Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Differential Immunoregulation by Human Surfactant Protein A Variants Determines Severity of SARS-CoV-2-induced Lung Disease.

COVID-19 remains a significant threat to public health globally. Infection in some susceptible individuals causes life-threatening acute lung injury (ALI/ARDS) and/or death. Human surfactant protein A (SP-A) is a C-type lectin expressed in the lung and other mucosal tissues, and it plays a critical role in host defense against various pathogens. The human SP-A genes ( SFTPA1 and SFTPA2 ) are highly polymorphic and comprise several common genetic variants, i.e., SP-A1 (variants 6A 2 , 6A 4 ) and SP-A2 (variants 1A 0 , 1A 3 ). Here, we elucidated the differential antiviral and immunoregulatory roles of SP-A variants in response to SARS-CoV-2 infection in vivo . Six genetically-modified mouse lines, expressing both hACE2 (SARS-CoV-2 receptor) and individual SP-A variants: (hACE2/6A 2 (6A 2 ), hACE2/6A 4 (6A 4 ), hACE2/1A 0 (1A 0 ), and hACE2/1A 3 (1A 3 ), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were challenged intranasally with 10 3 PFU SARS-CoV-2 or saline (Sham). Infected KO and 1A 0 mice had more weight loss and mortality compared to other mouse lines. Relative to other infected mouse lines, a more severe ALI was observed in KO, 1A 0 , and 6A 2 mice. Reduced viral titers were generally observed in the lungs of infected SP-A mice relative to KO mice. Transcriptomic analysis revealed an upregulation in genes that play central roles in immune responses such as MyD88 , Stat3 , IL-18 , and Jak2 in the lungs of KO and 1A 0 mice. However, Mapk1 was significantly downregulated in 6A 2 versus 1A 0 mice. Analysis of biological pathways identified those involved in lung host defense and innate immunity, including pathogen-induced cytokine, NOD1/2, and Trem1 signaling pathways. Consistent with the transcriptomic data, levels of cytokines and chemokines such as G-CSF, IL-6 and IL-1ß were comparatively higher in the lungs and sera of KO and 1A 0 mice with the highest mortality rate. These findings demonstrate that human SP-A variants differentially modulate SARS-CoV-2-induced lung injury and disease severity by differentially inhibiting viral infectivity and regulating immune-related gene expressions.

The Lone Star tick, Amblyomma americanum, salivary factors exacerbate the clinical outcome of Heartland virus disease in a small animal model.

Heartland virus was first isolated in 2009 from two patients in Missouri and is transmitted by the Lone Star tick, Amblyomma americanum. To understand disease transmission and pathogenesis, it is necessary to develop an animal model which utilizes the natural route of transmission and manifests in a manner similar to documented human cases. Herein we describe our investigations on identifying A129 mice as the most appropriate small animal model for HRTV pathogenesis that mimics human clinical outcomes. We further investigated the impact of tick saliva in enhancing pathogen transmission and clinical outcomes. Our investigations revealed an increase in viral load in the groups of mice that received both virus and tick salivary gland extract (SGE). Spleens of all infected mice showed extramedullary hematopoiesis (EH), depleted white pulp, and absence of germinal centers. This observation mimics the splenomegaly observed in natural human cases. In the group that received both HRTV and tick SGE, the clinical outcome of HRTV infection was exacerbated compared to HRTV only infection. EH scores and the presence of viral antigens in spleen were higher in mice that received both HRTV and tick SGE. In conclusion, we have developed a small animal model that mimics natural human infection and also demonstrated the impact of tick salivary factors in exacerbating the HRTV infection.

The Lone Star Tick, Amblyomma americanum, salivary factors exacerbate the clinical outcome of Heartland virus disease in a small animal model.

Heartland virus was first isolated in 2009 from two patients in Missouri and is transmitted by the lone star tick, Amblyomma americanum. To understand disease transmission and pathogenesis, it is necessary to develop an animal model that utilizes the natural transmission route and manifests in a manner similar to documented human cases. Herein we describe our investigations on identifying A129 mice as the most appropriate small animal model for HRTV pathogenesis that mimics human clinical outcomes. We further investigated the impact of tick saliva in enhancing pathogen transmission and clinical outcomes. Our investigations revealed an increase in viral load in the groups of mice that received both virus and tick salivary gland extract (SGE). Spleens of all infected mice showed extramedullary hematopoiesis (EH), depleted white pulp, and absence of germinal centers. This observation mimics the splenomegaly observed in natural human cases. In the group that received both HRTV and tick SGE, the clinical outcome of HRTV infection was exacerbated compared to HRTV-only infection. EH scores and viral antigens in the spleen were higher in mice that received both HRTV and tick SGE. In conclusion, we have developed a small animal model that mimics natural human infection and also demonstrated the impact to tick salivary factors in exacerbating the HRTV infection.

Quantification and persistence of COVID-19 virus in recently deceased individuals before and after embalming.

The COVID-19 pandemic severely affected the medical education worldwide. The infection risk for medical students and healthcare personnel who work with COVID-19 positive cadavers or tissues remains unclear. Moreover, COVID-19 positive cadavers have been rejected by medical schools, adversely impacting the continuum of medical education. Herein, the viral genome abundance in tissues from four COVID-19 positive donors before and after embalming were compared. Tissue samples were collected from the lungs, liver, spleen, and brain both pre- and postembalming. The possible presence of infectious COVID-19 was determined by inoculating human tissue homogenates onto a monolayer of human A549-hACE2 cells and observing for cytopathic effects up to 72 h postinoculation. A real- time quantitative reverse transcription polymerase chain reaction was performed to quantify COVID-19 present in culture supernatants. Fully intact viral genome sequence was possible to obtain in samples with higher levels of virus, even several days postmortem. The embalming procedure described above substantially reduces the abundance of viable COVID-19 genomes in all tissues, sometimes even to undetectable levels. However, in some cases, COVID-19 RNA can still be detected, and a cytopathic effect can be seen both pre- and postembalmed tissues. This study suggests that embalmed COVID-19 positive cadavers might be used safely with appropriate precautions followed in gross anatomy laboratories and in clinical and scientific research. Deep lung tissue is the best specimen to test for the virus. If the tests on the lung tissues are negative, there is a very low likelihood that other tissues will show positive results.

Human attachment site preferences of ticks parasitizing in New York.

Ticks transmit several arthropod-borne pathogens in New York State. The primary human-biting ticks in this region are Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis. Body regions where tick bites human vary depending on the tick species and life stage, and clothing worn by the host. A community tick submission system was used to acquire information about bite-site location prior to pathogen testing to understand species and life stage-specific body-segment preferences. These data resulted in the identification of species-specific preferences for location, with D. variabilis preferentially biting the head and neck and A. americanum preferring the thighs, groin, and abdomen. Ixodes scapularis was found across the body, although it showed a significant life stage difference with adults preferring the head, midsection, and groin, while nymphs/larvae preferred the extremities. Infection with Borrelia burgdorferi resulted in a significant change in attachment site. This provides an assessment of which body region ticks of the most common species in New York are likely to be found.

Salivary gland extract from the deer tick, Ixodes scapularis, facilitates neuroinvasion by Powassan virus in BALB/c mice.

Powassan virus (POWV) is a neuroinvasive flavivirus transmitted to mammals by the bite of ixodid ticks. In this study, we sought to investigate the impact of tick salivary gland extract (SGE) on POWV neuroinvasion. BALB/c mice were footpad inoculated with either a high dose or a low dose of POWV, with and without Ixodes scapularis salivary gland extract. Brain and spinal cord were extracted daily, and immunohistochemical techniques were used for temporal tracking of POWV antigen. The temporal pattern of POWV staining showed a caudal to rostral spread of POWV in the brains of mice from both high dose infection groups. For the high dose infection groups, the presence of tick SGE did not influence the spread of POWV in the brain. Mice infected with the low dose of virus alone did not present POWV staining in the brain; however, in the presence of SGE, low dose infected mice presented scattered foci of POWV-infected cells throughout the brain. This study shows that tick SGE facilitates POWV neuroinvasion when mice are infected with the lower dose of POWV. We also found two patterns of central nervous system invasion that were directly influenced by the dose of POWV administered.

Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/µL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome.

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.

Detection of Rickettsiae, Borreliae, and Ehrlichiae in Ticks Collected from Walker County, Texas, 2017-2018.

Cases of tick-borne diseases, including spotted fever rickettsioses, borreliosis, babesiosis, anaplasmosis and ehrlichiosis, in the United States and territories have more than doubled from 2004 to 2016 and account for 77% of all vector-borne disease reports. In an effort to inform control efforts, the presence of tick-borne pathogens and their vectors was assessed in a recreational park in Walker County, Texas. Here we report data from questing ticks collected on three dates from June 2017 to June 2018. The majority of ticks collected were Amblyomma americanum (96.69%) followed by three additional tick species: Dermacentor variabilis (2.59%), Ixodes scapularis (0.52%), and A. maculatum (0.21%). Ticks were pooled and tested for molecular evidence of bacterial and viral pathogens, respectively. All of the 68 pools of A. americanum had molecular evidence of the spotted fever group rickettsia, Rickettsia amblyommatis. Additionally, six (8.82%) of the A. americanum pools contained sequences matching Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis, and 11 (16.18%) for E. ewingii. Three of the A. americanum pools demonstrated evidence of Borrelia lonestari. The presence of etiologic agents of known human disease in this study merits the continued surveillance efforts of ticks and their pathogens in areas where they could pose risks to public health.

An Overview of Animal Models for Arthropod-Borne Viruses.

Arthropod-borne viruses (arboviruses) have continued to emerge in recent years, posing a significant health threat to millions of people worldwide. The majority of arboviruses that are pathogenic to humans are transmitted by mosquitoes and ticks, but other types of arthropod vectors can also be involved in the transmission of these viruses. To alleviate the health burdens associated with arbovirus infections, it is necessary to focus today's research on disease control and therapeutic strategies. Animal models for arboviruses are valuable experimental tools that can shed light on the pathophysiology of infection and will enable the evaluation of future treatments and vaccine candidates. Ideally an animal model will closely mimic the disease manifestations observed in humans. In this review, we outline the currently available animal models for several viruses vectored by mosquitoes, ticks, and midges, for which there are no standardly available vaccines or therapeutics.

Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir.

Heartland virus (HRTV) is an emerging tick-borne virus (Bunyaviridae, Phlebovirus) that has caused sporadic cases of human disease in several central and mid-eastern states of America. Animal models of HRTV disease are needed to gain insights into viral pathogenesis and advancing antiviral drug development. Presence of clinical disease following HRTV challenge in hamsters deficient in STAT2 function underscores the important role played by type I interferon-induced antiviral responses. However, the recovery of most of the infected animals suggests that other mechanisms to control infection and limit disease offer substantial protection. The most prominent disease sign with HRTV infection in STAT2 knockout hamsters was dramatic weight loss with clinical laboratory and histopathology demonstrating acute inflammation in the spleen, lymph node, liver and lung. Finally, we show that HRTV disease in hamsters can be prevented by the use of favipiravir, a promising broad-spectrum antiviral in clinical development for the treatment of influenza.