Experimental Infection of Amblyomma americanum (Acari: Ixodidae) With Bourbon Virus (Orthomyxoviridae: Thogotovirus).

Following the recent discovery of Bourbon virus (BRBV) as a human pathogen, and the isolation of the virus from Amblyomma americanum (L.) collected near the location of a fatal human case, we undertook a series of experiments to assess the laboratory vector competence of this tick species for BRBV. Larval ticks were infected using an immersion technique, and transstadial transmission of virus to the nymphal and then to the adult stages was demonstrated. Transstadially infected nymphs transmitted virus to adult ticks at very high rates during cofeeding, indicating the presence of infectious virus in the saliva of engorging ticks. Vertical transmission by transstadially infected females to their progeny occurred, but at a low rate. Rabbits fed on by infected ticks of all active life stages developed high titers of antibody to the virus, demonstrating host exposure to BRBV antigens/live virus during tick blood feeding. These results demonstrate that A. americanum is a competent vector of BRBV and indicate that cofeeding could be critical for enzootic maintenance.

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.

Powassan Virus Infection Likely Acquired Through Blood Transfusion Presenting as Encephalitis in a Kidney Transplant Recipient.

A kidney transplant patient without known tick exposure developed encephalitis 3 weeks after transplantation. During the transplant hospitalization, the patient had received a blood transfusion from an asymptomatic donor later discovered to have been infected with Powassan virus. Here, we describe a probable instance of transfusion-transmitted Powassan virus infection.

Duration of seropositivity following yellow fever vaccination in U.S. military service members.

BACKGROUND: The United States military regularly deploys thousands of service members throughout areas of South America and Africa that are endemic for yellow fever (YF) virus. To determine if booster doses might be needed for service members who are repetitively or continually deployed to YF endemic areas, we evaluated seropositivity among US military personnel receiving a single dose of YF vaccine based on time post-vaccination. METHODS: Serum antibodies were measured using a plaque reduction neutralization test with 50% cutoff in 682 military personnel at 5-39 years post-vaccination. We determined noninferiority of immune response by comparing the proportion seropositive among those vaccinated 10-14 years previously with those vaccinated 5-9 years previously. Noninferiority was supported if the lower-bound of the 2-tailed 95% CI for p10-14years - p5-9years was ≥-0.10. Additionally, the geometric mean antibody titer (GMT) at various timepoints following vaccination were compared to the GMT at 5-9 years. RESULTS: The proportion of military service members with detectable neutralizing antibodies 10-14 years after a single dose of YF vaccine (95.8%, 95% CI 91.2-98.1%) was non-inferior to the proportion 5-9 years after vaccination (97.8%, 95% CI 93.7-99.3%). Additionally, GMT among vaccine recipients at 10-14 years post vaccination (99, 95% CI 82-121) was non-inferior to GMT in YF vaccine recipients at 5-9 years post vaccination (115, 95% CI 96-139). The proportion of vaccinees with neutralizing antibodies remained high, and non-inferior, among those vaccinated 15-19 years prior (98.5%, 95%CI 95.5-99.7%). Although the proportion seropositive decreased among vaccinees ≥ 20 years post vaccination, >90% remained seropositive. CONCLUSIONS: Neutralizing antibodies were present in > 95% of vaccine recipients for at least 19 years after vaccination, suggesting that booster doses every 10 years are not essential for most U.S. military personnel.

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.

Heartland virus infection in a heart transplant recipient from the Heartland.

Tick-borne infections represent a significant health risk each year in the United States. Immunocompromised patients are typically at risk of more severe disease manifestations than their immunocompetent counterparts. Here we report a case of a newly emerging phlebovirus, Heartland virus, in a heart transplant recipient.

Seroprevalence of Heartland Virus Antibodies in Blood Donors, Northwestern Missouri, USA.

We estimated the seroprevalence of Heartland virus antibodies to be 0.9% (95% CI 0.4%-4.2%) in a convenience sample of blood donors from northwestern Missouri, USA, where human cases and infected ticks have been identified. Although these findings suggest that some past human infections were undetected, the estimated prevalence is low.

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.

Seroprevalence and Symptomatic Attack Rate of Chikungunya Virus Infection, United States Virgin Islands, 2014-2015.

When introduced into a naïve population, chikungunya virus generally spreads rapidly, causing large outbreaks of fever and severe polyarthralgia. We randomly selected households in the U.S. Virgin Islands (USVI) to estimate seroprevalence and symptomatic attack rate for chikungunya virus infection at approximately 1 year following the introduction of the virus. Eligible household members were administered a questionnaire and tested for chikungunya virus antibodies. Estimated proportions were calibrated to age and gender of the population. We enrolled 509 participants. The weighted infection rate was 31% (95% confidence interval [CI]: 26-36%). Among those with evidence of chikungunya virus infection, 72% (95% CI: 65-80%) reported symptomatic illness and 31% (95% CI: 23-38%) reported joint pain at least once per week approximately 1 year following the introduction of the virus to USVI. Comparing rates from infected and noninfected study participants, 70% (95% CI: 62-79%) of fever and polyarthralgia and 23% (95% CI: 9-37%) of continuing joint pain in patients infected with chikungunya virus were due to their infection. Overall, an estimated 43% (95% CI: 33-52%) of the febrile illness and polyarthralgia in the USVI population during the outbreak was attributable to chikungunya virus and only 12% (95% CI: 7-17%) of longer term joint pains were attributed to chikungunya virus. Although the rates of infection, symptomatic disease, and longer term joint symptoms identified in USVI are similar to other outbreaks of the disease, a lower proportion of acute fever and joint pain was found to be attributable to chikungunya virus.

Incorporation of IgG Depletion in a Neutralization Assay Facilitates Differential Diagnosis of Zika and Dengue in Secondary Flavivirus Infection Cases.

Zika virus (ZIKV) has emerged as a major global public health concern due to its link as a causative agent of human birth defects. Laboratory diagnosis of suspected ZIKV infections by serological testing of specimens collected a week or more after symptom onset primarily relies on detection of anti-ZIKV-specific IgM antibodies by enzyme-linked immunosorbent assay coupled with detection of ZIKV-specific neutralizing antibody by neutralization tests. A definitive diagnosis based on serological assays is possible during primary ZIKV infections; however, due to the cross-reactivity of antibodies elicited during flaviviral infections, a definitive diagnosis is not always possible, especially among individuals who have previously been exposed to closely related flaviviruses, such as dengue virus (DENV). Here, we investigated the neutralizing IgM antibody profiles of 33 diagnostic specimens collected from individuals with suspected primary and secondary flaviviral infections acquired when visiting areas experiencing active ZIKV transmission in 2015 and 2016. Specimens collected between 1 day and 3 months postexposure were tested for ZIKV and dengue virus type 1 (DENV1) and type 2 (DENV2) by the plaque reduction neutralization test (PRNT) before and after IgG depletion. We found that IgG depletion prior to neutralization testing had little effect in differentiating samples from individuals with secondary infections taken less than 3 weeks postexposure; however, IgG depletion significantly reduced the cross-reactive neutralizing antibody titers and increased the percentage of cases discernible by PRNT from 15.4% (95% confidence interval [CI], 4.3 to 42.2%) to 76.9% (95% CI, 49.7 to 91.8%) for samples collected between roughly 3 and 12 weeks postexposure. These results highlight the potential of IgG depletion to improve the specificity of PRNT for better confirmation and differential diagnosis of flavivirus infections.

Ability To Serologically Confirm Recent Zika Virus Infection in Areas with Varying Past Incidence of Dengue Virus Infection in the United States and U.S. Territories in 2016.

Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections.

Investigation of a Guillain-Barré syndrome cluster in the Republic of Fiji.

BACKGROUND: In 2014, we investigated a cluster of Guillain-Barre syndrome (GBS) in Fiji that occurred during a dengue epidemic. We designed a case-control study to determine the etiology. METHODS: Cases were patients meeting Brighton Collaboration criteria for GBS with onset from February 2014 to May 2014. Controls were persons without symptoms of GBS who were matched by age group and location. We collected information on demographics and potential exposures. Serum samples were tested for evidence of recent arboviral or Leptospira spp. infections. RESULTS: Nine cases of GBS were identified for an incidence of five cases per 100,000 population/year. Median age of cases was 27years (range: 0.8-52); five (56%) were male. Six (67%) reported an acute illness prior to GBS onset. Among the 9 cases and 28 controls enrolled, odds ratios for reported exposures or antibodies against various arboviruses or Leptospira spp. were not statistically significant. CONCLUSIONS: No clear etiologies were identified for this unusual GBS cluster. There was a temporal association between the GBS cluster and a dengue epidemic, but we were unable to substantiate an epidemiologic or laboratory association. Further study is needed to explore potential associations between arboviral infections and GBS.

Zika Virus Disease in Travelers Returning to the United States, 2010-2014.

Zika virus is an emerging mosquito-borne flavivirus that typically causes a mild febrile illness with rash, arthralgia, or conjunctivitis. Zika virus has recently caused large outbreaks of disease in southeast Asia, Pacific Ocean Islands, and the Americas. We identified all positive Zika virus test results performed at U.S. Centers for Disease Control and Prevention from 2010 to 2014. For persons with test results indicating a recent infection with Zika virus, we collected information on demographics, travel history, and clinical features. Eleven Zika virus disease cases were identified among travelers returning to the United States. The median age of cases was 50 years (range: 29-74 years) and six (55%) were male. Nine (82%) cases had their illness onset from January to April. All cases reported a travel history to islands in the Pacific Ocean during the days preceding illness onset, and all cases were potentially viremic while in the United States. Public health prevention messages about decreasing mosquito exposure, preventing sexual exposure, and preventing infection in pregnant women should be targeted to individuals traveling to or living in areas with Zika virus activity. Health-care providers and public health officials should be educated about the recognition, diagnosis, and prevention of Zika virus disease.

Chikungunya and Dengue Virus Infections Among United States Community Service Volunteers Returning from the Dominican Republic, 2014.

Chikungunya spread throughout the Dominican Republic (DR) after the first identified laboratory-confirmed cases were reported in April 2014. In June 2014, a U.S.-based service organization operating in the DR reported chikungunya-like illnesses among several staff. We assessed the incidence of chikungunya virus (CHIKV) and dengue virus (DENV) infection and illnesses and evaluated adherence to mosquito avoidance measures among volunteers/staff deployed in the DR who returned to the United States during July-August 2014. Investigation participants completed a questionnaire that collected information on demographics, medical history, self-reported illnesses, and mosquito exposures and avoidance behaviors and provided serum for CHIKV and DENV diagnostic testing by reverse transcription polymerase chain reaction and IgM enzyme-linked immunosorbent assay. Of 102 participants, 42 (41%) had evidence of recent CHIKV infection and two (2%) had evidence of recent DENV infection. Of the 41 participants with evidence of recent CHIKV infection only, 39 (95%) reported fever, 37 (90%) reported rash, and 37 (90%) reported joint pain during their assignment. All attended the organization's health trainings, and 89 (87%) sought a pretravel health consultation. Most (∼95%) used insect repellent; however, only 30% applied it multiple times daily and < 5% stayed in housing with window/door screens. In sum, CHIKV infections were common among these volunteers during the 2014 chikungunya epidemic in the DR. Despite high levels of preparation, reported adherence to mosquito avoidance measures were inconsistent. Clinicians should discuss chikungunya with travelers visiting areas with ongoing CHIKV outbreaks and should consider chikungunya when diagnosing febrile illnesses in travelers returning from affected areas.

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.

Novel thogotovirus associated with febrile illness and death, United States, 2014.

A previously healthy man from eastern Kansas, USA, sought medical care in late spring because of a history of tick bite, fever, and fatigue. The patient had thrombocytopenia and leukopenia and was given doxycycline for a presumed tickborne illness. His condition did not improve. Multiorgan failure developed, and he died 11 days after illness onset from cardiopulmonary arrest. Molecular and serologic testing results for known tickborne pathogens were negative. However, testing of a specimen for antibodies against Heartland virus by using plaque reduction neutralization indicated the presence of another virus. Next-generation sequencing and phylogenetic analysis identified the virus as a novel member of the genus Thogotovirus.

Notes from the field: Heartland virus disease - United States, 2012-2013.

Heartland virus is a newly identified phlebovirus that was first isolated from two northwestern Missouri farmers hospitalized with fever, leukopenia, and thrombocytopenia in 2009. Based on the patients' clinical findings and their reported exposures, the virus was suspected to be transmitted by ticks. After this discovery, CDC worked with state and local partners to define the ecology and modes of transmission of Heartland virus, develop diagnostic assays, and identify additional cases to describe the epidemiology and clinical disease. From this work, it was learned that Heartland virus is found in the Lone Star tick (Amblyomma americanum). Six additional cases of Heartland virus disease were identified during 2012-2013; four of those patients were hospitalized, including one with comorbidities who died.

Isolation of a novel orthobunyavirus (Brazoran virus) with a 1.7 kb S segment that encodes a unique nucleocapsid protein possessing two putative functional domains.

In July, 2012 three isolations were made from mosquitoes collected in Brazoria, Orange and Montgomery counties, Texas, USA. Data from immunofluorescence testing suggested that these isolates are members of the genus Orthobunyavirus. Expanded analyses confirmed that these isolates comprise three independent isolations of the same virus; a novel orthobunyavirus. The genetic organization of the M and L segments of this virus is similar to that of other orthobunyaviruses. However, the S segment (~1.7 kb) is nearly twice the length of known orthobunyavirus S segments, encoding a significantly larger nucleocapsid, N (~50 kDa) and putative non-structural NSs (~20 kDa) proteins in a novel strategy by which the NSs ORF precedes the N ORF. The N protein appears to consist of two functional domains; an amino portion that possesses motifs similar to other orthobunyavirus N proteins and a carboxyl portion that possesses a glutamine-rich domain with no known homologue among Bunyaviridae.

Detection of anti-yellow fever virus immunoglobulin m antibodies at 3-4 years following yellow fever vaccination.

The duration of anti-yellow fever (YF) virus immunoglobulin M (IgM) antibodies following YF vaccination is unknown, making it difficult to interpret positive IgM antibody results in previously vaccinated travelers. We evaluated the frequency and predictors of YF IgM antibody positivity 3-4 years following YF vaccination. Twenty-nine (73%) of 40 participants had YF IgM antibodies 3-4 years postvaccination. No demographic or exposure variables were predictive of YF IgM positivity. However, persons who were YF IgM positive at 3-4 years postvaccination had earlier onset viremia and higher neutralizing antibody geometric mean titers at 1 month and 3-4 years postvaccination compared with persons who were YF IgM negative. Detection of YF IgM antibodies several years postvaccination might reflect remote YF vaccination rather than recent YF vaccination or YF virus infection.

Immunogenicity of one dose of Vero cell culture-derived Japanese encephalitis (JE) vaccine in adults previously vaccinated with mouse brain-derived JE vaccine.

BACKGROUND: There are no data on the use of inactivated Vero cell culture-derived Japanese encephalitis (JE) vaccine (JE-VC) as a booster among individuals who previously received inactivated mouse brain-derived JE vaccine (JE-MB). METHODS: Military personnel who received ≥3 doses of JE-MB or were JE vaccine-naïve were vaccinated with 2 doses of JE-VC on days 0 and 28. Serum neutralizing antibodies were measured pre-vaccination and 28 days after each dose. Non-inferiority was evaluated for seroprotection rate and geometric mean titer (GMT) between previously vaccinated participants post-dose 1 and vaccine-naïve participants post-dose 2. RESULTS: Fifty-three previously vaccinated and 70 JE vaccine-naïve participants were enrolled. Previously vaccinated participants had significantly higher GMTs pre-vaccination, post-dose 1, and post-dose 2. Seroprotection rates among previously vaccinated participants post-dose 1 (44/44, 100%) were noninferior to those achieved in previously naïve participants post-dose 2 (53/57, 93%). The GMT was significantly higher in previously vaccinated participants post-dose 1 (GMT 315; 95% CI 191-520) compared to previously naïve participants post-dose 2 (GMT 79; 95% CI 54-114). CONCLUSIONS: Among military personnel previously vaccinated with ≥3 doses of JE-MB, a single dose of JE-VC adequately boosts neutralizing antibody levels and provides at least short-term protection. Additional studies are needed to confirm these findings in other populations and determine the duration of protection following a single dose of JE-VC in prior recipients of JE-MB.