West Nile Virus Screening Research Articles

West Nile Virus Seroprevalence in an Outdoor Nonhuman Primate Breeding Colony in South Florida.

West Nile virus (WNV) was first detected in Florida in July 2001, with 404 human cases reported to the Centers for Disease Control and Prevention as of February 2020. The subtropical climate of Florida is ideal for the mosquitoes that transmit WNV. We investigated the WNV seroprevalence in 3 NHP species housed outdoors at The Mannheimer Foundation in South Florida. From January to December 2016, 520 3 to 30 y old NHP were sampled at our 2 closed sites in Homestead and LaBelle: 200 rhesus macaques (Macaca mulatta), 212 cynomolgus macaques (Macaca fascicularis), and 108 hamadryas baboons (Papio hamadryas hamadryas). The presence of WNV IgG antibodies in these animals was determined by serum neutralization assays, which found a total seroprevalence of 14%. Seroprevalence was significantly higher in the baboons (29%) than the rhesus (11%) and cynomolgus (9%) macaques. The probability of seropositivity significantly increased with age, but sex and site did not significantly affect seroprevalence. The frequency of WNV seropositivity detected in these outdoor-housed NHP suggests that screening for WNV and other vector-borne diseases may be necessary prior to experimental use, particularly for infectious disease studies in which viremia or viral antibodies could confound results, and especially for populations housed outdoors in warm, wet climates. As no seropositive subjects demonstrated clinical signs of WNV and WNV exposure did not appear to significantly impact colony health, routine testing is likely unnecessary for most NHP colonies. However, WNV infection should still be considered as a differential diagnosis for any NHP presenting with nonspecific neurologic signs. Mosquito abatement plans and vigilant sanitation practices to further decrease mosquito and avian interaction with research NHP should also be considered.

Donation testing and transfusion transmissible infections

Donation testing and transfusion transmissible infections

West Nile Virus Seroprevalence Among Blood Donors in Hungary.

Background and Objectives: West Nile virus (WNV) is one of the most important viral zoonotic infections in Hungary; however, no transfusion-transmitted WNV infections have been confirmed so far. In 2016, the number of clinical cases of WNV reported was 44, but the seasonal WNV screening of whole-blood donors has not yet been implemented. Our aims were to assess the WNV RNA reactivity and the prevalence of WNV-specific antibodies in the samples of blood donors collected in 2016. Materials and Methods: WNV RNA with Cobas TaqScreen and anti-WNV antibody determination from plasma samples of 2112 donors was performed. Cross-reactivity to tick-borne encephalitis virus was excluded. WNV neutralization test was used for the confirmation of anti-WNV IgG reactive results, and the presence of anti-WNV IgM antibodies was also determined. Results: None of the samples showed WNV RNA reactivity. The total weighted anti-WNV IgG prevalence was 2.34% (95% confidence interval 1.65-3.03), and in addition, three donors were found to be IgM positive. There was a comparable tendency between the data of WNV seroprevalence and cumulative incidence in six out of seven statistical regions in Hungary. Conclusion: Our results show a comparable data with publications that estimated the WNV seroprevalence in some other European endemic areas. As protective measures, both the 30-day deferral of blood donors who spent at least 24 h in WNV-exposed areas and the exclusion of affected Hungarian territories from blood donation are enforced by the Hungarian National Blood Transfusion Service. Our study is the first comprehensive serological survey to obtain actual data about WNV seroprevalence in the Hungarian human population.

West Nile and Usutu Virus Infections and Challenges to Blood Safety in the European Union.

West Nile virus (WNV) and Usutu virus (USUV) circulate in several European Union (EU) countries. The risk of transfusion-transmitted West Nile virus (TT-WNV) has been recognized, and preventive blood safety measures have been implemented. We summarized the applied interventions in the EU countries and assessed the safety of the blood supply by compiling data on WNV positivity among blood donors and on reported TT-WNV cases. The paucity of reported TT-WNV infections and the screening results suggest that blood safety interventions are effective. However, limited circulation of WNV in the EU and presumed underrecognition or underreporting of TT-WNV cases contribute to the present situation. Because of cross-reactivity between genetically related flaviviruses in the automated nucleic acid test systems, USUV-positive blood donations are found during routine WNV screening. The clinical relevance of USUV infection in humans and the risk of USUV to blood safety are unknown.

Proceedings of the Food and Drug Administration public workshop on pathogen reduction technologies for blood safety 2018 (Commentary, p. 3026).

On November 29, 2018, experts in the field of infectious diseases, pathogen reduction technologies (PRTs) and other participants from blood centers, academia, and industry gathered at the Food and Drug Administration (FDA) White Oak Campus in Silver Spring, Maryland, for a 2‐day public workshop entitled “Pathogen Reduction Technologies for Blood Safety.” The workshop opened with welcome remarks from Dr. Nicole Verdun, Director, Office of Blood Research and Review (OBRR), Center for Biologics Evaluation and Research (CBER), FDA, followed by introductory remarks from Dr. Peter Marks, Director, CBER, FDA. The first day of the workshop focused on blood‐borne infectious agents and their impact on blood safety, experiences of the American Red Cross, and other blood establishments in implementing FDA‐approved pathogen inactivation (PI) technology for plasma and platelets (PLTs) in the United States and novel PRTs under consideration for whole blood (WB) and red blood cells (RBCs). The second day opened with welcome remarks from Dr. Chintamani Atreya, Associate Director for Research, OBRR, CBER, FDA. The focus was on emerging innovations relevant to PRTs and potential alternatives to PRTs. The workshop concluded with remarks on insights for future research and development in this area for blood and blood product safety from infectious agents. A brief introduction of each session by the session moderator followed by a summary of the speaker presentation as submitted by the moderator and speaker are reported here.

Expanding Usutu virus circulation in Italy: detection in the Lazio region, central Italy, 2017 to 2018.

Blood donation screening for West Nile virus (WNV) was mandatory in the Lazio region in 2017 and 2018 (June-November) according to the national surveillance plan. In these years, all five donations reactive in WNV nucleic acid amplification tests harboured instead Usutu virus (USUV). Clade ‘Europe 2’ was identified in four blood donations and a 2018 mosquito pool. The cocirculation of WNV and USUV in Lazio warrants increased laboratory support and awareness of possible virus misidentification.

Development of a stable Japanese encephalitis virus replicon cell line for antiviral screening.

Japanese encephalitis virus (JEV), an important pathogen in Eastern and Southern Asia and the Pacific, has spread to Australia and other territories in recent years. Although the vaccine for JEV has been used in some countries, development of efficient antiviral drugs is still an urgent requirement. Replicon systems have been widely used in the research of viral replication and antiviral screening for West Nile virus (WNV), yellow fever virus (YFV) and dengue virus (DENV). Here, a novel JEV replicon harboring the Rluc and Pac gene (JEV-Pac-Rluc-Rep) was constructed. Furthermore, we established a BHK-21 cell line harboring JEV-Pac-Rluc-Rep (BHK-21/PAC/Rluc cell line) through continuous puromycin selection. Characterization of cell line stability showed that the replicon RNA could persistently replicate in this cell line for at least up to 10 rounds of passage. Using a known flavivirus inhibitor, the JEV replicon cell line was validated for antiviral screening. The JEV replicon cell line will be a valuable tool for both compound screening and viral replication studies.

A nationwide seroprevalence screening for West Nile virus and Tick-borne encephalitis virus in the population of Bulgaria.

To assess local circulation and risk for human infections with West Nile virus (WNV) and Tick-borne encephalitis virus (TBEV) in Bulgaria, a nationwide seroprevalence study was conducted. In total, 1451 residents of all 28 districts in Bulgaria were tested for WNV-specific and TBEV-specific IgG antibodies. The survey found overall seroprevalence of 1.5% and 0.6%, respectively. The highest WNV seroprevalence was found in Sofia Province and districts near the river Danube. TBEV circulation was detected among residents of six districts. The results showed that the two virus infections seem to be more wide-spread in the country as has been described.

Implementation of NAT Screening for West Nile Virus and Experience with Seasonal Testing in Germany

Background: West Nile virus (WNV) can be transmitted by transfusion through infected blood components. In Germany, a 28-day deferral for blood donors of therapeutic blood components who had spent at least 2 days in WNV-endemic areas from June 1 to November 30, 2014 was enforced. Otherwise, screening of blood donors for WNV RNA or the application of pathogen reduction techniques are appropriate alternatives. Methods: In the present study, we evaluated NAT screening for the detection of WNV in blood components. A total of 58 minipools consisting of 357 individual blood donors were screened for the presence of WNV RNA employing an automated high-volume extraction method using the RealStar WNV RT-PCR Kit. Additionally, different WNV reference reagents were quantified to prove the status quo of standardization. Four different WNV real-time NAT kits were compared using samples of an external quality assessment panel. Results: The 95% lower detection limit of the WNV MP-NAT was determined to 30.2 copies/ml (95% CI 24.2-45.4 copies/ml). No WNV RNA-positive minipool was detected. Quantification of WNV reference reagents revealed shortcomings in standardization. Comparison of several WNV NAT assays showed considerable differences in assay sensitivities and particularly a missing detection of WNV lineage 2. Implementation of seasonal WNV MP-NAT screening was demonstrated. Conclusion: Actually, WNV infections in Germany are rare events introduced by returning travelers, but surveillance of these emerging infections is important for safety in blood supply. The validation study pointed out the need for standardization of WNV NAT because of current lack of an international standard for WNV RNA.

Current practices and evaluation of screening solid organ donors for West Nile virus

The first cases of West Nile virus (WNV) transmitted through solid organ transplantation (SOT) were identified in 2002. Subsequently, 5 additional clusters have been reported to public health officials in the United States. Based upon a limited number of known cases, patients who acquire WNV from infected donor organs might be at higher risk for severe neurologic disease and death, compared with patients infected through mosquito bites. In response, some organ procurement organizations (OPOs) have instituted pre-transplant screening of organ donors for WNV infection. We evaluated the current practices, concerns, and challenges related to screening organ donors for WNV in the United States by reviewing the relevant medical literature and interviewing key stakeholders. Screening organ donors for WNV is not required by national policy. In 2008, 11 (19%) of 58 OPOs performed WNV screening using nucleic acid amplification testing (NAT). These OPOs differ in their screening strategies, NAT performed, and logistical challenges. Concerns of delays in receiving NAT results before transplant and potential false-positive results leading to organ wasting are limitations to more widespread screening. Furthermore, it is unknown if WNV screening practices decrease SOT-related morbidity and mortality, or if screening is cost-effective. Additional data are needed to assess and improve transplant outcomes related to WNV.

West Nile virus transmission through organ transplantation in north-eastern Italy: a case report and implications for pre-procurement screening

West Nile virus (WNV) transmission through organ transplantation occurs rarely and screening of organ donors for WNV infection remains controversial. This report describes the case of WNV encephalitis in a kidney recipient and the case of asymptomatic WNV infection in the organ donor, both observed at Treviso Hospital, northeastern Italy. After briefly reviewing the literature, we discuss the implications for WNV screening. We reviewed medical, laboratory and epidemiological records at our hospital, and the literature concerning cases of organ-transmitted WNV infections and WNV screening of organ donors in Italy and worldwide. The kidney recipient was the first confirmed case of WNV infection notified in northeastern Italy in 2011, and the first case of WNV infection in a cluster of four transplant recipients who acquired the infection from a common organ donor. The organ donor, whose WNV infection was only retrospectively diagnosed by IgM detection, represents the index case of a WNV outbreak in the Treviso Province. Screening of her blood prior to organ recovery did not show detectable levels of WNV nucleic acid with the use of quantitative real-time polymerase chain reaction. This report emphasizes that transplant-acquired WNV neuroinvasive disease can be particularly severe. We suggest that pre-procurement screening of organ donors by testing blood with both WNV IgM capture ELISA and a sensitive nucleic acid testing should be adopted during the transmission season in the present Italian epidemiological setting.

Application of a full-genome microarray-based assay for the study of genetic variability of West Nile virus

Viral adaptation through fixation of spontaneous mutations is an important factor potentially associated with reoccurrence of West Nile virus (WNV) outbreaks in the New World. The emergence of new genetic variants of WNV represents an important public health issue because it may affect the sensitivity of WNV screening and diagnostic assays, as well as the development and efficacy of WNV vaccines and anti-viral drugs. A microarray assay was developed and optimized to enable simple monitoring of WNV genetic variability and rapid detection of any nucleotide mutations within the entire viral genome. The assay was validated using 11 WNV isolates from the 2007 and 2009 U.S. epidemics. The developed microarray system can potentially serve as a high throughput, rapid, and effective approach for the detection of circulating WNV genetic variants.

West Nile virus infection in blood donors in the New York City area during the 2010 seasonal epidemic

A uniform threshold strategy for converting from minipool (MP)-nucleic acid testing (NAT) to individual donation (ID)-NAT screening for acute West Nile virus (WNV) infection among blood donors is lacking. We report on WNV screening at the New York Blood Center during the 2010 seasonal WNV epidemic, the most severe epidemic in that state since the original outbreak in 1999. Between July 1 and October 31, 2010, blood donations were screened by MP-NAT or ID-NAT and the presence of anti-WNV immunoglobulin (Ig)M and IgG was evaluated among NAT-positive donations. Twenty presumed viremic donations were identified for a frequency of 0.0129% (1 in 7752 donations). Nine donations that could have been missed by MP-NAT were identified. Two of these donations were both IgM and IgG negative, one of which would have been missed if more than one positive donation was required for initiating ID-NAT. Retrospective ID-NAT revealed two positive donations. The majority of the NAT-positive donations in New York (16/19) were from donors who lived in counties that had the highest incidence of human WNV cases in the state. Our data details the identification of WNV NAT-positive blood donations during a severe seasonal epidemic in the New York area. By initiating ID-NAT after one positive donation, using retrospective testing, and triggering ID-NAT regionally, we were able to prevent the release of presumably infectious donations. The detection of NAT-positive donations with retrospective testing, however, may indicate the need for changes in our trigger criteria.

Species Composition and WNV Screening of Mosquitoes from Lagoons in a Wetland Area of the Algarve, Portugal.

The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO2, and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance.

Comparison of immunohistochemistry and polymerase chain reaction for detection of West Nile virus in naturally infected dead birds

Credible vector-borne disease surveillance programs, especially in developing countries with limited resources, must include diagnostic tests that are efficient, inexpensive and simple and safe to administer while maintaining high levels of sensitivity and specificity. Since immunohistochemistry (IHC) includes most of these features, its sensitivity, specificity, predictive positive value (PPV) and predictive negative value (PNV) for West Nile virus (WNv) screening were compared to those of the gold standard, RT-PCR testing of kidney tissue in dead birds. IHC and RT-PCR were performed for WNv antigen on 41 dead birds (belonging to five orders) collected from the northwest region of the Riverside County of California. Fixed tissue sections were screened by IHC using polyclonal antibodies, and frozen kidney tissues were tested with RT-PCR. Kidney screening with IHC showed sensitivity, specificity, PPV and NPV of 95.45%, 73.68%, 80.77% and 93.33%, respectively. Based on WNv screening of kidney tissue, IHC and RT-PCR were in agreement with 95.45% (21/22) for positive dead birds and were in 100% (22/22) agreement when multi-organ screening by IHC was performed. The present study showed that IHC is as equally effective as RT-PCR in screening for WNv in dead birds. Therefore, IHC can effectively serve as a competent screening technique for those disease surveillance agencies that lack expensive RT-PCR technology while promoting safer biohazardous conditions, except at the initial stage of tissue collection.

Regarding unexpected severe and life-threatening donor-transmitted viral infections and use of high-risk behavior donors

Regarding unexpected severe and life-threatening donor-transmitted viral infections and use of high-risk behavior donors

Feasibility of routine individual donation testing for West Nile virus RNA during epidemic season using the investigational Roche cobas TaqScreen West Nile virus test and cobas s 201 system prototype

Minipool (MP) screening for West Nile virus (WNV) RNA may fail to detect presumptive viremic donations (PVDs) detectable by individual donation screening (IDS). Most blood centers switch collection regions to IDS when PVD detection by MP screening reaches a certain frequency. Use of IDS for all donations during WNV season was assessed during a clinical trial of the Roche cobas TaqScreen WNV test. Also evaluated was whether PVD detection reliably identifies regions that should be targeted for IDS. Test results, deviation reports, and service records were reviewed for 13.5 weeks of IDS in 2006 and 11.5 weeks of IDS in 2007. Numbers of PVDs and clinical WNV cases were obtained from public health and AABB Web sites and regional donor centers. Approximately 1000 donations were tested per week divided in six test runs. Each run required 1.2 shifts of technologists plus volunteers. A total of 7.2 percent of samples were initially unreportable in 2006 and 4.8 percent in 2007. Of 26,952 donations screened by IDS, none were reactive for WNV. A comparison of PVD and clinical case reports indicates that PVD detection in areas with intermediate or high clinical case prevalence may not reach commonly used criteria for triggering testing to IDS. Seasonal IDS was feasible using the cobas TaqScreen WNV test on the s 201, although staffing was impacted and a relatively high number of samples required retesting because of error messages. Seasonal IDS utilizing this highly specific assay may be a reasonable alternative to IDS triggered by regional PVD detection.

The Food and Drug Administration Approves Second West Nile Virus Screening Test for Donated Blood and Organs

The US Food and Drug Administration (FDA) announced approval on August 28, 2007, of a second test for detection of West Nile virus (WNV) in blood and organs. The cobas TaqScreen WNV test is an automated test that is able to detect viral RNA early during infection. Such nucleic acid testing improves blood and organ safety, detecting whether donated blood and organs have been infected, even before the donor’s immune system has begun to produce antibodies against the virus. Most often, WNV is transmitted to humans by mosquitoes; however, this pathogen can also be transmitted by blood transfusion or organ transplantation from infected donors. Although WNV infection is common in Africa, Asia, and the Middle East, it did not appear in the United States until 1999. Since then, WNV has become endemic in most of this country, with from 1–3 million cases reported between 1999 and 2006, according to the Centers for Disease Control and Prevention (CDC). “This action is the culmination of the dedicated efforts of FDA, our sister agencies, blood establishments, and manufacturers to bring donor screening tests to market for this increasingly common virus,” said Dr Jesse L. Goodman, director of the FDA’s Center for Biologics Evaluation and Research. “As a result, blood centers and hospitals now have a choice of 2 FDA approved tests to screen for West Nile Virus in donated blood and organs.” Most people infected with WNV show no signs of the disease but about 1 in 150 to 1 in 350 infected people develop serious symptoms, including encephalitis. Since introduction of the virus, the reported number of human cases of serious WNV in the United States has grown steadily from 62 in 1999 to 4269 in 2006. WNV has been particularly virulent this year. Although it is still early in the WNV season, 58 blood donors who are potentially positive for the virus have been reported to the CDC as of August 21, 2007. The cobas TaqScreen WNV test is approved for detection of the virus in plasma specimens from human donors of whole blood and blood components (plasma, red or white cells, platelets) and living donors of cells, reproductive cells, and other tissues. It is also intended for use in testing plasma specimens of organ donors when specimens are obtained while the donor’s organs are still viable. The test is not intended for use on samples of cord blood or as an aid in the diagnosis of WNV infection (Figure 1). Approval comes as the FDA is preparing guidance on the use of licensed WNV screening tests for blood donors. The test is manufactured by Roche Molecular Systems Inc (Pleasanton, CA). In December 2005, Procleix WNV Assay, from Gen-Probe Inc (San Diego, CA), became the first FDA-approved WNV genetic test for blood and organs.

Fluorescent Antibody Test for Rapid Detection of West Nile Virus Antigen in Avian Tissues

An indirect fluorescent antibody test (IFA) was developed to detect West Nile virus (WNV) antigens in tissues from avian species. The test samples used in the study consisted of 100 sets of tissues from dead crows that had been collected during the 2001 surveillance in Connecticut. The test tissues were punctured with a fine point Dacron cotton-tipped applicator and smeared in duplicate on 10-well diagnostic printed glass slides. Among several fixatives tested, 4% paraformaldehyde was the best. Reagent calibration for the IFA test was done in WNV-infected Vero cells and control uninfected Vero cells. Optimized antibody and fluorescent conjugate concentrations were then applied for the detection of WNV antigen on fixed tissue impression smears. Several tissues, including brain, heart, liver, kidney, and spleen were tested by the IFA test. The brain and heart seemed to be unsuitable for the test because of excessive background. Both virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) were used for validation, with the latter technique having a higher sensitivity. Therefore, IFA results were compared with RT-PCR results. The diagnostic sensitivity was 96.8% for liver, 96.4% for kidney, and 100% for spleen. The diagnostic specificity was 69% for liver, 95.3% for kidney and 95.8 for spleen. The IFA test performed best with spleen and kidney. The IFA test described here is a useful, practical, and rapid test for screening for WNV.

Transfusion‐associated transmission of West Nile virus, United States 2003 through 2005

National blood donation screening for West Nile virus (WNV) started in June 2003, after the documentation of WNV transfusion-associated transmission (TAT) in 2002. Blood donations were screened with investigational nucleic acid amplification assays in minipool formats. Blood collection agencies (BCAs) reported screening results to state and local public health authorities. Donor test results and demographic information were forwarded to CDC via ArboNET, the national electronic arbovirus surveillance system. State health departments and BCAs also reported suspect WNV TATs to CDC, which investigated these reports to confirm WNV infection in blood transfusion recipients in the absence of likely mosquito exposure. During 2003 to 2005, a total of 1,425 presumptive viremic donors were reported to CDC from 41 states. Of 36 investigations of suspected WNV TAT in 2003, 6 cases were documented. Estimated viremia levels were available for donations implicated in four TAT cases; the median estimated viremia was 0.1 plaque-forming units (PFUs) per mL (range, 0.06-0.50 PFU/mL; 1 PFU equals approximately 400 copies/mL). National blood screening for WNV identified and removed more than 1,400 potentially infectious blood donations in 2003 through 2005. Despite the success of screening in 2003, some residual WNV TAT risk remained due to donations containing very low levels of virus. Screening algorithms employing selected individual-donation testing were designed to address this residual risk and were fully implemented in 2004 and 2005. Continued vigilance for TAT will evaluate the effectiveness of these strategies.