Stability of Aerosolized SARS-CoV-2 on Masks and Transfer to Skin.

The potential for masks to act as fomites in the transmission of SARS-CoV-2 has been suggested but not demonstrated experimentally or observationally. In this study, we aerosolized a suspension of SARS-CoV-2 in saliva and used a vacuum pump to pull the aerosol through six different types of masks. After 1 h at 28 °C and 80% RH, SARS-CoV-2 infectivity was not detectable on an N95 and surgical mask, was reduced by 0.7 log10 on a nylon/spandex mask, and was unchanged on a polyester mask and two different cotton masks when recovered by elution in a buffer. SARS-CoV-2 RNA remained stable for 1 h on all masks. We pressed artificial skin against the contaminated masks and detected the transfer of viral RNA but no infectious virus to the skin. The potential for masks contaminated with SARS-CoV-2 in aerosols to act as fomites appears to be less than indicated by studies involving SARS-CoV-2 in very large droplets.

Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania.

Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.

Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of Droplets.

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 µL) and relative humidity (RH; 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage Phi6 (a common surrogate for enveloped viruses), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e., a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with the initial droplet volume. Virus decay rates differed between the wet phase, while the droplets were still evaporating, and the dry phase. For Phi6, decay was faster in the wet phase than in the dry phase under most conditions. For H1N1pdm09, decay rates between the two phases were distinct and initial droplet volume had an effect on virus viability within 2 h. Importantly, we observed differences in virus decay characteristics by droplet size and virus. In general, influenza virus and SARS-CoV-2 decayed similarly, whereas Phi6 decayed more rapidly under certain conditions. Overall, this study suggests that virus decay in media is related to the extent of droplet evaporation, which is controlled by RH. Importantly, accurate assessment of transmission risk requires the use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. IMPORTANCE During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments with SARS-CoV-2 in different droplet volumes under various humidity conditions. Our study, the first of its kind, provides a model for the decay of multiple enveloped RNA viruses in cell culture medium deposited in 50-, 5-, and 1-µL droplets at 40%, 65%, and 85% RH over time. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets may overestimate transmission risk for contaminated surfaces, as observed during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

North American House Sparrows Are Competent for Usutu Virus Transmission.

Usutu virus (USUV, Flaviviridae) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log10 PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

Environmental Stability of Enveloped Viruses is Impacted by the Initial Volume and Evaporation Kinetics of Droplets.

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 µL) and relative humidity (RH: 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage, Phi6, a common surrogate for enveloped viruses, and SARS-CoV-2 under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e. a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with initial droplet volume. We observed more rapid virus decay when the droplets were still wet and undergoing evaporation, and slower decay after the droplets had dried. Initial droplet volume had a major effect on virus viability over the first few hours; whereby the decay rate of influenza virus was faster in smaller droplets. In general, influenza virus and SARS-CoV-2 decayed similarly. Overall, this study suggests that virus decay in media is closely correlated with the extent of droplet evaporation, which is controlled by RH. Taken together, these data suggest that decay of different viruses is more similar at higher RH and in smaller droplets and is distinct at lower RH and in larger droplets. Importantly, accurate assessment of transmission risk requires use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. Funding: National Institute of Allergy and Infectious Diseases, National Institute of Neurological Disorders and Stroke, National Institutes of Health; Department of Health and Human Services; Flu Lab. Importance: During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments involving SARS-CoV-2 applied to surfaces in large droplets at various humidity conditions. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets likely over-estimates transmission risk for contaminated surfaces, as occurred during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

Association Between Prolonged Shedding of Zika Virus in Human Semen and Male Reproductive Tract Inflammation.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes congenital defects. Sexual transmission of ZIKV was confirmed in a recent epidemic; however, mechanisms behind ZIKV infection and persistence in the male reproductive tract (MRT) are unknown. Previously, we found that approximately 33% of men with symptomatic ZIKV infections shed ZIKV RNA in semen, and some men shed ZIKV RNA for >3 months. Here, we evaluated the semen of 49 ZIKV-infected men to identify immune factors correlating with long-term ZIKV shedding in semen and ZIKV-infected cell types in semen. We found that prolonged ZIKV RNA shedding in semen was associated with MRT inflammation, indicated by higher leukocyte counts and inflammatory cytokine concentrations in semen of long-term versus short-term shedders. In addition, we found ZIKV RNA in seminal leukocytes and epithelial cells. This study of human semen from ZIKV-infected men provides critical insights into the effects of ZIKV on MRT health.

Correction: Differential pathogenesis of Usutu virus isolates in mice.

[This corrects the article DOI: 10.1371/journal.pntd.0008765.].

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1-/- mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1-/- model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.

Infectious SARS-CoV-2 Is Emitted in Aerosol Particles.

Respiratory viruses such as SARS-CoV-2 are transmitted in respiratory droplets and aerosol particles, which are released during talking, breathing, coughing, and sneezing. Noncontact transmission of SARS-CoV-2 has been demonstrated, suggesting transmission via virus carried through the air. Here, we demonstrate that golden Syrian hamsters produce infectious SARS-CoV-2 in aerosol particles prior to and concurrent with the onset of mild clinical signs of disease. The average emission rate in this study was 25 infectious virions/hour on days 1 and 2 postinoculation, with average viral RNA levels 200-fold higher than infectious virus in aerosol particles. The majority of virus was contained within particles <5 µm in size. Thus, we provide direct evidence that, in hamsters, SARS-CoV-2 is an airborne virus. IMPORTANCE SARS-CoV-2 is a respiratory virus and has been isolated from the air near COVID-19 patients. Here, using a hamster model of infection, we demonstrate that SARS-CoV-2 is emitted in aerosol particles prior to and concurrent with the onset of mild disease. Virus is contained primarily within aerosol particles <5 µm in size, which can remain airborne and be inhaled. These findings indicate that SARS-CoV-2 is an airborne virus and support the use of ventilation to reduce SARS-CoV-2 transmission.

Modeling the dynamics of Usutu virus infection in birds.

Usutu virus is an emerging zoonotic flavivirus causing high avian mortality rates and occasional severe neurological disorders in humans. Several virus strains are co-circulating and the differences in their characteristics and avian pathogenesis levels are still unknown. In this study, we use within-host mathematical models to characterize the mechanisms responsible for virus expansion and clearance in juvenile chickens challenged with four Usutu virus strains. We find heterogeneity between the virus strains, with the time between cell infection and viral production varying between 16 h and 23 h, the infected cell lifespan varying between 48 min and 9.5 h, and the basic reproductive number R0 varying between 12.05 and 19.49. The strains with high basic reproductive number have short infected cell lifespan, indicative of immune responses. The virus strains with low basic reproductive number have lower viral peaks and longer lasting viremia, due to lower infection rates and high infected cell lifespan. We discuss how the host and virus heterogeneities may differently impact the public health threat presented by these virus strains.

Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis.

The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.

Persistence of Zika virus RNA in the epididymis of the murine male reproductive tract.

Zika virus (ZIKV) can infect developing fetuses in utero and cause severe congenital defects independent of route of maternal infection. Infected men can shed ZIKV RNA in semen for over six months. Whether prolonged viral RNA shedding in semen indicates a persistent infection in the male reproductive tract is unknown. We hypothesized that if ZIKV establishes a persistent infection in the male reproductive tract (MRT), then immunosuppressant treatment should stimulate ZIKV replication and seminal shedding. Male mice were infected with ZIKV and immunosuppressed when they shed viral RNA but not infectious virus in ejaculates. Following immunosuppression, we did not detect infectious virus in ejaculates. However, we did detect ZIKV positive and negative sense RNA in the epididymal lumens of mice treated with cyclophosphamide, suggesting that ZIKV persists in the epididymis. This study provides insight into the mechanisms behind ZIKV sexual transmission, which may inform public health decisions regarding ZIKV risks.

Pathogenesis and shedding of Usutu virus in juvenile chickens.

Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Development and characterization of infectious clones of two strains of Usutu virus.

Usutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. Here, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in mammalian and insect cells. Additionally, we observed similar levels of replication and disease in two mouse models. These clones will aid the study of USUV infection, transmission, diagnostics, and vaccines.

Differential pathogenesis of Usutu virus isolates in mice.

Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/ß receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern.

On the Fly: Interactions Between Birds, Mosquitoes, and Environment That Have Molded West Nile Virus Genomic Structure Over Two Decades.

West Nile virus (WNV) was first identified in North America almost 20 yr ago. In that time, WNV has crossed the continent and established enzootic transmission cycles, resulting in intermittent outbreaks of human disease that have largely been linked with climatic variables and waning avian seroprevalence. During the transcontinental dissemination of WNV, the original genotype has been displaced by two principal extant genotypes which contain an envelope mutation that has been associated with enhanced vector competence by Culex pipiens L. (Diptera: Culicidae) and Culex tarsalis Coquillett vectors. Analyses of retrospective avian host competence data generated using the founding NY99 genotype strain have demonstrated a steady reduction in viremias of house sparrows over time. Reciprocally, the current genotype strains WN02 and SW03 have demonstrated an inverse correlation between house sparrow viremia magnitude and the time since isolation. These data collectively indicate that WNV has evolved for increased avian viremia while house sparrows have evolved resistance to the virus such that the relative host competence has remained constant. Intrahost analyses of WNV evolution demonstrate that selection pressures are avian species-specific and purifying selection is greater in individual birds compared with individual mosquitoes, suggesting that the avian adaptive and/or innate immune response may impose a selection pressure on WNV. Phylogenomic, experimental evolutionary systems, and models that link viral evolution with climate, host, and vector competence studies will be needed to identify the relative effect of different selective and stochastic mechanisms on viral phenotypes and the capacity of newly evolved WNV genotypes for transmission in continuously changing landscapes.

Duration of seminal Zika viral RNA shedding in immunocompetent mice inoculated with Asian and African genotype viruses.

Prior to the emergence of Asian genotype Zika virus (ZIKV) in the Western hemisphere, sexual transmission in humans was documented. Sexual transmission by African genotype ZIKVs has not been assessed in laboratory animal models, due to rapid and high mortality rates of immunodeficient mice following inoculation. To overcome these limitations, immunocompetent C57Bl/6 mice were used to longitudinally assess Asian and African genotype ZIKV sexual transmission potential. Furthermore, to determine if enhanced pathogenesis of African genotype ZIKVs is due to structural determinants, PRVABC59 prM/E was replaced with African MR766 prM/E (chimeric ZIKV). The African genotype and chimeric ZIKV elicited greater pathogenic effects in the male reproductive tract and generated higher viremias. Yet, the duration, magnitude and efficiency of seminal shedding of infectious virus and viral RNA were similar between chimeric-, African and Asian genotype ZIKV-inoculated mice. These data show that increased male reproductive tract pathology does not increase sexual transmission potential.

Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in mice.

Zika virus (ZIKV) infection can result in neurological disorders including Congenital Zika Syndrome in infants exposed to the virus in utero. Pregnant women can be infected by mosquito bite as well as by sexual transmission from infected men. Herein, the variants of ZIKV within the male reproductive tract and ejaculates were assessed in inoculated mice. We identified two non-synonymous variants at positions E-V330L and NS1-W98G. These variants were also present in the passage three PRVABC59 isolate and infectious clone relative to the patient serum PRVABC59 sequence. In subsequent studies, ZIKV E-330L was less pathogenic in mice than ZIKV E-330V as evident by increased average survival times. In Vero cells, ZIKV E-330L/NS1-98G outcompeted ZIKV E-330V/NS1-98W within 3 passages. These results suggest that the E-330L/NS1-98G variants are attenuating in mice and were enriched during cell culture passaging. Cell culture propagation of ZIKV could significantly affect animal model development and vaccine efficacy studies.

Heartland Virus Epidemiology, Vector Association, and Disease Potential.

First identified in two Missouri farmers exhibiting low white-blood-cell and platelet counts in 2009, Heartland virus (HRTV) is genetically closely related to severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne phlebovirus producing similar symptoms in China, Korea, and Japan. Field isolations of HRTV from several life stages of unfed, host-seeking Amblyomma americanum, the lone star tick, implicated it as a putative vector capable of transstadial transmission. Laboratory vector competence assessments confirmed transstadial transmission of HRTV, demonstrated vertical infection, and showed co-feeding infection between A. americanum. A vertical infection rate of 33% from adult females to larvae in the laboratory was observed, while only one of 386 pools of molted nymphs (1930) reared from co-feeding larvae was positive for HRTV (maximum-likelihood estimate of infection rate = 0.52/1000). Over 35 human HRTV cases, all within the distribution range of A. americanum, have been documented. Serological testing of wildlife in areas near the index human cases, as well as in widely separated regions of the eastern United States where A. americanum occur, indicated many potential hosts such as raccoons and white-tailed deer. Attempts, however, to experimentally infect mice, rabbits, hamsters, chickens, raccoons, goats, and deer failed to produce detectable viremia. Immune-compromised mice and hamsters are the only susceptible models. Vertical infection augmented by co-feeding transmission could play a role in maintaining the virus in nature. A more complete assessment of the natural transmission cycle of HRTV coupled with serosurveys and enhanced HRTV disease surveillance are needed to better understand transmission dynamics and human health risks.