Human Orthohantavirus disease prevalence and genotype distribution in the U.S., 2008-2020: a retrospective observational study.

Background: In the United States (U.S.), hantavirus pulmonary syndrome (HPS) and non-HPS hantavirus infection are nationally notifiable diseases. Criteria for identifying human cases are based on clinical symptoms (HPS or non-HPS) and acute diagnostic results (IgM+, rising IgG+ titers, RT-PCR+, or immunohistochemistry (IHC)+). Here we provide an overview of diagnostic testing and summarize human Hantavirus disease occurrence and genotype distribution in the U.S. from 2008 to 2020. Methods: Epidemiological data from the national hantavirus registry was merged with laboratory diagnostic testing results performed at the CDC. Residual hantavirus-positive specimens were sequenced, and the available epidemiological and genetic data sets were linked to conduct a genomic epidemiological study of hantavirus disease in the U.S. Findings: From 1993 to 2020, 833 human hantavirus cases have been identified, and from 2008 to 2020, 335 human cases have occurred. Among New World (NW) hantavirus cases detected at the CDC diagnostic laboratory (representing 29.2% of total cases), most (85.0%) were detected during acute disease, however, some convalescent cases were detected in states not traditionally associated with hantavirus infections (Connecticut, Missouri, New Jersey, Pennsylvania, Tennessee, and Vermont). From 1993 to 2020, 94.9% (745/785) of U.S. hantaviruses cases were detected west of the Mississippi with 45.7% (359/785) in the Four Corners region of the U.S. From 2008 to 2020, 67.7% of NW hantavirus cases were detected between the months of March and August. Sequencing of RT-PCR-positive cases demonstrates a geographic separation of Orthohantavirus sinnombreense species [Sin Nombre virus (SNV), New York virus, and Monongahela virus]; however, there is a large gap in viral sequence data from the Northwestern and Central U.S. Finally, these data indicate that commercial IgM assays are not concordant with CDC-developed assays, and that "concordant positive" (i.e., commercial IgM+ and CDC IgM+ results) specimens exhibit clinical characteristics of hantavirus disease. Interpretation: Hantaviral disease is broadly distributed in the contiguous U.S, viral variants are localised to specific geographic regions, and hantaviral disease infrequently detected in most Southeastern states. Discordant results between two diagnostic detection methods highlight the need for an improved standardised testing plan in the U.S. Hantavirus surveillance and detection will continue to improve with clearly defined, systematic reporting methods, as well as explicit guidelines for clinical characterization and diagnostic criteria. Funding: This work was funded by core funds provided to the Viral Special Pathogens Branch at CDC.

Sosuga Virus Detected in Egyptian Rousette Bats (Rousettus aegyptiacus) in Sierra Leone.

Sosuga virus (SOSV), a rare human pathogenic paramyxovirus, was first discovered in 2012 when a person became ill after working in South Sudan and Uganda. During an ecological investigation, several species of bats were sampled and tested for SOSV RNA and only one species, the Egyptian rousette bat (ERBs; Rousettus aegyptiacus), tested positive. Since that time, multiple other species have been sampled and ERBs in Uganda have continued to be the only species of bat positive for SOSV infection. Subsequent studies of ERBs with SOSV demonstrated that ERBs are a competent host for SOSV and shed this infectious virus while exhibiting only minor infection-associated pathology. Following the 2014 Ebola outbreak in West Africa, surveillance efforts focused on discovering reservoirs for zoonotic pathogens resulted in the capture and testing of many bat species. Here, SOSV RNA was detected by qRT-PCR only in ERBs captured in the Moyamba District of Sierra Leone in the central region of the country. These findings represent a substantial range extension from East Africa to West Africa for SOSV, suggesting that this paramyxovirus may occur in ERB populations throughout its sub-Saharan African range.

Coordinated inflammatory responses dictate Marburg virus control by reservoir bats.

Bats are increasingly recognized as reservoirs of emerging zoonotic pathogens. Egyptian rousette bats (ERBs) are the known reservoir of Marburg virus (MARV), a filovirus that causes deadly Marburg virus disease (MVD) in humans. However, ERBs harbor MARV asymptomatically, likely due to a coadapted and specific host immunity-pathogen relationship. Recently, we measured transcriptional responses in MARV-infected ERB whole tissues, showing that these bats possess a disease tolerant strategy that limits pro-inflammatory gene induction, presumably averting MVD-linked immunopathology. However, the host resistant strategy by which ERBs actively limit MARV burden remains elusive, which we hypothesize requires localized inflammatory responses unresolvable at bulk-tissue scale. Here, we use dexamethasone to attenuate ERB pro-inflammatory responses and assess MARV replication, shedding and disease. We show that MARV-infected ERBs naturally mount coordinated pro-inflammatory responses at liver foci of infection, comprised of recruited mononuclear phagocytes and T cells, the latter of which proliferate with likely MARV-specificity. When pro-inflammatory responses are diminished, ERBs display heightened MARV replication, oral/rectal shedding and severe MVD-like liver pathology, demonstrating that ERBs balance immunoprotective tolerance with discreet MARV-resistant pro-inflammatory responses. These data further suggest that natural ERB immunomodulatory stressors like food scarcity and habitat disruption may potentiate viral shedding, transmission and therefore outbreak risk.

Micro‒Global Positioning Systems for Identifying Nightly Opportunities for Marburg Virus Spillover to Humans by Egyptian Rousette Bats.

Marburg virus disease, caused by Marburg and Ravn orthomarburgviruses, emerges sporadically in sub-Saharan Africa and is often fatal in humans. The natural reservoir is the Egyptian rousette bat (ERB), which sheds virus in saliva, urine, and feces. Frugivorous ERBs discard test-bitten and partially eaten fruit, potentially leaving infectious virus behind that could be consumed by other susceptible animals or humans. Historically, 8 of 17 known Marburg virus disease outbreaks have been linked to human encroachment on ERB habitats, but no linkage exists for the other 9 outbreaks, raising the question of how bats and humans might intersect, leading to virus spillover. We used micro‒global positioning systems to identify nightly ERB foraging locations. ERBs from a known Marburg virus‒infected population traveled long distances to feed in cultivated fruit trees near homes. Our results show that ERB foraging behavior represents a Marburg virus spillover risk to humans and plausibly explains the origins of some past outbreaks.

Tick salivary gland components dampen Kasokero virus infection and shedding in its vertebrate reservoir, the Egyptian rousette bat (Rousettus aegyptiacus).

BACKGROUND: The human-pathogenic Kasokero virus (KASV) circulates in an enzootic transmission cycle between Egyptian rousette bats (ERBs; Rousettus aegyptiacus) and their argasid tick ectoparasites, Ornithodoros (Reticulinasus) faini. Although tick salivary gland components have been shown to potentiate virus infection in vertebrate non-reservoirs (i.e. incidental hosts or small animal models of disease), there is a lack of information on the effect of tick salivary gland components on viral infection and shedding in vertebrate reservoirs. METHODS: To determine the impact of tick salivary gland components on KASV infection and shedding in ERBs, KASV loads were quantified in blood, oral swab, rectal swab, and urine specimens collected daily through 18 days post inoculation from groups of ERBs intradermally inoculated with KASV or KASV + O. (R.) faini tick salivary gland extract (SGE). RESULTS: Bats inoculated with KASV + tick SGE had significantly lower peak and cumulative KASV viremias and rectal shedding loads compared to bats inoculated with KASV only. CONCLUSIONS: We report for the first time to our knowledge that tick salivary gland components dampen arbovirus infection and shedding in a vertebrate reservoir. This study advances our understanding of biological factors underlying arbovirus maintenance in nature.

Pathogenesis of Kasokero virus in experimentally infected Egyptian rousette bats (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs; Rousettus aegyptiacus; family Pteropodidae) are associated with a growing number of bunyaviruses of public health importance, including Kasokero virus (KASV), which was first identified as a zoonosis in Uganda in 1977. In this study, formalin-fixed paraffin-embedded tissues from a previous experiment in which KASV infection was confirmed in 18 experimentally infected ERBs were used for an in-depth analysis using histopathology, in situ hybridization (ISH) for detection of viral RNA, immunohistochemistry (IHC) to assess the mononuclear phagocyte system response, and quantitative digital image analysis to investigate virus clearance from the liver and spleen within a spatial context. Significant gross and histological lesions were limited to the liver, where KASV-infected bats developed mild to moderate, acute viral hepatitis, which was first observed at 3 days postinfection (DPI), peaked at 6 DPI, and was resolved by 20 DPI. A subset of bats had glycogen depletion (n = 10) and hepatic necrosis (n = 3), rarely with intralesional bacteria (n = 1). Virus replication was confirmed by ISH in the liver, spleen, lymph nodes, and tongue. In the liver, KASV replicated in the cytoplasm of hepatocytes, to a lesser extent in mononuclear phagocytes, and rarely in presumptive endothelial cells. Most KASV RNA, as detected by ISH, was cleared from the spleen and liver by 6 DPI. It is concluded that ERBs have effective mechanisms to respond to this virus, clearing it without evidence of clinical disease.

Peripheral immune responses to filoviruses in a reservoir versus spillover hosts reveal transcriptional correlates of disease.

Several filoviruses, including Marburg virus (MARV), cause severe disease in humans and nonhuman primates (NHPs). However, the Egyptian rousette bat (ERB, Rousettus aegyptiacus), the only known MARV reservoir, shows no overt illness upon natural or experimental infection, which, like other bat hosts of zoonoses, is due to well-adapted, likely species-specific immune features. Despite advances in understanding reservoir immune responses to filoviruses, ERB peripheral blood responses to MARV and how they compare to those of diseased filovirus-infected spillover hosts remain ill-defined. We thus conducted a longitudinal analysis of ERB blood gene responses during acute MARV infection. These data were then contrasted with a compilation of published primate blood response studies to elucidate gene correlates of filovirus protection versus disease. Our work expands on previous findings in MARV-infected ERBs by supporting both host resistance and disease tolerance mechanisms, offers insight into the peripheral immunocellular repertoire during infection, and provides the most direct known cross-examination between reservoir and spillover hosts of the most prevalently-regulated response genes, pathways and activities associated with differences in filovirus pathogenesis and pathogenicity.

Exposure of Egyptian Rousette Bats (Rousettus aegyptiacus) and a Little Free-Tailed Bat (Chaerephon pumilus) to Alphaviruses in Uganda.

The reservoir for zoonotic o'nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats.

Natural reservoir Rousettus aegyptiacus bat host model of orthonairovirus infection identifies potential zoonotic spillover mechanisms.

The human-pathogenic Kasokero virus (KASV; genus Orthonairovirus) has been isolated from the sera of Egyptian rousette bats (ERBs; Rousettus aegyptiacus) captured in Uganda and unengorged Ornithodoros (Reticulinasus) faini ticks collected from the rock crevices of ERB colonies in South Africa and Uganda. Although evidence suggests that KASV is maintained in an enzootic transmission cycle between O. (R.) faini ticks and ERBs with potential for incidental virus spillover to humans through the bite of an infected tick, the vertebrate reservoir status of ERBs for KASV has never been experimentally evaluated. Furthermore, the potential for bat-to-bat and bat-to-human transmission of KASV is unknown. Herein, we inoculate two groups of ERBs with KASV; one group of bats is serially sampled to assess viremia, oral, fecal, and urinary shedding and the second group of bats is serially euthanized to assess virus-tissue tropism. Throughout the study, none of the bats exhibit overt signs of clinical disease. Following the detection of high KASV loads of long duration in blood, oral, fecal, and urine specimens collected from ERBs in the serial sampling group, all bats seroconvert to KASV. ERBs from the serial euthanasia group exhibit high KASV loads indicative of virus replication in the skin at the inoculation site, spleen, and inguinal lymph node tissue, and histopathology and in situ hybridization reveal virus replication in the liver and self-limiting, KASV-induced lymphohistiocytic hepatitis. The results of this study suggest that ERBs are competent, natural vertebrate reservoir hosts for KASV that can sustain viremias of appropriate magnitude and duration to support virus maintenance through bat-tick-bat transmission cycles. Viral shedding data suggests that KASV might also be transmitted bat-to-bat and highlights the potential for KASV spillover to humans through contact with infectious oral secretions, feces, or urine.

GPS Tracking of Free-Roaming Cats (Felis catus) on SARS-CoV-2-Infected Mink Farms in Utah.

Zoonotic transmission of SARS-CoV-2 from infected humans to other animals has been documented around the world, most notably in mink farming operations in Europe and the United States. Outbreaks of SARS-CoV-2 on Utah mink farms began in late July 2020 and resulted in high mink mortality. An investigation of these outbreaks revealed active and past SARS-CoV-2 infections in free-roaming and in feral cats living on or near several mink farms. Cats were captured using live traps, were sampled, fitted with GPS collars, and released on the farms. GPS tracking of these cats show they made frequent visits to mink sheds, moved freely around the affected farms, and visited surrounding residential properties and neighborhoods on multiple occasions, making them potential low risk vectors of additional SARS-CoV-2 spread in local communities.