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.

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.

Human-Pathogenic Kasokero Virus in Field-Collected Ticks.

Kasokero virus (KASV; genus Orthonairovirus) was first isolated in 1977 at Uganda Virus Research Institute from serum collected from Rousettus aegyptiacus bats captured at Kasokero Cave, Uganda. During virus characterization studies at the institute, 4 laboratory-associated infections resulted in mild to severe disease. Although orthonairoviruses are typically associated with vertebrate and tick hosts, a tick vector of KASV never has been reported. We tested 786 Ornithodoros (Reticulinasus) faini tick pools (3,930 ticks) for KASV. The ticks were collected from a large R. aegyptiacus bat roosting site in western Uganda. We detected KASV RNA in 43 tick pools and recovered 2 infectious isolates, 1 of which was derived from host blood-depleted ticks. Our findings suggest that KASV is maintained in an enzootic transmission cycle involving O. (R.) faini ticks and R. aegyptiacus bats and has the potential for incidental virus spillover to humans.

Antibody-Mediated Virus Neutralization Is Not a Universal Mechanism of Marburg, Ebola, or Sosuga Virus Clearance in Egyptian Rousette Bats.

Although bats are increasingly being recognized as natural reservoir hosts of emerging zoonotic viruses, little is known about how they control and clear virus infection in the absence of clinical disease. Here, we test >50 convalescent sera from Egyptian rousette bats (ERBs) experimentally primed or prime-boosted with Marburg virus, Ebola virus, or Sosuga virus for the presence of virus-specific neutralizing antibodies, using infectious reporter viruses. After serum neutralization testing, we conclude that antibody-mediated virus neutralization does not contribute significantly to the control and clearance of Marburg virus, Ebola virus, or Sosuga virus infection in ERBs.

Humanized Mouse Model of Ebola Virus Disease Mimics the Immune Responses in Human Disease.

Animal models recapitulating human Ebola virus disease (EVD) are critical for insights into virus pathogenesis. Ebola virus (EBOV) isolates derived directly from human specimens do not, without adaptation, cause disease in immunocompetent adult rodents. Here, we describe EVD in mice engrafted with human immune cells (hu-BLT). hu-BLT mice developed EVD following wild-type EBOV infection. Infection with high-dose EBOV resulted in rapid, lethal EVD with high viral loads, alterations in key human antiviral immune cytokines and chemokines, and severe histopathologic findings similar to those shown in the limited human postmortem data available. A dose- and donor-dependent clinical course was observed in hu-BLT mice infected with lower doses of either Mayinga (1976) or Makona (2014) isolates derived from human EBOV cases. Engraftment of the human cellular immune system appeared to be essential for the observed virulence, as nonengrafted mice did not support productive EBOV replication or develop lethal disease. hu-BLT mice offer a unique model for investigating the human immune response in EVD and an alternative animal model for EVD pathogenesis studies and therapeutic screening.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015.

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens.

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 10(7) to 4 × 10(2) 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 10(2) TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD.

Molecular evolution of viruses of the family Filoviridae based on 97 whole-genome sequences.

Viruses in the Ebolavirus and Marburgvirus genera (family Filoviridae) have been associated with large outbreaks of hemorrhagic fever in human and nonhuman primates. The first documented cases occurred in primates over 45 years ago, but the amount of virus genetic diversity detected within bat populations, which have recently been identified as potential reservoir hosts, suggests that the filoviruses are much older. Here, detailed Bayesian coalescent phylogenetic analyses are performed on 97 whole-genome sequences, 55 of which are newly reported, to comprehensively examine molecular evolutionary rates and estimate dates of common ancestry for viruses within the family Filoviridae. Molecular evolutionary rates for viruses belonging to different species range from 0.46 × 10(-4) nucleotide substitutions/site/year for Sudan ebolavirus to 8.21 × 10(-4) nucleotide substitutions/site/year for Reston ebolavirus. Most recent common ancestry can be traced back only within the last 50 years for Reston ebolavirus and Zaire ebolavirus species and suggests that viruses within these species may have undergone recent genetic bottlenecks. Viruses within Marburg marburgvirus and Sudan ebolavirus species can be traced back further and share most recent common ancestors approximately 700 and 850 years before the present, respectively. Examination of the whole family suggests that members of the Filoviridae, including the recently described Lloviu virus, shared a most recent common ancestor approximately 10,000 years ago. These data will be valuable for understanding the evolution of filoviruses in the context of natural history as new reservoir hosts are identified and, further, for determining mechanisms of emergence, pathogenicity, and the ongoing threat to public health.

Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human infection.

Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ~2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (~six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

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.

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.

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.

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.

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.

Histopathologic and Immunohistochemical Evaluation of Induced Lesions, Tissue Tropism and Host Responses following Experimental Infection of Egyptian Rousette Bats (Rousettus aegyptiacus) with the Zoonotic Paramyxovirus, Sosuga Virus.

Ecological and experimental infection studies have identified Egyptian rousette bats (ERBs; Rousettus aegyptiacus: family Pteropodidae) as a reservoir host for the zoonotic rubula-like paramyxovirus Sosuga virus (SOSV). A serial sacrifice study of colony-bred ERBs inoculated with wild-type, recombinant SOSV identified small intestines and salivary gland as major sites of viral replication. In the current study, archived formalin-fixed paraffin-embedded (FFPE) tissues from the serial sacrifice study were analyzed in depth-histologically and immunohistochemically, for SOSV, mononuclear phagocytes and T cells. Histopathologic lesion scores increased over time and viral antigen persisted in a subset of tissues, indicating ongoing host responses and underscoring the possibility of chronic infection. Despite the presence of SOSV NP antigen and villus ulcerations in the small intestines, there were only mild increases in mononuclear phagocytes and T cells, a host response aligned with disease tolerance. In contrast, there was a statistically significant, robust and targeted mononuclear phagocyte cell responses in the salivary glands at 21 DPI, where viral antigen was sparse. These findings may have broader implications for chiropteran-paramyxovirus interactions, as bats are hypothesized to be the ancestral hosts of this diverse virus family and for ERB immunology in general, as this species is also the reservoir host for the marburgviruses Marburg virus (MARV) and Ravn virus (RAVV) (family Filoviridae).

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.

Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

Subgenomic flavivirus RNA (sfRNA) associated with Asian lineage Zika virus identified in three species of Ugandan bats (family Pteropodidae).

Serological cross-reactivity among flaviviruses makes determining the prior arbovirus exposure of animals challenging in areas where multiple flavivirus strains are circulating. We hypothesized that prior infection with ZIKV could be confirmed through the presence of subgenomic flavivirus RNA (sfRNA) of the 3' untranslated region (UTR), which persists in tissues due to XRN-1 stalling during RNA decay. We amplified ZIKV sfRNA but not NS5 from three experimentally-infected Jamaican fruit bats, supporting the hypothesis of sfRNA tissue persistence. Applying this approach to 198 field samples from Uganda, we confirmed presence of ZIKV sfRNA, but not NS5, in four bats representing three species: Eidolon helvum (n = 2), Epomophorus labiatus (n = 1), and Rousettus aegyptiacus (n = 1). Amplified sequence was most closely related to Asian lineage ZIKV. Our results support the use of sfRNA as a means of identifying previous flavivirus infection and describe the first detection of ZIKV RNA in East African bats.

Asymptomatic Infection of Marburg Virus Reservoir Bats Is Explained by a Strategy of Immunoprotective Disease Tolerance.

Marburg virus (MARV) is among the most virulent pathogens of primates, including humans. Contributors to severe MARV disease include immune response suppression and inflammatory gene dysregulation ("cytokine storm"), leading to systemic damage and often death. Conversely, MARV causes little to no clinical disease in its reservoir host, the Egyptian rousette bat (ERB). Previous genomic and in vitro data suggest that a tolerant ERB immune response may underlie MARV avirulence, but no significant examination of this response in vivo yet exists. Here, using colony-bred ERBs inoculated with a bat isolate of MARV, we use species-specific antibodies and an immune gene probe array (NanoString) to temporally characterize the transcriptional host response at sites of MARV replication relevant to primate pathogenesis and immunity, including CD14+ monocytes/macrophages, critical immune response mediators, primary MARV targets, and skin at the inoculation site, where highest viral loads and initial engagement of antiviral defenses are expected. Our analysis shows that ERBs upregulate canonical antiviral genes typical of mammalian systems, such as ISG15, IFIT1, and OAS3, yet demonstrate a remarkable lack of significant induction of proinflammatory genes classically implicated in primate filoviral pathogenesis, including CCL8, FAS, and IL6. Together, these findings offer the first in vivo functional evidence for disease tolerance as an immunological mechanism by which the bat reservoir asymptomatically hosts MARV. More broadly, these data highlight factors determining disparate outcomes between reservoir and spillover hosts and defensive strategies likely utilized by bat hosts of other emerging pathogens, knowledge that may guide development of effective antiviral therapies.