Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in a Georgia School District-United States, December 2020-January 2021.

BACKGROUND: To inform prevention strategies, we assessed the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and settings in which transmission occurred in a Georgia public school district. METHODS: During 1 December 2020-22 January 2021, SARS-CoV-2-infected index cases and their close contacts in schools were identified by school and public health officials. For in-school contacts, we assessed symptoms and offered SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) testing; performed epidemiologic investigations and whole-genome sequencing to identify in-school transmission; and calculated secondary attack rate (SAR) by school setting (eg, sports, elementary school classroom), index case role (ie, staff, student), and index case symptomatic status. RESULTS: We identified 86 index cases and 1119 contacts, 688 (61.5%) of whom received testing. Fifty-nine of 679 (8.7%) contacts tested positive; 15 of 86 (17.4%) index cases resulted in ≥2 positive contacts. Among 55 persons testing positive with available symptom data, 31 (56.4%) were asymptomatic. Highest SARs were in indoor, high-contact sports settings (23.8% [95% confidence interval {CI}, 12.7%-33.3%]), staff meetings/lunches (18.2% [95% CI, 4.5%-31.8%]), and elementary school classrooms (9.5% [95% CI, 6.5%-12.5%]). The SAR was higher for staff (13.1% [95% CI, 9.0%-17.2%]) vs student index cases (5.8% [95% CI, 3.6%-8.0%]) and for symptomatic (10.9% [95% CI, 8.1%-13.9%]) vs asymptomatic index cases (3.0% [95% CI, 1.0%-5.5%]). CONCLUSIONS: Indoor sports may pose a risk to the safe operation of in-person learning. Preventing infection in staff members, through measures that include coronavirus disease 2019 vaccination, is critical to reducing in-school transmission. Because many positive contacts were asymptomatic, contact tracing should be paired with testing, regardless of symptoms.

Divergent Rabies Virus Variant of Probable Bat Origin in 2 Gray Foxes, New Mexico, USA.

In the Western Hemisphere, bat-associated rabies viruses (RABVs) have established independent transmission cycles in multiple mammal hosts, forming genetically distinct lineages. In New Mexico, USA, skunks, bats, and gray foxes are rabies reservoir hosts and represent a public health risk because of encounters with humans. During 2015 and 2019, two previously undescribed RABVs were detected in 2 gray foxes (Urocyon cinereoargenteus) in Lincoln County, New Mexico. Phylogenetic analysis of the nucleoprotein gene indicated that the isolates are a novel RABV variant. These 2 cases probably represent repeated spillover events from an unknown bat reservoir to gray foxes. Molecular analysis of rabies cases across New Mexico identified that other cross-species transmission events were the result of viral variants previously known to be enzootic to New Mexico. Despite a robust rabies public health surveillance system in the United States, advances in testing and surveillance techniques continue to identify previously unrecognized zoonotic pathogens.

Host-pathogen evolutionary signatures reveal dynamics and future invasions of vampire bat rabies.

Anticipating how epidemics will spread across landscapes requires understanding host dispersal events that are notoriously difficult to measure. Here, we contrast host and virus genetic signatures to resolve the spatiotemporal dynamics underlying geographic expansions of vampire bat rabies virus (VBRV) in Peru. Phylogenetic analysis revealed recent viral spread between populations that, according to extreme geographic structure in maternally inherited host mitochondrial DNA, appeared completely isolated. In contrast, greater population connectivity in biparentally inherited nuclear microsatellites explained the historical limits of invasions, suggesting that dispersing male bats spread VBRV between genetically isolated female populations. Host nuclear DNA further indicated unanticipated gene flow through the Andes mountains connecting the VBRV-free Pacific coast to the VBRV-endemic Amazon rainforest. By combining Bayesian phylogeography with landscape resistance models, we projected invasion routes through northern Peru that were validated by real-time livestock rabies mortality data. The first outbreaks of VBRV on the Pacific coast of South America could occur by June 2020, which would have serious implications for agriculture, wildlife conservation, and human health. Our results show that combining host and pathogen genetic data can identify sex biases in pathogen spatial spread, which may be a widespread but underappreciated phenomenon, and demonstrate that genetic forecasting can aid preparedness for impending viral invasions.

Variable evolutionary routes to host establishment across repeated rabies virus host shifts among bats.

Determining the genetic pathways that viruses traverse to establish in new host species is crucial to predict the outcome of cross-species transmission but poorly understood for most host-virus systems. Using sequences encoding 78% of the rabies virus genome, we explored the extent, repeatability and dynamic outcome of evolution associated with multiple host shifts among New World bats. Episodic bursts of positive selection were detected in several viral proteins, including regions associated with host cell interaction and viral replication. Host shifts involved unique sets of substitutions, and few sites exhibited repeated evolution across adaptation to many bat species, suggesting diverse genetic determinants over host range. Combining these results with genetic reconstructions of the demographic histories of individual viral lineages revealed that although rabies viruses shared consistent three-stage processes of emergence in each new bat species, host shifts involving greater numbers of positively selected substitutions had longer delays between cross-species transmission and enzootic viral establishment. Our results point to multiple evolutionary routes to host establishment in a zoonotic RNA virus that may influence the speed of viral emergence.

Rates of viral evolution are linked to host geography in bat rabies.

Rates of evolution span orders of magnitude among RNA viruses with important implications for viral transmission and emergence. Although the tempo of viral evolution is often ascribed to viral features such as mutation rates and transmission mode, these factors alone cannot explain variation among closely related viruses, where host biology might operate more strongly on viral evolution. Here, we analyzed sequence data from hundreds of rabies viruses collected from bats throughout the Americas to describe dramatic variation in the speed of rabies virus evolution when circulating in ecologically distinct reservoir species. Integration of ecological and genetic data through a comparative bayesian analysis revealed that viral evolutionary rates were labile following historical jumps between bat species and nearly four times faster in tropical and subtropical bats compared to temperate species. The association between geography and viral evolution could not be explained by host metabolism, phylogeny or variable selection pressures, and instead appeared to be a consequence of reduced seasonality in bat activity and virus transmission associated with climate. Our results demonstrate a key role for host ecology in shaping the tempo of evolution in multi-host viruses and highlight the power of comparative phylogenetic methods to identify the host and environmental features that influence transmission dynamics.

Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009.

In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001-2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.

Rabies death attributed to exposure in Central America with symptom onset in a U.S. detention facility - Texas, 2013.

On June 7, 2013, a man was diagnosed in a Texas hospital with rabies. He had been detained in a U.S. detention facility during his infectious period. To identify persons exposed to rabies who might require rabies postexposure prophylaxis (PEP), CDC and the Texas Department of State Health Services (DSHS) conducted investigations at four detention facilities, one medical clinic, and two hospitals. In all, 25 of 742 persons assessed for rabies exposure were advised to receive PEP. Early diagnosis of rabies is essential for implementation of appropriate hospital infection control measures and for rapid assessment of potential contacts for PEP recommendations.

Metabolomics of cerebrospinal fluid from humans treated for rabies.

Rabies is a rapidly progressive lyssavirus encephalitis that is statistically 100% fatal. There are no clinically effective antiviral drugs for rabies. An immunologically naïve teenager survived rabies in 2004 through improvised supportive care; since then, 5 additional survivors have been associated with use of the so-called Milwaukee Protocol (MP). The MP applies critical care focused on the altered metabolic and physiologic states associated with rabies. The aim of this study was to examine the metabolic profile of cerebrospinal fluid (CSF) from rabies patients during clinical progression of rabies encephalitis in survivors and nonsurvivors and to compare these samples with control CSF samples. Unsupervised clustering algorithms distinguished three stages of rabies disease and identified several metabolites that differentiated rabies survivors from those who subsequently died, in particular, metabolites related to energy metabolism and cell volume control. Moreover, for those patients who survived, the trajectory of their metabolic profile tracked toward the control profile and away from the rabies profile. NMR metabolomics of human rabies CSF provide new insights into the mechanisms of rabies pathogenesis, which may guide future therapy of this disease.

Enzootic and epizootic rabies associated with vampire bats, peru.

During the past decade, incidence of human infection with rabies virus (RABV) spread by the common vampire bat (Desmodus rotundus) increased considerably in South America, especially in remote areas of the Amazon rainforest, where these bats commonly feed on humans. To better understand the epizootiology of rabies associated with vampire bats, we used complete sequences of the nucleoprotein gene to infer phylogenetic relationships among 157 RABV isolates collected from humans, domestic animals, and wildlife, including bats, in Peru during 2002-2007. This analysis revealed distinct geographic structuring that indicates that RABVs spread gradually and involve different vampire bat subpopulations with different transmission cycles. Three putative new RABV lineages were found in 3 non-vampire bat species that may represent new virus reservoirs. Detection of novel RABV variants and accurate identification of reservoir hosts are critically important for the prevention and control of potential virus transmission, especially to humans.

Raccoon rabies virus variant transmission through solid organ transplantation.

IMPORTANCE: The rabies virus causes a fatal encephalitis and can be transmitted through tissue or organ transplantation. In February 2013, a kidney recipient with no reported exposures to potentially rabid animals died from rabies 18 months after transplantation. OBJECTIVES: To investigate whether organ transplantation was the source of rabies virus exposure in the kidney recipient, and to evaluate for and prevent rabies in other transplant recipients from the same donor. DESIGN: Organ donor and all transplant recipient medical records were reviewed. Laboratory tests to detect rabies virus-specific binding antibodies, rabies virus neutralizing antibodies, and rabies virus antigens were conducted on available specimens, including serum, cerebrospinal fluid, and tissues from the donor and the recipients. Viral ribonucleic acid was extracted from tissues and amplified for nucleoprotein gene sequencing for phylogenetic comparisons. MAIN OUTCOMES AND MEASURES: Determination of whether the donor died from undiagnosed rabies and whether other organ recipients developed rabies. RESULTS: In retrospect, the donor's clinical presentation (which began with vomiting and upper extremity paresthesias and progressed to fever, seizures, dysphagia, autonomic dysfunction, and brain death) was consistent with rabies. Rabies virus antigen was detected in archived autopsy brain tissue collected from the donor. The rabies viruses infecting the donor and the deceased kidney recipient were consistent with the raccoon rabies virus variant and were more than 99.9% identical across the entire N gene (1349/1350 nucleotides), thus confirming organ transplantation as the route of transmission. The 3 other organ recipients remained asymptomatic, with rabies virus neutralizing antibodies detected in their serum after completion of postexposure prophylaxis (range, 0.3-40.8 IU/mL). CONCLUSIONS AND RELEVANCE: Unlike the 2 previous clusters of rabies virus transmission through solid organ transplantation, there was a long incubation period in the recipient who developed rabies, and survival of 3 other recipients without pretransplant rabies vaccination. Rabies should be considered in patients with acute progressive encephalitis of unexplained etiology, especially for potential organ donors. A standard evaluation of potential donors who meet screening criteria for infectious encephalitis should be considered, and risks and benefits for recipients of organs from these donors should be evaluated.

Revealing the complexity of vampire bat rabies "spillover transmission".

BACKGROUND: The term virus 'spillover' embodies a highly complex phenomenon and is often used to refer to viral transmission from a primary reservoir host to a new, naïve yet susceptible and permissive host species. Spillover transmission can result in a virus becoming pathogenic, causing disease and death to the new host if successful infection and transmission takes place. MAIN TEXT: The scientific literature across diverse disciplines has used the terms virus spillover, spillover transmission, cross-species transmission, and host shift almost indistinctly to imply the complex process of establishment of a virus from an original host (source/donor) to a naïve host (recipient), which have close or distant taxonomic or evolutionary ties. Spillover transmission may result in unsuccessful onward transmission, if the virus dies off before propagation. Alternatively, successful viral establishment in the new host can occur if subsequent secondary transmission among individuals of the same novel species and among other sympatric susceptible species occurred. As such, virus spillover transmission is a common yet highly complex phenomenon that encompasses multiple subtle stages that can be deconstructed to be studied separately to better understand the drivers of disease emergence. Rabies virus (RABV) is a well-documented viral pathogen which still inflicts heavy impact on humans, companion animals, wildlife, and livestock throughout Latin America due substantial spatial temporal and ecological-natural and expansional-overlap with several virus reservoir hosts. Thereby, the rabies disease system represents a robust avenue through which the drivers and uncertainties surrounding spillover transmission can be unravel at its different subtle stages to better understand how they may be affected by coarse, medium, and fine scale variables. CONCLUSIONS: The continued study of viral spillover transmission necessitates the elucidation of its complexities to better assess the cross-scale impacts of ecological forces linked to the propensity of spillover success. Improving capacities to reconstruct and predict spillover transmission would prevent public health impacts on those most at risk populations across the globe.

The Importance of Accurate Host Species Identification in the Framework of Rabies Surveillance, Control and Elimination.

Accurate host identification is paramount to understand disease epidemiology and to apply appropriate control measures. This is especially important for multi-host pathogens such as the rabies virus, a major and almost invariably fatal zoonosis that has mobilized unanimous engagement at an international level towards the final goal of zero human deaths due to canine rabies. Currently, diagnostic laboratories implement a standardized identification using taxonomic keys. However, this method is challenged by high and undiscovered biodiversity, decomposition of carcasses and subjective misevaluation, as has been attested to by findings from a cohort of 242 archived specimens collected across Sub-Saharan Africa and submitted for rabies diagnosis. We applied two simple and cheap methods targeting the Cytochrome b and Cytochrome c oxidase subunit I to confirm the initial classification. We therefore suggest prioritizing a standardized protocol that includes, as a first step, the implementation of taxonomic keys at a family or subfamily level, followed by the molecular characterization of the host species.

A recombinant rabies vaccine that prevents viral shedding in rabid common vampire bats (Desmodus rotundus).

Vampire bat transmitted rabies (VBR) is a continuing burden to public health and agricultural sectors in Latin America, despite decades-long efforts to control the disease by culling bat populations. Culling has been shown to disperse bats, leading to an increased spread of rabies. Thus, non-lethal strategies to control VBR, such as vaccination, are desired. Here, we evaluated the safety and efficacy of a viral-vectored recombinant mosaic glycoprotein rabies vaccine candidate (RCN-MoG) in vampire bats (Desmodus rotundus) of unknown history of rabies exposure captured in México and transported to the United States. Vaccination with RCN-MoG was demonstrated to be safe, even in pregnant females, as no evidence of lesions or adverse effects were observed. We detected rabies neutralizing antibodies in 28% (8/29) of seronegative bats post-vaccination. Survival proportions of adult bats after rabies virus (RABV) challenge ranged from 55-100% and were not significantly different among treatments, pre- or post-vaccination serostatus, and route of vaccination, while eight pups (1-2.5 months of age) used as naïve controls all succumbed to challenge (P<0.0001). Importantly, we found that vaccination with RCN-MoG appeared to block viral shedding, even when infection proved lethal. Using real-time PCR, we did not detect RABV nucleic acid in the saliva samples of 9/10 vaccinated bats that succumbed to rabies after challenge (one was inconclusive). In contrast, RABV nucleic acid was detected in saliva samples from 71% of unvaccinated bats (10/14 sampled, plus one inconclusive) that died of the disease, including pups. Low seroconversion rates post-vaccination and high survival of non-vaccinated bats, perhaps due to earlier natural exposure, limited our conclusions regarding vaccine efficacy. However, our findings suggest a potential transmission-blocking effect of vaccination with RCN-MoG that could provide a promising strategy for controlling VBR in Latin America beyond longstanding culling programs.

Detection of North American orthopoxviruses by real time-PCR.

The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad.

A Cross Sectional Sampling Reveals Novel Coronaviruses in Bat Populations of Georgia.

Mammal-associated coronaviruses have a long evolutionary history across global bat populations, which makes them prone to be the most likely ancestral origins of coronavirus-associated epidemics and pandemics globally. Limited coronavirus research has occurred at the junction of Europe and Asia, thereby investigations in Georgia are critical to complete the coronavirus diversity map in the region. We conducted a cross-sectional coronavirus survey in bat populations at eight locations of Georgia, from July to October of 2014. We tested 188 anal swab samples, remains of previous pathogen discovery studies, for the presence of coronaviruses using end-point pan-coronavirus RT-PCR assays. Samples positive for a 440 bp amplicon were Sanger sequenced to infer coronavirus subgenus or species through phylogenetic reconstructions. Overall, we found a 24.5% positive rate, with 10.1% for Alphacoronavirus and 14.4% for Betacoronavirus. Albeit R. euryale, R. ferrumequinum, M. blythii and M. emarginatus were found infected with both CoV genera, we could not rule out CoV co-infection due to limitation of the sequencing method used and sample availability. Based on phylogenetic inferences and genetic distances at nucleotide and amino acid levels, we found one putative new subgenus and three new species of Alphacoronavirus, and two new species of Betacoronavirus.

Use of partial N-gene sequences as a tool to monitor progress on rabies control and elimination efforts in Ethiopia.

Ethiopia is one of the African countries most affected by rabies. A coarse catalog of rabies viruses (RABV) was created as a benchmark to assess the impact of control and elimination activities. We evaluated a 726 bp amplicon at the end of the N-gene to infer viral lineages in circulation using maximum likelihood and Bayesian methods for phylogenetic reconstruction. We sequenced 228 brain samples from wild and domestic animals collected in five Ethiopian regions during 2010-2017. Results identified co-circulating RABV lineages that are causing recurrent spillover infections into wildlife and domestic animals. We found no evidence of importation of RABVs from other African countries or vaccine-induced cases in the area studied. A divergent RABV lineage might be involved in an independent rabies cycle in jackals. This investigation provides a feasible approach to assess rabies control and elimination efforts in resource-limited countries.

Ferret badger rabies origin and its revisited importance as potential source of rabies transmission in Southeast China.

BACKGROUND: The frequent occurrence of ferret badger-associated human rabies cases in southeast China highlights the lack of laboratory-based surveillance and urges revisiting the potential importance of this animal in rabies transmission. To determine if the ferret badgers actually contribute to human and dog rabies cases, and the possible origin of the ferret badger-associated rabies in the region, an active rabies survey was conducted to determine the frequency of rabies infection and seroprevalence in dogs and ferret badgers. METHODS: A retrospective survey on rabies epidemics was performed in Zhejiang, Jiangxi and Anhui provinces in southeast China. The brain tissues from ferret badgers and dogs were assayed by fluorescent antibody test. Rabies virus was isolated and sequenced for phylogenetic analysis. The sera from ferret badgers and dogs were titrated using rabies virus neutralizing antibodies (VNA) test. RESULTS: The ferret badgers presented a higher percentage of rabies seroconversion than dogs did in the endemic region, reaching a maximum of 95% in the collected samples. Nine ferret badger-associated rabies viruses were isolated, sequenced, and were phylogenetically clustered as a separate group. Nucleotide sequence revealed 99.4-99.8% homology within the ferret badger isolates, and 83-89% homology to the dog isolates in the nucleoprotein and glycoprotein genes in the same rabies endemic regions. CONCLUSIONS: Our data suggest ferret badger-associated rabies has likely formed as an independent enzootic originating from dogs during the long-term rabies infestation in southeast China. The eventual role of FB rabies in public health remains unclear. However, management of ferret badger bites, rabies awareness and control in the related regions should be an immediate need.

Abortive vampire bat rabies infections in Peruvian peridomestic livestock.

Rabies virus infections normally cause universally lethal encephalitis across mammals. However, 'abortive infections' which are resolved prior to the onset of lethal disease have been described in bats and a variety of non-reservoir species. Here, we surveyed rabies virus neutralizing antibody titers in 332 unvaccinated livestock of 5 species from a vampire bat rabies endemic region of southern Peru where livestock are the main food source for bats. We detected rabies virus neutralizing antibody titers in 11, 5 and 3.6% of cows, goats and sheep respectively and seropositive animals did not die from rabies within two years after sampling. Seroprevalence was correlated with the number of local livestock rabies mortalities reported one year prior but also one year after sample collection. This suggests that serological status of livestock can indicate the past and future levels of rabies risk to non-reservoir hosts. To our knowledge, this is the first report of anti-rabies antibodies among goats and sheep, suggesting widespread abortive infections among livestock in vampire bat rabies endemic areas. Future research should resolve the within-host biology underlying clearance of rabies infections. Cost-effectiveness analyses are also needed to evaluate whether serological monitoring of livestock can be a viable complement to current monitoring of vampire bat rabies risk based on animal mortalities alone.

Evaluation of species identification and rabies virus characterization among bat rabies cases in the United States.

OBJECTIVE: To evaluate species identification and rabies virus (RABV) characterization among samples from bats submitted for rabies testing in the United States and assess whether a standardized approach to specimen selection for RABV characterization could enhance detection of a sentinel event in virus dissemination among bats. SAMPLE: United States public health rabies surveillance system data collected in January 2010 through December 2015. PROCEDURES: The number of rabies-tested bats for which species was reported and the number of RABV-positive samples for which virus characterization would likely provide information regarding introduction of novel RABV variants and translocation and host-shift events were calculated. These specimens were designated as specimens of epizootiological importance (SEIs). Additionally, the estimated test load that public health laboratories could expect if all SEIs underwent RABV characterization was determined. RESULTS: Species was reported for 74,928 of 160,017 (47%) bats submitted for rabies testing. Identified SEIs were grouped in 3 subcategories, namely nonindigenous bats; bats in southern border states, Florida, Puerto Rico, and the US Virgin Islands; and bats of species that are not commonly found to be inflected with RABV. Annually, 692 (95% CI, 600 to 784) SEIs were identified, of which only 295 (95% CI, 148 to 442) underwent virus characterization. Virus characterization of all SEIs would be expected to increase public health laboratories' overall test load by 397 (95% CI, 287 to 506) samples each year. CONCLUSIONS AND CLINICAL RELEVANCE: Species identification and RABV characterization may aid detection of a sentinel event in bat RABV dissemination. With additional resources, RABV characterization of all SEIs as a standardized approach to testing could contribute to knowledge of circulating bat RABV variants.