Transmission of yellow fever vaccine virus through blood transfusion and organ transplantation in the USA in 2021: report of an investigation.

BACKGROUND: In 2021, four patients who had received solid organ transplants in the USA developed encephalitis beginning 2-6 weeks after transplantation from a common organ donor. We describe an investigation into the cause of encephalitis in these patients. METHODS: From Nov 7, 2021, to Feb 24, 2022, we conducted a public health investigation involving 15 agencies and medical centres in the USA. We tested various specimens (blood, cerebrospinal fluid, intraocular fluid, serum, and tissues) from the organ donor and recipients by serology, RT-PCR, immunohistochemistry, metagenomic next-generation sequencing, and host gene expression, and conducted a traceback of blood transfusions received by the organ donor. FINDINGS: We identified one read from yellow fever virus in cerebrospinal fluid from the recipient of a kidney using metagenomic next-generation sequencing. Recent infection with yellow fever virus was confirmed in all four organ recipients by identification of yellow fever virus RNA consistent with the 17D vaccine strain in brain tissue from one recipient and seroconversion after transplantation in three recipients. Two patients recovered and two patients had no neurological recovery and died. 3 days before organ procurement, the organ donor received a blood transfusion from a donor who had received a yellow fever vaccine 6 days before blood donation. INTERPRETATION: This investigation substantiates the use of metagenomic next-generation sequencing for the broad-based detection of rare or unexpected pathogens. Health-care workers providing vaccinations should inform patients of the need to defer blood donation for at least 2 weeks after receiving a yellow fever vaccine. Despite mitigation strategies and safety interventions, a low risk of transfusion-transmitted infections remains. FUNDING: US Centers for Disease Control and Prevention (CDC), the Biomedical Advanced Research and Development Authority, and the CDC Epidemiology and Laboratory Capacity Cooperative Agreement for Infectious Diseases.

Increase in Colorado Tick Fever Virus Disease Cases and Effect of COVID-19 Pandemic on Behaviors and Testing Practices, Montana, 2020.

In 2020, Montana, USA, reported a large increase in Colorado tick fever (CTF) cases. To investigate potential causes of the increase, we conducted a case-control study of Montana residents who tested positive or negative for CTF during 2020, assessed healthcare providers' CTF awareness and testing practices, and reviewed CTF testing methods. Case-patients reported more time recreating outdoors on weekends, and all reported finding a tick on themselves before illness. No consistent changes were identified in provider practices. Previously, only CTF serologic testing was used in Montana. In 2020, because of SARS-CoV-2 testing needs, the state laboratory sent specimens for CTF testing to the Centers for Disease Control and Prevention, where more sensitive molecular methods are used. This change in testing probably increased the number of CTF cases detected. Molecular testing is optimal for CTF diagnosis during acute illness. Tick bite prevention measures should continue to be advised for persons doing outdoor activities.

Severe Arboviral Neuroinvasive Disease in Patients on Rituximab Therapy: A Review.

With increasing use of rituximab and other B-cell depleting monoclonal antibodies for multiple indications, infectious complications are being recognized. We summarize clinical findings of patients on rituximab with arboviral diseases identified through literature review or consultation with the Centers for Disease Control and Prevention. We identified 21 patients on recent rituximab therapy who were diagnosed with an arboviral disease caused by West Nile, tick-borne encephalitis, eastern equine encephalitis, Cache Valley, Jamestown Canyon, and Powassan viruses. All reported patients had neuroinvasive disease. The diagnosis of arboviral infection required molecular testing in 20 (95%) patients. Median illness duration was 36 days (range, 12 days to 1 year), and 15/19 (79%) patients died from their illness. Patients on rituximab with arboviral disease can have a severe or prolonged course with an absence of serologic response. Patients should be counseled about mosquito and tick bite prevention when receiving rituximab and other B-cell depleting therapies.

Transfusion-Transmitted Cache Valley Virus Infection in a Kidney Transplant Recipient With Meningoencephalitis.

BACKGROUND: Cache Valley virus (CVV) is a mosquito-borne virus that is a rare cause of disease in humans. In the fall of 2020, a patient developed encephalitis 6 weeks following kidney transplantation and receipt of multiple blood transfusions. METHODS: After ruling out more common etiologies, metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) was performed. We reviewed the medical histories of the index kidney recipient, organ donor, and recipients of other organs from the same donor and conducted a blood traceback investigation to evaluate blood transfusion as a possible source of infection in the kidney recipient. We tested patient specimens using reverse-transcription polymerase chain reaction (RT-PCR), the plaque reduction neutralization test, cell culture, and whole-genome sequencing. RESULTS: CVV was detected in CSF from the index patient by mNGS, and this result was confirmed by RT-PCR, viral culture, and additional whole-genome sequencing. The organ donor and other organ recipients had no evidence of infection with CVV by molecular or serologic testing. Neutralizing antibodies against CVV were detected in serum from a donor of red blood cells received by the index patient immediately prior to transplant. CVV neutralizing antibodies were also detected in serum from a patient who received the co-component plasma from the same blood donation. CONCLUSIONS: Our investigation demonstrates probable CVV transmission through blood transfusion. Clinicians should consider arboviral infections in unexplained meningoencephalitis after blood transfusion or organ transplantation. The use of mNGS might facilitate detection of rare, unexpected infections, particularly in immunocompromised patients.

Correction: Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics.

[This corrects the article DOI: 10.1371/journal.pntd.0010487.].

Laboratory evaluation of RealStar Yellow Fever Virus RT-PCR kit 1.0 for potential use in the global yellow fever laboratory network.

BACKGROUND: Early detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0. METHODOLOGY AND PRINCIPAL FINDINGS: Specific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly. CONCLUSIONS AND SIGNIFICANCE: The RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

Colorado Tick Fever Virus in the Far West: Forgotten, but Not Gone.

In the past few decades, reported human cases of Colorado tick fever in the western United States have decreased dramatically. The goal of this study was to conduct surveillance for Colorado tick fever virus (CTFV) in Dermacentor ticks in recreational sites in Colorado, Wyoming, and California to determine whether the virus is still present in Dermacentor ticks from these states. Surveillance focused on regions where surveys had been conducted in the 1950s, 1960s, and 1970s. Adult Rocky Mountain wood ticks (Dermacentor andersoni), Pacific Coast ticks (Dermacentor occidentalis), and winter ticks (Dermacentor albipictus) were tested by PCR. A subset of PCR-positive D. andersoni ticks (n = 7) were cultured in Vero cells. CTFV-positive Rocky Mountain wood ticks were found in all states: Colorado (58% prevalence), Wyoming (21%), and California (4%). Although no winter ticks tested positive, Pacific Coast ticks tested positive in one county (Siskiyou County, 15% prevalence) and were positive only in a location that also maintained Rocky Mountain wood ticks and golden mantled ground squirrels, a known CTFV host. In summary, CTFV is prevalent in D. andersoni and D. occidentalis in regions where they are sympatric in California and in D. andersoni in Colorado and Wyoming. Although the number of human CTFV cases has declined dramatically, this decrease in reported disease does not appear to be due to the disappearance or even the decline in prevalence of this virus in ticks in historically endemic regions of the country.

Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics.

Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.

West Nile Virus Transmission by Solid Organ Transplantation and Considerations for Organ Donor Screening Practices, United States.

West Nile virus (WNV) is the most common domestic arbovirus in the United States. During 2018, WNV was transmitted through solid organ transplantation to 2 recipients who had neuroinvasive disease develop. Because of increased illness and death in transplant recipients, organ procurement organizations should consider screening during region-specific WNV transmission months.

Genomic Evaluation of the Genus Coltivirus Indicates Genetic Diversity among Colorado Tick Fever Virus Strains and Demarcation of a New Species.

The type species of the genus Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.

Fatal Human Infection with Evidence of Intrahost Variation of Eastern Equine Encephalitis Virus, Alabama, USA, 2019.

Eastern equine encephalitis virus (EEEV) is an arbovirus in the family Togaviridae, genus Alphavirus, found in North America and associated with freshwater/hardwood swamps in the Atlantic, Gulf Coast, and Great Lakes regions. EEEV disease in humans is rare but causes substantial illness and death. To investigate the molecular epidemiology and microevolution of EEEV from a fatal case in Alabama, USA, in 2019, we used next-generation sequencing of serum and cerebrospinal fluid (CSF). Phylogenetic inference indicated that the infecting strain may be closely related to isolates from Florida detected during 2010-2014, suggesting potential seeding from Florida. EEEV detected in serum displayed a higher degree of variability with more single-nucleotide variants than that detected in the CSF. These data refine our knowledge of EEEV molecular epidemiologic dynamics in the Gulf Coast region and demonstrate potential quasispecies bottlenecking within the central nervous system of a human host.

Reassortant Cache Valley Virus Associated With Acute Febrile, Nonneurologic Illness, Missouri.

An adult male from Missouri sought care for fever, fatigue, and gastrointestinal symptoms. He had leukopenia and thrombocytopenia and was treated for a presumed tickborne illness. His condition deteriorated with respiratory and renal failure, lactic acidosis, and hypotension. Next-generation sequencing and phylogenetic analysis identified a reassortant Cache Valley virus.

Development of diagnostic microsphere-based immunoassays for Heartland virus.

BACKGROUND: Heartland virus (HRTV), a recently reclassified member of the genus Bandavirus, family Phenuiviridae, was first isolated in 2009 from a Missouri farmer exhibiting leukopenia and thrombocytopenia with suspected ehrlichiosis. Since then, more HRTV cases have been diagnosed, and firstline laboratory diagnostic assays are needed to identify future infections Objectives. We sought to develop rapid and reliable IgM and IgG microsphere immunoassays (MIAs) to test sera of patients suspected of having HRTV infection, and to distinguish between recent and past infections. STUDY DESIGN: Heartland virus antigen was captured by an anti-HRTV monoclonal antibody covalently bound to microspheres. Antibodies in human sera from confirmed HRTV-positive and negative cases were reacted with the microsphere complexes and detected using a BioPlex® 200 instrument. Assay cutoffs were determined by receiver operator characteristic analysis of the normalized test output values, equivocal zones for each assay were defined, and sensitivities, specificities, accuracies, and imprecision values were calculated. RESULTS: Sensitivities, specificities and accuracies of the IgM and IgG MIAs were all >95 %. Both tests were precise within and between assay plates, and cross-reactivity with other arboviruses was not observed. CONCLUSIONS: HRTV IgM and IgG MIAs are accurate and rapid first-line methods to serologically identify recent and past HRTV infections.

Investigation of Heartland Virus Disease Throughout the United States, 2013-2017.

BACKGROUND: Heartland virus (HRTV) was first described as a human pathogen in 2012. From 2013 to 2017, the Centers for Disease Control and Prevention (CDC) implemented a national protocol to evaluate patients for HRTV disease, better define its geographic distribution, epidemiology, and clinical characteristics, and develop diagnostic assays for this novel virus. METHODS: Individuals aged ≥12 years whose clinicians contacted state health departments or the CDC about testing for HRTV infections were screened for recent onset of fever with leukopenia and thrombocytopenia. A questionnaire was administered to collect data on demographics, risk factors, and signs and symptoms; blood samples were tested for the presence of HRTV RNA and neutralizing antibodies. RESULTS: Of 85 individuals enrolled and tested, 16 (19%) had evidence of acute HRTV infection, 1 (1%) had past infection, and 68 (80%) had no infection. Patients with acute HRTV disease were residents of 7 states, 12 (75%) were male, and the median age (range) was 71 (43-80) years. Illness onset occurred from April to September. The majority reported fatigue, anorexia, nausea, headache, confusion, arthralgia, or myalgia. Fourteen (88%) cases were hospitalized; 2 (13%) died. Fourteen (88%) participants reported finding a tick on themselves in the 2 weeks before illness onset. HRTV-infected individuals were significantly older (P < .001) and more likely to report an attached tick (P = .03) than uninfected individuals. CONCLUSIONS: Health care providers should consider HRTV disease testing in patients with an acute febrile illness with either leukopenia or thrombocytopenia not explained by another condition or who were suspected to have a tickborne disease but did not improve following appropriate treatment.

Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management.

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Persistence of yellow fever virus-specific neutralizing antibodies after vaccination among US travellers.

Background: Few studies have assessed the duration of humoral immunity following yellow fever (YF) vaccination in a non-endemic population. We evaluated seropositivity among US resident travellers based on time post-vaccination. Methods: We identified serum samples from US travellers with YF virus-specific plaque reduction neutralization testing (PRNT) performed at CDC from 1988 to 2016. Analyses were conducted to assess the effect of time since vaccination on neutralizing antibody titer counts. Results: Among 234 travellers who had neutralizing antibody testing performed on a specimen obtained ≥1 month after vaccination, 13 received multiple YF vaccinations and 221 had one dose of YF vaccine reported. All 13 who received more than one dose of YF vaccine had a positive PRNT regardless of the amount time since most recent vaccination. Among the 221 travellers with one reported dose of YF vaccine, 155 (70%) were vaccinated within 10 years (range 1 month-9 years) and 66 (30%) were vaccinated ≥10 years (range 10-53 years) prior to serum collection. Among the 155 individuals vaccinated, <10 years prior to serum collection, 146 (94%) had a positive PRNT compared with 82% (54/66) of individuals vaccinated ≥10 years prior to serum collection (P = 0.01). Post-vaccination PRNT titers showed a time-dependent decrease. Individuals with immunocompromising conditions were less likely to have a positive PRNT (77%) compared with those who were not immunocompromised (92%; P = 0.04). Conclusion: Although the percentage of vaccinees with a positive PRNT and antibody titers decreased over time, a single dose of YF vaccine provided long-lasting protection in the majority of US travellers. A booster dose could be considered for certain travellers who are planning travel to a high risk area based on immune competence and time since vaccination.

Serological Survey for Antibodies to Mosquito-Borne Bunyaviruses Among US National Park Service and US Forest Service Employees.

Serum samples from 295 employees of Great Smoky Mountains National Park (GRSM), Rocky Mountain National Park (ROMO), and Grand Teton National Park with adjacent Bridger-Teton National Forest (GRTE-BTNF) were subjected to serological analysis for mosquito-borne bunyaviruses. The sera were analyzed for neutralizing antibodies against six orthobunyaviruses: La Crosse virus (LACV), Jamestown Canyon virus (JCV), snowshoe hare virus (SSHV), California encephalitis virus, and Trivittatus virus (TVTV) belonging to the California serogroup and Cache Valley virus (CVV) belonging to the Bunyamwera serogroup. Sera were also tested for immunoglobulin (Ig) G antibodies against LACV and JCV by enzyme-linked immunosorbent assay (ELISA). The proportion of employees with neutralizing antibodies to any California serogroup bunyavirus was similar in all three sites, with the prevalence ranging from 28% to 36%. The study demonstrated a seroprevalence of 3% to CVV across the three parks. However, proportions of persons with antibodies to specific viruses differed between parks. Participants residing in the eastern regions had a higher seroprevalence to LACV, with 24% (18/75) GRSM employees being seropositive. In contrast, SSHV seroprevalence was limited to employees from the western sites, with 1.7% (1/60) ROMO and 3.8% (6/160) GRTE-BTNF employees being positive. Seroprevalence to JCV was noted in employees from all sites at rates of 6.7% in GRSM, 21.7% in ROMO, and 15.6% in GRTE-BTNF. One employee each from ROMO (1.7%) and GRTE-BTNF (1.9%) were positive for TVTV. This study also has illustrated the greater sensitivity and specificity of plaque reduction neutralization test compared to IgG ELISA in conducting serosurveys for LACV and JCV.

Molecular, serological and in vitro culture-based characterization of Bourbon virus, a newly described human pathogen of the genus Thogotovirus.

BACKGROUND: In June of 2014, a previously healthy man from Kansas with a recent history of tick exposure died from complications related to an illness marked by fever, thrombocytopenia and leukopenia. An isolate was derived from the blood of this patient during the course of diagnostic testing. This isolate was subsequently identified as a novel orthomyxovirus of the genus Thogotovirus by next generation sequencing and was named Bourbon virus after the patient's county of residence. OBJECTIVES: To support research and diagnostic aims, we provide a basic description of Bourbon virus at both the molecular and serological levels. Furthermore, to preliminarily identify potential host and vector range associations we have characterized the growth kinetics of Bourbon virus in a variety of vertebrate and invertebrate cell lines. STUDY DESIGN: Bourbon virus was subjected to next generation-high throughput sequencing, phylogenetic, and basic structural protein analyses as well as 2-way plaque reduction neutralization assays. Also, we inoculated a variety of cell types with Bourbon virus and evaluated the growth kinetics by determining viral titers in the supernatants taken from infected cells over time. RESULTS: Bourbon virus possesses 24-82% identity at the amino acid sequence level and low serological cross-reactivity with other Thogotoviruses. In vitro growth kinetics reveal robust replication of Bourbon virus in mammalian and tick cells. CONCLUSIONS: Molecular and serological characterizations identify Bourbon virus as a novel member of the genus Thogotovirus. Results from cell culture analyses suggest an association between Bourbon virus and mammalian and tick hosts.

Comparative sequence analyses of La Crosse virus strain isolated from patient with fatal encephalitis, Tennessee, USA.

We characterized a La Crosse virus (LACV) isolate from the brain of a child who died of encephalitis-associated complications in eastern Tennessee, USA, during summer 2012. We compared the isolate with LACV sequences from mosquitoes collected near the child's home just after his postmortem diagnosis. In addition, we conducted phylogenetic analyses of these and other sequences derived from LACV strains representing varied temporal, geographic, and ecologic origins. Consistent with historical findings, results of these analyses indicate that a limited range of LACV lineage I genotypes is associated with severe clinical outcomes.