Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009-2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs.

BACKGROUND: Neuroinvasive infection with Francisella tularensis, the causative agent of tularemia, is rare. Establishing clinical suspicion is challenging if risk factors or clinical features classically associated with tularemia are absent. Tularemia is treatable with antibiotics; however, there are limited data to inform management of potentially fatal neuroinvasive infection. METHODS: We collected epidemiologic and clinical data on 2 recent US cases of neuroinvasive F. tularensis infection, and performed a literature review of cases of neuroinvasive F. tularensis infection published after 1950. RESULTS: One patient presented with focal neurologic deficits and brain lesions; broad-range molecular testing on resected brain tissue detected F. tularensis. The other patient presented with meningeal signs; tularemia was suspected based on animal exposure, and F. tularensis grew in cerebrospinal fluid (CSF) culture. Both patients received combination antibiotic therapy and recovered from infection. Among 16 published cases, tularemia was clinically suspected in 4 cases. CSF often displayed lymphocytic pleocytosis. Among cases with available data, CSF culture was positive in 13 of 16 cases, and F. tularensis antibodies were detected in 11 of 11 cases. Treatment typically included an aminoglycoside combined with either a tetracycline or a fluoroquinolone. Outcomes were generally favorable. CONCLUSIONS: Clinicians should consider neuroinvasive F. tularensis infection in patients with meningitis and signs suggestive of tularemia or compatible exposures, lymphocyte-predominant CSF, unrevealing standard microbiologic workup, or lack of response to empiric bacterial meningitis treatment. Molecular testing, culture, and serologic testing can reveal the diagnosis. Favorable outcomes can be achieved with directed antibiotic treatment.

Background Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pet cohabitants as a sub-study of an ongoing COVID-19 household transmission investigation.Methods Mammalian pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April–May 2020. Demographic/exposure information, oropharyngeal, nasal, rectal, and fur swabs, feces, and blood were collected from enrolled pets and tested by rRT-PCR and virus neutralization assays.Findings We enrolled 37 dogs and 19 cats from 34 of 41 eligible households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog’s fur swabs (2%) tested positive by rRT-PCR at the first animal sampling. Among 47 pets with serological results from 30 households, eight (17%) pets (4 dogs, 4 cats) from 6 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40–100%) compared to households with no seropositive pet (median 37%; range: 13–100%) (p=0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 hour) with the human index patient before the person’s COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the human index patient after diagnosis and none (0%) were seropositive; of the 19 (58%) pets with continued contact, 4 (21%) were seropositive.Interpretations Seropositive pets likely acquired infection from humans, which may occur more frequently than previously recognized. People with COVID-19 should restrict contact with animals.Funding Centers for Disease Control and Prevention, U.S. Department of AgricultureCompeting Interest StatementThe authors have declared no competing interest.

Soft tick relapsing fever (STRF) (also known as tickborne relapsing fever) is a rare infection caused by certain Borrelia spirochetes and transmitted to humans by soft-bodied Ornithodoros ticks. In the United States, acquisition of STRF is commonly associated with exposure to rustic cabins, camping, and caves. Antibiotic treatment is highly effective for STRF, but without timely treatment, STRF can result in severe complications, including death. No nationally standardized case definition for STRF exists; however, the disease is reportable in 12 states. This report summarizes demographic and clinical information for STRF cases reported during 2012-2021 from states where STRF is reportable. During this period, 251 cases were identified in 11 states. The median annual case count was 24. Most patients with STRF (55%) were hospitalized; no fatalities were reported. The geographic distribution and seasonal pattern of STRF have remained relatively constant since the 1990s. Persons should avoid rodent-infested structures and rodent habitats, such as caves, in areas where STRF is endemic. STRF surveillance, prevention, and control efforts would benefit from a standardized case definition and increased awareness of the disease among the public and clinicians.

This study reports on the validation of a real-time polymerase chain reaction test targeting the vomp region of Bartonella quintana. The assay displayed 100% sensitivity and specificity for the 52 bloods and 159 cultures tested. Molecular diagnosis of Bartonella quintana can aid clinical treatment during acute infection.

Infection with Borrelia miyamotoi in California, USA, has been suggested by serologic studies. We diagnosed B. miyamotoi infection in an immunocompromised man in California. Diagnosis was aided by plasma microbial cell-free DNA sequencing. We conclude that the infection was acquired in California.

Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.

While risk of fomite transmission of SARS-CoV-2 is considered low, there is limited environmental data within households. This January-April 2021 investigation describes frequency and types of surfaces positive for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction (RT-PCR) among residences with ≥1 SARS-CoV-2 infection, and associations of household characteristics with surface RT-PCR and viable virus positivity. Of 1232 samples from 124 households, 27.8% (n = 342) were RT-PCR positive with nightstands (44.1%) and pillows (40.9%) most frequently positive. SARS-CoV-2 lineage, documented household transmission, greater number of infected persons, shorter interval between illness onset and sampling, total household symptoms, proportion of infected persons ≤12 years old, and persons exhibiting upper respiratory symptoms or diarrhea were associated with more positive surfaces. Viable virus was isolated from 0.2% (n = 3 samples from one household) of all samples. This investigation suggests that while SARS-CoV-2 on surfaces is common, fomite transmission risk in households is low.

BACKGROUND: COVID-19 vaccination reduces SARS-CoV-2 infection and transmission. However, evidence is emerging on the degree of protection across variants and in high-transmission settings. To better understand the protection afforded by vaccination specifically in a high-transmission setting, we examined household transmission of SARS-CoV-2 during a period of high community incidence with predominant SARS-CoV-2 B.1.1.7 (Alpha) variant, among vaccinated and unvaccinated contacts. METHODS: We conducted a household transmission investigation in San Diego County, California, and Denver, Colorado, during January-April 2021. Households were enrolled if they had at least one person with documented SARS-CoV-2 infection. We collected nasopharyngeal swabs, blood, demographic information, and vaccination history from all consenting household members. We compared infection risks (IRs), RT-PCR cycle threshold values, SARS-CoV-2 culture results, and antibody statuses among vaccinated and unvaccinated household contacts. RESULTS: We enrolled 493 individuals from 138 households. The SARS-CoV-2 variant was identified from 121/138 households (88%). The most common variants were Alpha (75/121, 62%) and Epsilon (19/121, 16%). There were no households with discordant lineages among household members. One fully vaccinated secondary case was symptomatic (13%); the other 5 were asymptomatic (87%). Among unvaccinated secondary cases, 105/108 (97%) were symptomatic. Among 127 households with a single primary case, the IR for household contacts was 45% (146/322; 95% Confidence Interval [CI] 40-51%). The observed IR was higher in unvaccinated (130/257, 49%, 95% CI 45-57%) than fully vaccinated contacts (6/26, 23%, 95% CI 11-42%). A lower proportion of households with a fully vaccinated primary case had secondary cases (1/5, 20%) than households with an unvaccinated primary case (66/108, 62%). CONCLUSIONS: Although SARS-CoV-2 infections in vaccinated household contacts were reported in this high transmission setting, full vaccination protected against SARS-CoV-2 infection. These findings further support the protective effect of COVID-19 vaccination and highlight the need for ongoing vaccination among eligible persons.

IMPORTANCE: As self-collected home antigen tests become widely available, a better understanding of their performance during the course of SARS-CoV-2 infection is needed. OBJECTIVE: To evaluate the diagnostic performance of home antigen tests compared with reverse transcription-polymerase chain reaction (RT-PCR) and viral culture by days from illness onset, as well as user acceptability. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study was conducted from January to May 2021 in San Diego County, California, and metropolitan Denver, Colorado. The convenience sample included adults and children with RT-PCR-confirmed infection who used self-collected home antigen tests for 15 days and underwent at least 1 nasopharyngeal swab for RT-PCR, viral culture, and sequencing. EXPOSURES: SARS-CoV-2 infection. MAIN OUTCOMES AND MEASURES: The primary outcome was the daily sensitivity of home antigen tests to detect RT-PCR-confirmed cases. Secondary outcomes included the daily percentage of antigen test, RT-PCR, and viral culture results that were positive, and antigen test sensitivity compared with same-day RT-PCR and cultures. Antigen test use errors and acceptability were assessed for a subset of participants. RESULTS: This study enrolled 225 persons with RT-PCR-confirmed infection (median [range] age, 29 [1-83] years; 117 female participants [52%]; 10 [4%] Asian, 6 [3%] Black or African American, 50 [22%] Hispanic or Latino, 3 [1%] Native Hawaiian or Other Pacific Islander, 145 [64%] White, and 11 [5%] multiracial individuals) who completed 3044 antigen tests and 642 nasopharyngeal swabs. Antigen test sensitivity was 50% (95% CI, 45%-55%) during the infectious period, 64% (95% CI, 56%-70%) compared with same-day RT-PCR, and 84% (95% CI, 75%-90%) compared with same-day cultures. Antigen test sensitivity peaked 4 days after illness onset at 77% (95% CI, 69%-83%). Antigen test sensitivity improved with a second antigen test 1 to 2 days later, particularly early in the infection. Six days after illness onset, antigen test result positivity was 61% (95% CI, 53%-68%). Almost all (216 [96%]) surveyed individuals reported that they would be more likely to get tested for SARS-CoV-2 infection if home antigen tests were available over the counter. CONCLUSIONS AND RELEVANCE: The results of this cohort study of home antigen tests suggest that sensitivity for SARS-CoV-2 was moderate compared with RT-PCR and high compared with viral culture. The results also suggest that symptomatic individuals with an initial negative home antigen test result for SARS-CoV-2 infection should test again 1 to 2 days later because test sensitivity peaked several days after illness onset and improved with repeated testing.

BACKGROUND: In Spring 2021, SARS-CoV-2 B.1.1.7 (Alpha) became the predominant variant in the U.S. Research suggests that Alpha has increased transmissibility compared to non-Alpha lineages. We estimated household secondary infection risk (SIR), assessed characteristics associated with transmission, and compared symptoms of persons with Alpha and non-Alpha infections. METHODS: We followed households with SARS-CoV-2 infection for two weeks in San Diego County and metropolitan Denver, January to April 2021. We collected epidemiologic information and biospecimens for serology, RT-PCR, and whole genome sequencing. We stratified SIR and symptoms by lineage, and identified characteristics associated with transmission using Generalized Estimating Equations. RESULTS: We investigated 127 households with 322 household contacts; 72 households (56.7%) had member(s) with secondary infections. SIRs were not significantly higher for Alpha (61.0% [95% confidence interval (CI) 52.4-69.0%]) than non-Alpha (55.6% [CI 44.7-65.9%], P = 0.49). In households with Alpha, persons who identified as Asian or Hispanic/Latino had significantly higher SIRs than those who identified as White (P = 0.01 and 0.03, respectively). Close contact (e.g., kissing, hugging) with primary cases was associated with increased transmission for all lineages. Persons with Alpha infection were more likely to report constitutional symptoms than persons with non-Alpha (86.9% vs. 76.8%, P = 0.05). CONCLUSIONS: Household SIRs were similar for Alpha and non-Alpha. Comparable SIRs may be due to saturation of transmission risk in households owing to extensive close contact, or true lack of difference in transmission rates. Avoiding close contact within households may reduce SARS-CoV-2 transmission for all lineages among household members.

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

SARS-CoV-2 transmission from contaminated surfaces, or fomites, has been a concern during the COVID-19 pandemic. Households have been important sites of transmission throughout the COVID-19 pandemic, but there is limited information on SARS-CoV-2 contamination of surfaces in these settings. We describe environmental detection of SARS-CoV-2 in households of persons with COVID-19 to better characterize the potential risks of fomite transmission. Ten households with ≥1 person with laboratory-confirmed COVID-19 and with ≥2 members total were enrolled in Utah, U.S.A. Nasopharyngeal and anterior nasal swabs were collected from members and tested for the presence of SARS-CoV-2 by RT-PCR. Fifteen surfaces were sampled in each household and tested for presence and viability of SARS-CoV-2. SARS-CoV-2 RNA was detected in 23 (15%) of 150 environmental swab samples, most frequently on nightstands (4/6; 67%), pillows (4/23; 17%), and light switches (3/21; 14%). Viable SARS-CoV-2 was cultured from one sample. All households with SARS-CoV-2-positive surfaces had ≥1 person who first tested positive for SARS-CoV-2 ≤ 6 days prior to environmental sampling. SARS-CoV-2 surface contamination occurred early in the course of infection when respiratory transmission is most likely, notably on surfaces in close, prolonged contact with persons with COVID-19. While fomite transmission might be possible, risk is low.

During a recent outbreak of Bartonella quintana disease in Denver, 15% of 241 persons experiencing homelessness who presented for severe acute respiratory syndrome coronavirus 2 testing were seroreactive for Bartonella. Improved recognition of B quintana disease and prevention of louse infestation are critical for this vulnerable population.

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semi-multiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (B. hermsii and others), the emerging hard tick transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis) The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae, and multiple TaqMan probes to allow for detection of and differentiation among the three groups. The assay detects all RF borreliae tested with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single assay real-time PCR approach will help to improve diagnosis of RF by simplifying the selection of tests to aid in clinical management of acutely ill RF patients.

Virus shedding in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can occur before onset of symptoms; less is known about symptom progression or infectiousness associated with initiation of viral shedding. We investigated household transmission in 5 households with daily specimen collection for 5 consecutive days starting a median of 4 days after symptom onset in index patients. Seven contacts across 2 households implementing no precautionary measures were infected. Of these 7, 2 tested positive for SARS-CoV-2 by reverse transcription PCR on day 3 of 5. Both had mild, nonspecific symptoms for 1-3 days preceding the first positive test. SARS-CoV-2 was cultured from the fourth-day specimen in 1 patient and from the fourth- and fifth-day specimens in the other. We also describe infection control measures taken in the households that had no transmission. Persons exposed to SARS-CoV-2 should self-isolate, including from household contacts, wear a mask, practice hand hygiene, and seek testing promptly.

BACKGROUND: Detection of SARS-CoV-2 infection has principally been performed through the use of real-time reverse-transcription PCR (rRT-PCR) testing. Results of such tests can be reported as cycle threshold (Ct) values, which may provide semi-quantitative or indirect measurements of viral load. Previous reports have examined temporal trends in Ct values over the course of a SARS-CoV-2 infection. METHODS: Using testing data collected during a prospective household transmission investigation of outpatient and mild COVID-19 cases, we examined the relationship between Ct values of the viral RNA N1 target and demographic, clinical, and epidemiological characteristics collected through participant interviews and daily symptom diaries. RESULTS: We found Ct values are lowest (corresponding to higher viral RNA concentration) soon after symptom onset and are significantly correlated with time elapsed since onset (p<0.001); within 7 days after symptom onset, the median Ct value was 26.5 compared with a median Ct value of 35.0 occurring 21 days after onset. Ct values were significantly lower among participants under 18 years of age (p=0.01) and those reporting upper respiratory symptoms at the time of sample collection (p=0.001) and were higher among participants reporting no symptoms (p=0.05). CONCLUSIONS: These results emphasize the importance of early testing for SARS-CoV-2 among individuals with symptoms of respiratory illness and allows cases to be identified and isolated when their viral shedding may be highest.

Plague, an acute zoonosis caused by Yersinia pestis, is endemic in the West Nile region of northwestern Uganda and neighboring northeastern Democratic Republic of the Congo (DRC) (1-4). The illness manifests in multiple clinical forms, including bubonic and pneumonic plague. Pneumonic plague is rare, rapidly fatal, and transmissible from person to person via respiratory droplets. On March 4, 2019, a patient with suspected pneumonic plague was hospitalized in West Nile, Uganda, 4 days after caring for her sister, who had come to Uganda from DRC and died shortly thereafter, and 2 days after area officials received a message from a clinic in DRC warning of possible plague. The West Nile-based Uganda Virus Research Institute (UVRI) plague program, together with local health officials, commenced a multipronged response to suspected person-to-person transmission of pneumonic plague, including contact tracing, prophylaxis, and education. Plague was laboratory-confirmed, and no additional transmission occurred in Uganda. This event transpired in the context of heightened awareness of cross-border disease spread caused by ongoing Ebola virus disease transmission in DRC, approximately 400 km to the south. Building expertise in areas of plague endemicity can provide the rapid detection and effective response needed to mitigate epidemic spread and minimize mortality. Cross-border agreements can improve ability to respond effectively.

Two isolates of a Gram-negative, non-spore-forming coccobacillus cultured from the blood and cerebrospinal fluid of immunocompromised patients in the United States were described previously. Biochemical and phylogenetic analyses revealed that they belong to a novel species within the Francisella genus. Here we describe a third isolate of this species, recovered from blood of a febrile patient with renal failure, and formally name the Francisella species. Whole genome comparisons indicated the three isolates display greater than 99.9 % average nucleotide identity (ANI) to each other and are most closely related to the tick endosymbiont F. persica, with only 88.6-88.8 % ANI to the type strain of F. persica. Based on biochemical, metabolic and genomic comparisons, we propose that these three isolates should be recognized as Francisella opportunistica sp. nov, with the type strain of the species, PA05-1188(T), available through the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 107100) and the American Type Culture Collection (ATCC BAA-2974).

In response to the 2016 global public health emergency of international concern announced by the World Health Organization surrounding Zika virus (ZIKV) outbreaks, we developed a purified inactivated Zika virus vaccine (PIZV) candidate from ZIKV strain PRVABC59, isolated during the outbreak in 2015. The virus isolate was plaque purified, creating six sub-isolated virus stocks, two of which were selected to generate PIZV candidates for preclinical immunogenicity and efficacy evaluation in mice. The alum-adjuvanted PIZV candidates were highly immunogenic in both CD-1 and AG129 mice after a 2-dose immunization. Further, AG129 mice receiving 2 doses of PIZV formulated with alum were fully protected against lethal ZIKV challenge and mouse immune sera elicited by the PIZV candidates were capable of neutralizing ZIKVs of both African and Asian genetic lineages in vitro. Additionally, passive immunization of naive mice with ZIKV-immune serum showed strong positive correlation between neutralizing ZIKV antibody (NAb) titers and protection against lethal challenge. This study supported advancement of the PIZV candidate toward clinical development.

Although Arkansas lies within the geographic range of the principal Lyme disease tick vector, Ixodes scapularis, because of ecologic and entomologic factors, the risk for human infection is low, and no confirmed Lyme disease cases were reported in Arkansas during 2008–2014 (1). However, during 2015–2016, the Arkansas Department of Health (ADH) received several hundred potentially positive serologic laboratory reports for Lyme disease. Recommended serologic testing for Lyme disease is a two-tiered process; only if the first-tier enzyme immunoassay is positive or equivocal should the second-tier western blot be performed. A positive overall result can only be concluded when results of both individual tests are documented (2). Laboratory reports submitted to ADH during 2015–2016 did not always include complete or overall positive two-tiered serology results or associated clinical information needed to make a case determination. To facilitate Lyme disease surveillance in the setting of a high volume of reports and to ascertain whether local transmission of Lyme disease has occurred, ADH and CDC reviewed laboratory reports and clinical data, classified cases according to the surveillance definition, and investigated cases with potential for confirmation of Lyme disease.

Recombinant live-attenuated chimeric tetravalent dengue vaccine viruses, TDV-1, -2, -3, and -4, contain the premembrane and envelope genes of dengue virus serotypes 1-4 in the replicative background of the attenuated dengue virus type-2 (DENV-2) PDK-53 vaccine strain. Previous results have shown that these recombinant vaccine viruses demonstrate limited infection and dissemination in Aedes aegypti and are unlikely to be transmitted by the primary mosquito vector of DENVs. In this report, we expand this analysis by assessing vector competence of all four serotypes of the TDV virus in Aedes albopictus, the secondary mosquito vector of DENVs. Our results indicate that these vaccine viruses demonstrate incompetence or defective infection and dissemination in these mosquitoes and will likely not be transmissible.

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority ( approximately 75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development.

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7x more than sparrows, ~3.4x more than robins) and defective genomes (~1.4x greater than sparrows, ~2.7x greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6x lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

West Nile virus (WNV) replicates in a wide variety of avian species, which act as amplification hosts. In particular, WNV generates high titers and elicits severe pathology in American crows (AMCRs; Corvus brachyrhynchos), a species that has been used as a sentinel for WNV transmission. Although the specific cellular targets of WNV replication in AMCRs are not well defined, preliminary evidence suggests that leukocytes may be an important target of early replication. Therefore, development of a protocol for ex vivo culture of AMCR leukocytes as a model for assessing differential avian host susceptibility is described herein. WNV growth in these cultures mirrored in vivo viremia profiles. These data indicate that ex vivo leukocyte cultures can be used for preliminary pathological assessment of novel WNV strains and potentially of other flaviviruses that use avian reservoir hosts.