Nile virus surveillance in the European Union: relevance of equine data for blood safety.

West Nile virus (WNV) infection is notifiable in humans and equids in the European Union (EU). An area where a human case is detected is considered affected until the end of the mosquito transmission season (week 48) and blood safety measures have to be implemented. We used human and equine case notifications between 2013 and 2017 to define the WNV distribution in the EU and to investigate the relevance of using equine cases as a complementary trigger for blood safety measures. Adding areas with equine cases to the definition of an affected area would have a major impact on blood safety measures. Adding areas with equine cases where human cases have been reported in the past would increase the timeliness of blood safety measures with only a limited impact. Although the occurrence of human and/or equine cases confirms virus circulation in the EU, no evidence was found that occurrence of equine cases leads to human cases and vice versa. We conclude that information about equine data should contribute to raising awareness among public health experts and trigger enhanced surveillance. Further studies are required before extending the definition of affected areas to areas with human and/or equine cases.

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.

West Nile Virus Seroprevalence, Connecticut, USA, 2000-2014.

West Nile virus (WNV) infection is mainly asymptomatic but can be severe in elderly persons. As part of studies on immunity and aging in Connecticut, USA, we detected WNV seroconversion in 8.5% of nonimmunosuppressed and 16.8% of immunosuppressed persons. Age was not a significant seroconversion factor. Our findings suggest that immune factors affect seroconversion.

A probable case of West Nile virus transfusion transmission.

BACKGROUND: Transfusion-transmitted West Nile virus (WNV) infection is infrequent as a result of minipool (MP) and individual-donation (ID) nucleic acid testing (NAT) of blood donations. ID-NAT is triggered on the basis of local WNV activity identified by MP-NAT. STUDY DESIGN AND METHODS: A 78-year-old male patient who was treated for cardiac disease received 14 blood components from 30 donors in August 2016. He was discharged 7 days after aortic valve replacement and coronary bypass surgery, but was re-admitted on Day 12 with symptoms of viral infection, and eventually was diagnosed with aseptic meningitis. The patent died on Day 51. RESULTS: The patient was positive for WNV-immunoglobulin M (IgM) antibodies in his cerebrospinal fluid on Day 14 and was positive for WNV-IgM (on Days 14 and 16) and WNV-IgG antibodies (on Day 16) in his serum. All associated donations were nonreactive by MP-NAT or ID-NAT. However, one MP-NAT was noted to have an elevated (but negative) signal-to-cutoff ratio, and one donor from that MP was subsequently found positive for WNV-IgM and IgG antibodies; the donor was diagnosed with a WNV-like viral syndrome that had an onset 3 to 5 days postdonation. The donor's plasma was transfused 12 days before the patient's onset of WNV-meningoencephalitis. Conversion to ID-NAT was triggered for the region 7 days after the implicated donation, which was associated with the region's first human WNV case. CONCLUSION: Despite the possibility of mosquito-borne transmission, this was considered to be a case of transfusion-transmitted WNV infection from an MP-NAT-nonreactive donation collected just before triggering conversion to ID-NAT; a rare event recognized once in 84 million donations.

Characterizing environmental risk factors for West Nile virus in Quebec, Canada, using clinical data in humans and serology in pet dogs.

The identification of specific environments sustaining emerging arbovirus amplification and transmission to humans is a key component of public health intervention planning. This study aimed at identifying environmental factors associated with West Nile virus (WNV) infections in southern Quebec, Canada, by modelling and jointly interpreting aggregated clinical data in humans and serological data in pet dogs. Environmental risk factors were estimated in humans by negative binomial regression based on a dataset of 191 human WNV clinical cases reported in the study area between 2011 and 2014. Risk factors for infection in dogs were evaluated by logistic and negative binomial models based on a dataset including WNV serological results from 1442 dogs sampled from the same geographical area in 2013. Forested lands were identified as low-risk environments in humans. Agricultural lands represented higher risk environments for dogs. Environments identified as impacting risk in the current study were somewhat different from those identified in other studies conducted in north-eastern USA, which reported higher risk in suburban environments. In the context of the current study, combining human and animal data allowed a more comprehensive and possibly a more accurate view of environmental WNV risk factors to be obtained than by studying aggregated human data alone.

Incidence of West Nile virus infection in the Dallas-Fort Worth metropolitan area during the 2012 epidemic.

The 2012 West Nile virus (WNV) epidemic was the largest since 2003 and the North Texas region was the most heavily impacted. We conducted a serosurvey of blood donors from four counties in the Dallas-Fort Worth area to characterize the epidemic. Blood donor specimens collected in November 2012 were tested for WNV-specific antibodies. Donors positive for WNV-specific IgG, IgM, and neutralizing antibodies were considered to have been infected in 2012. This number was adjusted using a multi-step process that accounted for timing of IgM seroreversion determined from previous longitudinal studies of WNV-infected donors. Of 4971 donations screened, 139 (2·8%) were confirmed WNV IgG positive, and 69 (1·4%) had IgM indicating infection in 2012. After adjusting for timing of sampling and potential seroreversion, we estimated that 1·8% [95% confidence interval (CI) 1·5-2·2] of the adult population in the Dallas-Fort Worth area were infected during 2012. The resulting overall estimate for the ratio of infections to reported WNV neuroinvasive disease (WNND) cases was 238:1 (95% CI 192-290), with significantly increased risk of WNND in older age groups. These findings were very similar to previous estimates of infections per WNND case, indicating no change in virulence as WNV evolved into an endemic infection in the United States.

Seroprevalence of Japanese encephalitis virus & West Nile virus in Alappuzha district, Kerala.

BACKGROUND & OBJECTIVES: Several outbreaks of acute encephalitis syndrome (AES) have been reported in Alappuzha district, Kerala State, India, in the past. The aetiology of these outbreaks was either inconclusive or concluded as probable Japanese encephalitis virus (JEV) infection based on clinical presentation. The role of West Nile virus (WNV) in AES outbreaks was also determined. However, the extent of WNV infection has not been studied in this region previously. A population-based cross-sectional serosurvey study was undertaken to determine the seroprevalence of JEV and WNV in Alappuzha district. METHODS: A total of 30 clusters were identified from 12 blocks and five municipalities as per the probability proportional to size sampling method. A total of 1125 samples were collected from all age groups. A microneutralization assay was performed to estimate the prevalence of JEV and WNV neutralizing antibodies in the sample population. RESULTS: Of 1125 serum samples tested, 235 [21.5%, 95% confidence interval (CI): 15.2-27.8%] and 179 (15.9%, 95% CI: 9.6-22.3%) were positive for neutralizing antibodies against WNV and JEV, respectively. In addition, 411 (34.5%, 95% CI: 26.7-42.2%) were positive for cross-reactive antibodies against flaviviruses. INTERPRETATION & CONCLUSIONS: The study showed the seroprevalence of WNV and JEV antibodies in the surveyed area and the WNV seroprevalence was greater than JEV. It is necessary to create awareness in public and adopt suitable policy to control these diseases.

LOW PREVALENCE OF WEST NILE VIRUS ANTIBODIES IN SELECT NORTHERN CALIFORNIA OWL SPECIES (2007-2014).

The objective of this study was to determine evidence of previous West Nile virus (WNV) infection in northern California owls. Owl serum samples were collected from birds presenting to a veterinary medical teaching hospital between 2007 and 2014 and were screened for the presence of WNV antibodies by an indirect enzyme immunoassay (EIA). Only one of 71 samples (1.41%) tested was positive by EIA and confirmed by a plaque reduction neutralization test; it was the most recent sample collected. The reason for the low prevalence of WNV in these California owls despite a high prevalence in sympatric avian species in the same region is unknown and should be a topic for further research.

[Detection of West Nile virus in human samples: follow-up studies during the 2015 seasonal period]. / A nyugat-nílusi vírus kimutatása humán betegmintákból: nyomon követéses vizsgálatok a 2015. évi szezonális idoszakban.

INTRODUCTION: West Nile virus, a mosquito-borne viral zoonosis is responsible for human infections in Hungary. Laboratory diagnosis is based on serological tests, however the application of molecular methods has been appreciated. AIM: The aim of the study was to investigate blood, cerebrospinal-fluid and urine samples of acutely ill patients and to follow-up PCR positive cases to ascertain the length of virus excretion. METHOD: Clinical specimens were examined by indirect-immunofluorescent, haemagglutination-inhibition, two PCR tests and Sanger-sequencing. Virus isolation in case of two patients was successful. RESULTS: A follow-up study could be carried out in case of 5 patients. Viral nucleic acid was detectable in urine even for several weeks after symptom onset and viral RNA was present at higher concentration compared with other samples. CONCLUSIONS: PCR analysis of urine could provide useful epidemiological and diagnostic information. Therefore, it is recommended to collect urine samples in order to supplement the serological diagnosis. Orv Hetil. 2017; 158(20): 791-796.

Critical assessment of automated flow cytometry data analysis techniques.

Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.