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.

Detection of Sporadic Outbreaks of Rift Valley Fever in Uganda through the National Viral Hemorrhagic Fever Surveillance System, 2017-2020.

Rift Valley fever (RVF) is a zoonotic disease of public health and economic importance. Uganda has reported sporadic outbreaks of RVF in both humans and animals across the country, especially in the southwestern part of the "cattle corridor" through an established viral hemorrhagic fever surveillance system. We report 52 human cases of laboratory-confirmed RVF from 2017 to 2020. The case fatality rate was 42%. Among those infected, 92% were males and 90% were adults (≥ 18 years). Clinical symptoms were characterized by fever (69%), unexplained bleeding (69%), headache (51%), abdominal pain (49%), and nausea and vomiting (46%). Most of the cases (95%) originated from central and western districts that are part of the cattle corridor of Uganda, where the main risk factor was direct contact with livestock (P = 0.009). Other predictors of RVF positivity were determined to be male gender (P = 0.001) and being a butcher (P = 0.04). Next-generation sequencing identified the predominant Ugandan clade as Kenya-2, observed previously across East Africa. There is need for further investigation and research into the effect and spread of this neglected tropical disease in Uganda and the rest of Africa. Control measures such as promoting vaccination and limiting animal-human transmission could be explored to reduce the impact of RVF in Uganda and globally.

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.

Risk Factors for Ebola Virus Persistence in Semen of Survivors in Liberia.

BACKGROUND: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

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.

Chapare Hemorrhagic Fever and Virus Detection in Rodents in Bolivia in 2019.

BACKGROUND: In June 2019, the Bolivian Ministry of Health reported a cluster of cases of hemorrhagic fever that started in the municipality of Caranavi and expanded to La Paz. The cause of these cases was unknown. METHODS: We obtained samples for next-generation sequencing and virus isolation. Human and rodent specimens were tested by means of virus-specific real-time quantitative reverse-transcriptase-polymerase-chain-reaction assays, next-generation sequencing, and virus isolation. RESULTS: Nine cases of hemorrhagic fever were identified; four of the patients with this illness died. The etiologic agent was identified as Mammarenavirus Chapare mammarenavirus, or Chapare virus (CHAPV), which causes Chapare hemorrhagic fever (CHHF). Probable nosocomial transmission among health care workers was identified. Some patients with CHHF had neurologic manifestations, and those who survived had a prolonged recovery period. CHAPV RNA was detected in a variety of human body fluids (including blood; urine; nasopharyngeal, oropharyngeal, and bronchoalveolar-lavage fluid; conjunctiva; and semen) and in specimens obtained from captured small-eared pygmy rice rats (Oligoryzomys microtis). In survivors of CHHF, viral RNA was detected up to 170 days after symptom onset; CHAPV was isolated from a semen sample obtained 86 days after symptom onset. CONCLUSIONS: M. Chapare mammarenavirus was identified as the etiologic agent of CHHF. Both spillover from a zoonotic reservoir and possible person-to-person transmission were identified. This virus was detected in a rodent species, O. microtis. (Funded by the Bolivian Ministry of Health and others.).

Rapid establishment of a frontline field laboratory in response to an imported outbreak of Ebola virus disease in western Uganda, June 2019.

The Democratic Republic of the Congo (DRC) declared an Ebola virus disease (EVD) outbreak in North Kivu in August 2018. By June 2019, the outbreak had spread to 26 health zones in northeastern DRC, causing >2,000 reported cases and >1,000 deaths. On June 10, 2019, three members of a Congolese family with EVD-like symptoms traveled to western Uganda's Kasese District to seek medical care. Shortly thereafter, the Viral Hemorrhagic Fever Surveillance and Laboratory Program (VHF program) at the Uganda Virus Research Institute (UVRI) confirmed that all three patients had EVD. The Ugandan Ministry of Health declared an outbreak of EVD in Uganda's Kasese District, notified the World Health Organization, and initiated a rapid response to contain the outbreak. As part of this response, UVRI and the United States Centers for Disease Control and Prevention, with the support of Uganda's Public Health Emergency Operations Center, the Kasese District Health Team, the Superintendent of Bwera General Hospital, the United States Department of Defense's Makerere University Walter Reed Project, and the United States Mission to Kampala's Global Health Security Technical Working Group, jointly established an Ebola Field Laboratory in Kasese District at Bwera General Hospital, proximal to an Ebola Treatment Unit (ETU). The laboratory consisted of a rapid containment kit for viral inactivation of patient specimens and a GeneXpert Instrument for performing Xpert Ebola assays. Laboratory staff tested 76 specimens from alert and suspect cases of EVD; the majority were admitted to the ETU (89.3%) and reported recent travel to the DRC (58.9%). Although no EVD cases were detected by the field laboratory, it played an important role in patient management and epidemiological surveillance by providing diagnostic results in <3 hours. The integration of the field laboratory into Uganda's National VHF Program also enabled patient specimens to be referred to Entebbe for confirmatory EBOV testing and testing for other hemorrhagic fever viruses that circulate in Uganda.

Rift Valley Fever and Crimean-Congo Hemorrhagic Fever Viruses in Ruminants, Jordan.

The epidemiology of Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus (CCHFV) in Jordan is unknown. Our investigation showed 3% of 989 tested dairy cattle, sheep, and goats were RVFV seropositive and 14% were CCHFV seropositive. Ongoing surveillance is needed to assess risk to humans and protect public health.

Characteristics of Ebola Virus Disease Survivor Blood and Semen in Liberia: Serology and Reverse Transcription Polymerase Chain Reaction (RT-PCR).

INTRODUCTION: Ebola virus (EBOV), species Zaire ebolavirus, may persist in the semen of male survivors of Ebola virus disease (EVD). We conducted a study of male survivors of the 2014-2016 EVD outbreak in Liberia and evaluated their immune responses to EBOV. We report here findings from the serologic testing of blood for EBOV-specific antibodies, molecular testing for EBOV in blood and semen, and serologic testing of peripheral blood mononuclear cells (PBMCs) in a subset of study participants. METHODS: We tested for EBOV RNA in blood by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-EBOV-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) for 126 study participants. We performed PBMC analysis on a subgroup of 26 IgG-negative participants. RESULTS: All 126 participants tested negative for EBOV RNA in blood by qRT-PCR. The blood of 26 participants tested negative for EBOV-specific IgG antibodies by ELISA. PBMCs were collected from 23/26 EBOV IgG-negative participants. Of these, 1/23 participants had PBMCs that produced anti-EBOV-specific IgG antibodies upon stimulation with EBOV-specific glycoprotein (GP) and nucleoprotein (NP) antigens. CONCLUSIONS: The blood of EVD survivors, collected when they did not have symptoms meeting the case definition for acute or relapsed EVD, is unlikely to pose a risk for EBOV transmission. We identified 1 IgM/IgG negative participant who had PBMCs that produced anti-EBOV-specific antibodies upon stimulation. Immunogenicity following acute EBOV infection may exist along a spectrum, and absence of antibody response should not be exclusionary in determining an individual's status as a survivor of EVD.

Seoul Virus Infection and Spread in United States Home-Based Ratteries: Rat and Human Testing Results From a Multistate Outbreak Investigation.

BACKGROUND: During 2017, a multistate outbreak investigation occurred after the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested. METHODS: In addition to immunoglobulin (Ig)G and IgM serology and traditional reverse-transcription polymerase chain reaction (RT-PCR), novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed. RESULTS: Seventeen people had SEOV IgM, indicating recent infection; 7 reported symptoms and 3 were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with ≥10 rats tested, rat IgG prevalence ranged 2%-70% and SEOV RT-PCR positivity ranged 0%-70%. Human laboratory-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (P = .03 and P = .006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities. CONCLUSIONS: Pet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections.

Isolation of Angola-like Marburg virus from Egyptian rousette bats from West Africa.

Marburg virus (MARV) causes sporadic outbreaks of severe Marburg virus disease (MVD). Most MVD outbreaks originated in East Africa and field studies in East Africa, South Africa, Zambia, and Gabon identified the Egyptian rousette bat (ERB; Rousettus aegyptiacus) as a natural reservoir. However, the largest recorded MVD outbreak with the highest case-fatality ratio happened in 2005 in Angola, where direct spillover from bats was not  shown. Here, collaborative studies by the Centers for Disease Control and Prevention, Njala University, University of California, Davis USAID-PREDICT, and the University of Makeni identify MARV circulating in ERBs in Sierra Leone. PCR, antibody and virus isolation data from 1755 bats of 42 species shows active MARV infection in approximately 2.5% of ERBs. Phylogenetic analysis identifies MARVs that are similar to the Angola strain. These results provide evidence of MARV circulation in West Africa and demonstrate the value of pathogen surveillance to identify previously undetected threats.

Domestically Acquired Seoul Virus Causing Hemophagocytic Lymphohistiocytosis-Washington, DC, 2018.

Seoul orthohantavirus (SEOV) infections, uncommonly reported in the United States, often result in mild illness. We report a case of hemophagocytic lymphohistiocytosis secondary to SEOV infection that was domestically acquired in Washington, DC.

Marburg virus disease outbreak in Kween District Uganda, 2017: Epidemiological and laboratory findings.

INTRODUCTION: In October 2017, a blood sample from a resident of Kween District, Eastern Uganda, tested positive for Marburg virus. Within 24 hour of confirmation, a rapid outbreak response was initiated. Here, we present results of epidemiological and laboratory investigations. METHODS: A district task force was activated consisting of specialised teams to conduct case finding, case management and isolation, contact listing and follow up, sample collection and testing, and community engagement. An ecological investigation was also carried out to identify the potential source of infection. Virus isolation and Next Generation sequencing were performed to identify the strain of Marburg virus. RESULTS: Seventy individuals (34 MVD suspected cases and 36 close contacts of confirmed cases) were epidemiologically investigated, with blood samples tested for MVD. Only four cases met the MVD case definition; one was categorized as a probable case while the other three were confirmed cases. A total of 299 contacts were identified; during follow- up, two were confirmed as MVD. Of the four confirmed and probable MVD cases, three died, yielding a case fatality rate of 75%. All four cases belonged to a single family and 50% (2/4) of the MVD cases were female. All confirmed cases had clinical symptoms of fever, vomiting, abdominal pain and bleeding from body orifices. Viral sequences indicated that the Marburg virus strain responsible for this outbreak was closely related to virus strains previously shown to be circulating in Uganda. CONCLUSION: This outbreak of MVD occurred as a family cluster with no additional transmission outside of the four related cases. Rapid case detection, prompt laboratory testing at the Uganda National VHF Reference Laboratory and presence of pre-trained, well-prepared national and district rapid response teams facilitated the containment and control of this outbreak within one month, preventing nationwide and global transmission of the disease.

Infection with New York Orthohantavirus and Associated Respiratory Failure and Multiple Cerebral Complications.

We report a case of infection with New York orthohantavirus in a woman who showed renal impairment and hemorrhage, complicated by hydrocephalus, in Long Island, New York, USA. Phylogenetic analysis showed that this virus was genetically similar to a New York orthohantavirus isolated in the same region during 1993.

Revisiting the taxonomy of the genus Elizabethkingia using whole-genome sequencing, optical mapping, and MALDI-TOF, along with proposal of three novel Elizabethkingia species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii sp. nov., and Elizabethkingia occulta sp. nov.

The genus Elizabethkingia is genetically heterogeneous, and the phenotypic similarities between recognized species pose challenges in correct identification of clinically derived isolates. In addition to the type species Elizabethkingia meningoseptica, and more recently proposed Elizabethkingia miricola, Elizabethkingia anophelis and Elizabethkingia endophytica, four genomospecies have long been recognized. By comparing historic DNA-DNA hybridization results with whole genome sequences, optical maps, and MALDI-TOF mass spectra on a large and diverse set of strains, we propose a comprehensive taxonomic revision of this genus. Genomospecies 1 and 2 contain the type strains E. anophelis and E. miricola, respectively. Genomospecies 3 and 4 are herein proposed as novel species named as Elizabethkingia bruuniana sp. nov. (type strain, G0146T = DSM 2975T = CCUG 69503T = CIP 111191T) and Elizabethkingia ursingii sp. nov. (type strain, G4122T = DSM 2974T = CCUG 69496T = CIP 111192T), respectively. Finally, the new species Elizabethkingia occulta sp. nov. (type strain G4070T = DSM 2976T = CCUG 69505T = CIP 111193T), is proposed.

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.