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During 2017 – 2019, the Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6,855 animal samples for rabies using both the gold standard direct fluorescent antibody (DFA) test and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR). Two samples (0.03 %) were identified as LN34 RT-qPCR positive after failure to detect rabies virus antigen during initial DFA testing: an adult raccoon collected in 2017 and a juvenile raccoon collected in 2019. After the positive PCR result, additional tissues were collected and re-tested by DFA, where very sparse, disperse antigen was observed. Tissues from both animals were submitted to the Centers for Disease Control and Prevention (CDC) for confirmatory testing, and were confirmed positive. At both PABOL and CDC, rabies virus antigen and RNA levels were much lower than for a typical rabies case. In addition, rabies virus antigen and RNA levels were higher in brain stem and rostral spinal cord than cerebellum, hippocampus and cortex. Cross-contamination was ruled out in the case of the 2019 juvenile raccoon by sequencing, as nucleoprotein and glycoprotein gene sequences displayed >1% nucleotide differences to sequences from all positive samples processed at PABOL within two weeks of the juvenile raccoon. Taken together, the low level of rabies virus in the central nervous system combined with presence in more caudal brain structures suggest the possibility of an early infection in both cases. These two cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR in rabies diagnostics for the identification of low positive cases.

Rabies is an acute encephalitis that is nearly always fatal. It is caused by infection with viruses of the genus Lyssavirus, the most common of which is Rabies lyssavirus. The Council of State and Territorial Epidemiologists (CSTE) defines a confirmed human rabies case as an illness compatible with rabies that meets at least one of five different laboratory criteria.* Four of these criteria do not depend on the patient's rabies vaccination status; however, the remaining criterion, "identification of Lyssavirus-specific antibody (i.e. by indirect fluorescent antibody…test or complete [Rabies lyssavirus] neutralization at 1:5 dilution) in the serum," is only considered diagnostic in unvaccinated patients. Lyssavirus-specific antibodies include Rabies lyssavirus-specific binding immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies and Rabies lyssavirus neutralizing antibodies (RLNAs). This report describes six patients who were tested for rabies by CDC and who met CSTE criteria for confirmed human rabies because they had illnesses compatible with rabies, had not been vaccinated for rabies, and were found to have serum RLNAs (with complete Rabies lyssavirus neutralization at a serum dilution of 1:5). An additional four patients are described who were tested for rabies by CDC who were found to have serum RLNAs (with incomplete Rabies lyssavirus neutralization at a serum dilution of 1:5) despite having not been vaccinated for rabies. None of these 10 patients received a rabies diagnosis; rather, they were considered to have been passively immunized against rabies through recent receipt of intravenous immune globulin (IVIG). Serum RLNA test results should be interpreted with caution in patients who have not been vaccinated against rabies but who have recently received IVIG.

Haiti, a Caribbean country of 10.5 million people, is estimated to have the highest burden of canine-mediated human rabies deaths in the Western Hemisphere, and one of the highest rates of human rabies deaths in the world. Haiti is also the poorest country in the Western Hemisphere and has numerous economic and health priorities that compete for rabies-control resources. As a result, primary rabies-control actions, including canine vaccination programs, surveillance systems for human and animal rabies, and appropriate postbite treatment, have not been fully implemented at a national scale. After the 2010 earthquake that further hindered the development of public health program infrastructure and services, the U.S. Centers for Disease Control and Prevention worked with the Ministry of Public Health and Population and key health development partners (including the Pan-American Health Organization) to provide technical expertise and funding for general disease surveillance systems, laboratory capacity, and selected disease control programs; including rabies. In 2011, a cross-ministerial rabies consortium was convened with participation from multiple international rabies experts to develop a strategy for successful rabies control in Haiti. The consortium focused on seven pillars: 1) enhancement of laboratory diagnostic capacity, 2) development of comprehensive animal surveillance system, 3) development of comprehensive human rabies surveillance system, 4) educational outreach, 5) sustainable human rabies biologics supply, 6) achievement of sustained canine vaccination rates of ≥ 70%, and 7) finalization of a national rabies control strategy. From 2010 until 2015, Haiti has seen improvements in the program infrastructure for canine rabies control. The greatest improvements were seen in the area of animal rabies surveillance, in support of which an internationally recognized rabies laboratory was developed thereby leading to an 18-fold increase in the detection of rabid animals. Canine rabies vaccination practices also improved, from a 2010 level of approximately 12% to a 2015 dog population coverage level estimated to be 45%. Rabies vaccine coverage is still below the goal of 70%, however, the positive trend is encouraging. Gaps exist in the capacity to conduct national surveillance for human rabies cases and access to human rabies vaccine is lacking in many parts of the country. However, control has improved over the past 5 years as a result of the efforts of Haiti’s health and agriculture sectors with assistance from multiple international organizations. Haiti is well situated to eliminate canine-mediated human rabies deaths in the near future and should serve as a great example to many developing countries struggling with similar barriers and limitations.

An outbreak of rabies occurred in a captive colony of wild-caught big brown bats (Eptesicus fuscus). Five of 27 bats exhibited signs of rabies virus infection 22–51 d after capture or 18–...

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms., Author summary Although rabies is almost always fatal after the symptom onset phase, it can be prevented by timely administration of post-exposure prophylaxis (PEP), which involves passive antibody transfer and vaccination. One of the primary laboratory confirmatory tests for RABV infection is antigen detection, directed against the RABV encoded N protein using anti-N protein specific antibodies, in central nervous system (CNS) tissue samples of animals. This immuno-microscopy based detection utilizes either fluorescent tags (direct detection) or chromogenic substrates (indirect) in brain impressions from animals in which rabies is suspected. In this study, we explored the detection of N protein by a novel mass spectrometry (MS) based method that is label-free and does not require target amplification. The MS method specifically detected N protein in brain tissue and identified RABV variants based on amino acid sequence information. To our knowledge, this is the first report of an N protein detection method that does not utilize either antibodies or microscopy. This method provides an alternative platform for the development of future rabies diagnostic tests.

The rabies virus causes a fatal encephalitis and can be transmitted through organ transplantation. In 2013, a man developed rabies 18 months after receiving a kidney from a donor with rabies, who was not known to have been infected when the organs were procured. Three additional persons who received organs from the same donor (liver, kidney, heart), all of whom were not vaccinated for rabies before transplantation, received rabies post-exposure prophylaxis (PEP) with rabies immune globulin and 5 doses of rabies vaccine as soon as the diagnosis of rabies was made in the donor (18 months after their transplant surgeries). We describe their clinical management.As the 3 recipients were all on immunosuppressive medications, post-vaccination serologic testing was performed using the rapid fluorescent focus inhibition test to measure rabies virus neutralizing antibodies (RVNAs). An acceptable antibody response to administration of rabies vaccine was defined as detection of RVNAs at a concentration ≥0.1 IU/mL from a serum specimen collected ≥7 days after the fifth vaccine dose.All 3 recipients demonstrated an acceptable antibody response despite their immunosuppressed states. More than 36 months have passed since their transplant surgeries, and all 3 recipients have no evidence of rabies.The survival of 3 previously unvaccinated recipients of solid organs from a donor with rabies is unexpected. Although the precise factors that led to their survival remain unclear, our data suggest that PEP can possibly enhance transplant safety in settings in which donors are retrospectively diagnosed with rabies.

Rabies is an acute fatal encephalitis caused by all members of the Lyssavirus genus. The first human rabies survivor without benefit of prior vaccination was reported from Milwaukee in 2005. We report a second unvaccinated patient who showed early recovery from rabies and then died accidentally during convalescence, providing an unparalleled opportunity to examine the histopathology as well as immune and virological correlates of early recovery from human rabies.Case report, rapid fluorescent focus inhibition test, enzyme-linked immunosorbent assay, indirect and direct fluorescent antibody assays, reverse-transcriptase polymerase chain reaction, phylogenetic reconstruction, isolation in tissue culture, pathology and immunohistochemistry.The 9 year old died 76 days after presenting with rabies of vampire bat phylogeny transmitted by cat bite. Antibody response in serum and cerebrospinal fluid was robust and associated with severe cerebral edema. No rabies virus was cultured at autopsy. Rabies virus antigen was atypical in size and distribution. Rabies virus genome was present in neocortex but absent in brainstem.Clinical recovery was associated with detection of neutralizing antibody and clearance of infectious rabies virus in the central nervous system by 76 days but not clearance of detectable viral subcomponents such as nucleoprotein antigen or RNA in brain.

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay., Author Summary Rabies is a preventable disease–but is still responsible for approximately 70,000 human deaths worldwide each year. The majority of human deaths occur in Asia and Africa where there is a lack of diagnostic resources and expertise, making it difficult to develop effective prevention and control strategies. In recent years, several real-time RT-PCR based diagnostic assays have been introduced to many developing countries in an effort to control the H1N1 pandemic flu, Ebola outbreak, and other tropical viral infections. In an effort to further improve rabies diagnostics, we developed a pan-lyssavirus Taqman real-time RT-PCR assay called LN34 for the detection of all known RABV variants and other lyssavirus species. The LN34 assay uses a combination of degenerate nucleotides, multiplex primers and probes, and unique probe modifications to achieve superior sensitivity and specificity compared to previously published RT-PCR based rabies diagnostics. Equally important, the LN34 assay is simple to set up, high throughput, combines multiple standard controls and can be used directly in widely available real-time RT-PCR systems. The LN34 assay was validated using a broad and comprehensive panel of highly diverse RABV variants and other lyssaviruses. A validated universal rabies diagnostic assay will be important in regions where RABV and other lyssaviruses co-circulate and for establishing a widely accepted diagnostic protocol. Over 200 clinical samples (including ante-mortem, post-mortem, and field derived samples) were tested with the LN34 assay, and the assay achieved 100% diagnostic sensitivity and specificity in our laboratory. Over 300 published genome sequences from representatives of RABV and other lyssaviruses were found to contain the conserved LN34 primer and probe targeting sites in an in silico analysis. We are expanding the validation of the LN34 assay to multiple domestic and international laboratories and expect the LN34 assay will drastically improve rabies diagnostic capacities globally.

Summary—During 2013, 53 reporting jurisdictions reported 5,865 rabid animals and 3 human rabies cases to the CDC, representing a 4.8% decrease from the 6,162 rabid animals and 1 human case reported in 2012. Ninety-two percent of reported rabid animals were wildlife. Relative contributions by the major animal groups were as follows: 1,898 raccoons (32.4%), 1,598 bats (27.2%), 1,447 skunks (24.7%), 344 foxes (5.9%), 247 cats (4.2%), 86 cattle (1.5%), and 89 dogs (1.5%). One human case was reported from Maryland. The infection was determined to have been transmitted via organ transplantation. Infection in the organ donor, a North Carolina resident, was retrospectively diagnosed. Both the organ donor and the organ recipient were infected with the raccoon rabies virus variant. The third human case, reported by Texas, involved a Guatemalan resident who was detained while crossing the US border. The infection was determined to be caused by a canine rabies virus variant that circulates in Central America.

Cost effective diagnostic tests are needed in rabies virus (RABV) enzootic areas to study the prevalence, distribution, and transmission of rabies virus among reservoir hosts. To reduce the associated costs of acquiring and maintaining specialized laboratory equipment, an indirect rapid immunohistochemistry test (IRIT), for the detection and differentiation of RABV variants, was evaluated by traditional light microscopy. The IRIT utilizes fresh frozen brain touch impressions or cell culture monolayers fixed in buffered formalin, a panel of murine anti-nucleoprotein monoclonal antibodies (mAb-N) and commercially available biotin-labeled goat anti-mouse antibody. In this study, 96 RABV isolates, representing 20 RABV variants previously determined by antigenic typing using a panel of mAb-N and the indirect fluorescent antibody test (IFA), and genetic sequence analysis were characterized by IRIT and the results compared. The IRIT results revealed distinct reactivity patterns associated with current and historical RABV reservoir hosts similar to IFA test and genetic sequence analysis. Evaluation of suspected RABV samples through IRIT does not require specialized equipment and is possible to perform in a field setting. Additionally, commercially available labeled secondary antibodies permit the use of a standard panel of unlabeled primary mAbs, without the need for fluorescence microscopy, and should augment existing attempts at antigenic characterization during canine rabies elimination campaigns in developed and developing countries. These results are useful in studying the epizootiology of rabies and inferring the source of infection when unknown.

Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38–100% and 75.76–96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

Independent enzootics in wild terrestrial carnivores resulted from spillover events from long-term enzootics associated with dogs., To provide molecular and virologic evidence that domestic dog rabies is no longer enzootic to the United States and to identify putative relatives of dog-related rabies viruses (RVs) circulating in other carnivores, we studied RVs associated with recent and historic dog rabies enzootics worldwide. Molecular, phylogenetic, and epizootiologic evidence shows that domestic dog rabies is no longer enzootic to the United States. Nonetheless, our data suggest that independent rabies enzootics are now established in wild terrestrial carnivores (skunks in California and north-central United States, gray foxes in Texas and Arizona, and mongooses in Puerto Rico), as a consequence of different spillover events from long-term rabies enzootics associated with dogs. These preliminary results highlight the key role of dog RVs and human–dog demographics as operative factors for host shifts and disease reemergence into other important carnivore populations and highlight the need for the elimination of dog-related RVs worldwide.

Canine rabies virus variant has been eliminated in the United States and multiple other countries. Globally, however, dogs remain the principal source for human rabies infections. The World Health Organization recommends that when dogs cross international borders, national importing authorities should require an international veterinary certificate attesting that the animal did not show signs of rabies at the time of shipment, was permanently identified, vaccinated, or revaccinated, and had been subjected to a serologic test for rabies before shipment. On June 8, 2015, an adult female dog that had recently been picked up from the streets of Cairo, Egypt, and shipped by a U.S. animal rescue organization to the United States was confirmed to have rabies by the Virginia Department of General Services Division of Consolidated Laboratory Services (DCLS). This dog was part of a large shipment of dogs and cats from Egypt that rescue organizations had distributed to multiple states for adoption. During the investigation, public health officials learned that the rabies vaccination certificate used for entry of the rabid dog into the United States had intentionally been falsified to avoid exclusion of the dog from entry under CDC's current dog importation regulations. This report underscores the ongoing risk posed by U.S. importation of domestic animals that have not been adequately vaccinated against rabies.

The Republic of Haiti is one of only several countries in the Western Hemisphere in which canine rabies is still endemic. Estimation methods have predicted that 130 human deaths occur per year, yet existing surveillance mechanisms have detected few of these rabies cases. Likewise, canine rabies surveillance capacity has had only limited capacity, detecting only two rabid dogs per year, on average. In 2013, Haiti initiated a community-based animal rabies surveillance program comprised of two components: active community bite investigation and passive animal rabies investigation. From January 2013 –December 2014, 778 rabies suspect animals were reported for investigation. Rabies was laboratory-confirmed in 70 animals (9%) and an additional 36 cases were identified based on clinical diagnosis (5%), representing an 18-fold increase in reporting of rabid animals compared to the three years before the program was implemented. Dogs were the most frequent rabid animal (90%). Testing and observation ruled out rabies in 61% of animals investigated. A total of 639 bite victims were reported to the program and an additional 364 bite victims who had not sought medical care were identified during the course of investigations. Only 31% of people with likely rabies exposures had initiated rabies post-exposure prophylaxis prior to the investigation. Rabies is a neglected disease in-part due to a lack of surveillance and understanding about the burden. The surveillance methods employed by this program established a much higher burden of canine rabies in Haiti than previously recognized. The active, community-based bite investigations identified numerous additional rabies exposures and bite victims were referred for appropriate medical care, averting potential human rabies deaths. The use of community-based rabies surveillance programs such as HARSP should be considered in canine rabies endemic countries., Author Summary The Republic of Haiti has highest estimated burden of human rabies in the Western Hemisphere, at 130 estimated human deaths annually. Rabies surveillance systems in the majority of the developing world, including Haiti, are ineffective, resulting in underreporting of cases and contributing to the further neglect of this disease. In 2013 a passive rabies surveillance program was implemented in three of Haiti’s 140 communes near the nation’s capital city. Four animal rabies surveillance officers conducted 778 suspect animal rabies investigations in a two-year period and on average found a rabid animal for every 7.4 investigations. Prior to the implementation of this surveillance program Haiti reported an average of two canine and seven human rabies cases each year, for the entire country. This program identified 70 rabid animals and an additional 36 probable rabid animals in only a selected area of the country. These 106 cases represent an 18-fold increase in animal rabies reporting in Haiti. These findings support that canine rabies is a significant burden in Haiti and present data that can be used to improve human rabies burden estimations and enhance canine rabies control efforts.

Bats represent the major source of human rabies cases in the New World. In the USA, most cases are associated with species that are not commonly found or reported rabid. To understand better the epidemiology and public health significance of potentially important bat species, a molecular study was performed on samples collected from naturally infected rabid western pipistrelle (Pipistrellus hesperus), eastern pipistrelle (Pipistrellus subflavus) and silver-haired bats (Lasionycteris noctivagans) from different regions of their geographical distribution in the USA. A 264 bp fragment at the 5′ end of the N gene coding region was sequenced and analysed in comparison with rabies virus variants circulating within other North American mammals. Phylogenetic analysis demonstrated that P. hesperus bats maintain a unique rabies virus variant. Preliminary data also suggest that P. subflavus and Lasionycteris noctivagans may harbour two different rabies virus variants (Ps and Ln) that are likely to be maintained independently by each bat species, which recently appear to have emerged as major vectors of human disease.

Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus , Histiotus montanus , and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus . Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species.

Molecular epidemiological studies have linked many cryptic human rabies cases in the United States with exposure to rabies virus (RV) variants associated with insectivorous bats. In Colorado, bats accounted for 98% of all reported animal rabies cases between 1977 and 1996. The genetic divergence of RV was investigated in bat and terrestrial animal specimens that were submitted for rabies diagnosis to the Colorado Department of Public Health and Environment (CDPHE), Colorado, USA. RV isolates from animal specimens across the United States were also included in the analysis. Phylogenetic analyses were performed on partial nucleoprotein (N) gene sequences, which revealed seven principal clades. RV associated with the colonial big brown bat, Eptesicus fuscus, an bats of the genus Myotis were found to segregate into two distinct clades (I and IV). Clade I was harbored by E. fuscus and Myotis species, but was also identified in terrestrial animals such as domestic cats and striped skunks (Mephitis mephitis). Clade IV was divided into subclades IVA, IVB, and IVC; IVA was identified in E. fuscus, and Myotis species bats, and also in a fox; subclades IVB and IVC circulated predominantly in E. fuscus. Clade II was formed by big free-tailed bat (Nyctinomops macrotis) and striped skunk (Mephitis mephitis) samples. Clade III included RVs that are maintained by generally solitary, migratory bats such as the silver-haired bat (Lasionycteris noctivagans) and bats of the genus Lasiurus. Big brown bats were found to harbor this RV variant. None of the Colorado specimens segregated with clades V and VII that harbor RVs associated with terrestrial animals. Different species of bats had the same RV variant, indicating active inter-species rabies transmission. In Colorado, animal rabies occurs principally in bats, and the identification of bat RVs in cat, gray fox Urocyon cinereoargenteus), and striped skunks demonstrated the importance of rabies spillover from bats to domestic and terrestrial wildlife species.

Epizootiological patterns of rabies are described, using antigenic and genetic analysis of samples obtained from infected domestic and wild mammals in 20 Mexican states during 1976-2002. Two independent origins are suggested for rabies in Mexican carnivores. One group shares ancestry with canine rabies, while the other group appears to share a common origin with bat rabies in North America. More than 12 sublineages were found in rabid dog populations, suggesting at least six major spatio-temporal foci. Coyote rabies was found as independent enzootic foci that probably emerged via spillover from dog rabies, translocated from major foci in the southcentral and western regions of Mexico. One focus of gray fox rabies was widely distributed in northwestern Mexico, overlapping with a focus in the same species in the southwestern United States. A skunk rabies focus distributed in the northcentral Mexican states appears to share a common origin with bat rabies foci in North America, and is a close relative of southcentral skunk and raccoon rabies in the United States. Two other skunk foci share a common ancestor with canine rabies and were distributed in northwest Mexico and Yucatan.

The direct fluorescent antibody test is a sensitive and specific procedure used in the routine diagnosis of rabies. However, given the critical role of the rabies diagnostic laboratory in patient management and public health decision-making, the use of a standardized national rabies diagnostic procedure is highly recommended. Seemingly small variations in test procedures may have dramatic effects on sensitivity. For example, two independent reports of diminished staining performance of two lots of a commercial anti-rabies conjugate were investigated in this study. The diminished staining occurred only with a single rabies-virus variant, associated with big brown bats, Eptesicus fuscus, in the southwestern United States. Similarly diluted and prepared diagnostic reagents provided bright staining on all other variants of rabies-virus tested. Subsequent evaluation disclosed that the phenomenon was associated with the relative concentrations of glycerol used in the mounting media by the reporting laboratories. These findings, related to the proper selection of an optimal cover-glass mountant for use in the immunofluorescence procedure, demonstrate the potential for erroneous results with severe implications for patient health, when uncontrolled variations in protocol occur. This paper underscores the necessity for all rabies diagnostic laboratories to follow one standard protocol. Such a protocol has been placed on the websites maintained by the Centers for Disease Control and Prevention: .

Throughout North America, rabies virus (RV) is endemic in bats. Distinct RV variants exist that are closely associated with infection of individual host species, such that there is little or no sustained spillover infection away from the primary host. Using Bayesian methodology, nucleotide substitution rates were estimated from alignments of partial nucleoprotein (N) gene sequences of nine distinct bat RV variants from North America. Substitution rates ranged from 2·32×10−4to 1·38×10−3substitutions per site per year. A maximum-likelihood (ML) molecular clock model was rejected for only two of the nine datasets. In addition, using sequences from bat RV variants across the Americas, the evolutionary rate for the complete N gene was estimated to be 2·32×10−4. This rate was used to scale trees using Bayesian and ML methods, and the time of the most recent common ancestor for current bat RV variant diversity in the Americas was estimated to be 1660 (range 1267–1782) and 1651 (range 1254–1773), respectively. Our reconstructions suggest that RV variants currently associated with infection of bats from Latin America (DesmodusandTadarida) share the earliest common ancestor with the progenitor RV. In addition, from the ML tree, times were estimated for the emergence of the three major lineages responsible for bat rabies cases in North America. Adaptation to infection of the colonial bat species analysed (Eptesicus fuscus,Myotisspp.) appears to have occurred much quicker than for the solitary species analysed (Lasionycteris noctivagans,Pipistrellus subflavus,Lasiurus borealis,Lasiurus cinereus), suggesting that the process of virus adaptation may be dependent on host biology.

Bat lyssaviruses Aravan and Khujand were isolated in southern Kyrgyzstan in 1991 and in northern Tajikistan in 2001, respectively. Preliminary studies with anti-nucleocapsid monoclonal antibodies suggested that the viruses were distinct from other lyssavirus serotypes. These data were supported by sequencing of the N gene of Aravan virus. In the present study, we sequenced the entire N, P and G genes of both Aravan and Khujand viruses and compared them with respective sequences of other lyssaviruses available from GenBank. The results suggested that each virus should be considered as a newly recognized genotype according to the current approaches for genotype definition (amount of nucleotide identity of the N gene and bootstrap support of joining to certain phylogenetic groups). Use of different phylogenetic methods and comparison of different parts of the genomes generally suggested that Khujand virus was mainly related to genotype 6, while Aravan virus, on the one hand, was related to Khujand virus, and, on the other hand, demonstrated moderate similarity to genotypes 4, 5 and 6. The potential significance of these new lyssaviruses for veterinary and public health should not be underestimated.

Most human rabies deaths in the United States can be attributed to unrecognized exposures to rabies viruses associated with bats, particularly those associated with two infrequently encountered bat species (Lasionycteris noctivagans and Pipistrellus subflavus). These human rabies cases tend to cluster in the southeastern and northwestern United States. In these regions, most rabies deaths associated with bats in nonhuman terrestrial mammals are also associated with virus variants specific to these two bat species rather than more common bat species; outside of these regions, more common bat rabies viruses contribute to most transmissions. The preponderance of rabies deaths connected with the two uncommon L. noctivagans and P. subflavus bat rabies viruses is best explained by their evolution of increased viral infectivity.

This study investigated the safety, efficacy, and clearance of SAG-2, an attentuated rabies virus, after oral vaccination in dogs. Nineteen dogs consumed baits containing lyophilized vaccine, but residual SAG-2 virus was recovered in only one of 57 oral swabs, collected one hour post-vaccination. Seven vaccinates were euthanized between 24 and 96 h after consuming a bait. Rabies virus RNA was detected in tonsils from all seven dogs by nested RT-PCR, with primers to the viral glycoprotein. Genomic, sense-transcripts, and m-RNAs were detected in five of seven tonsil samples using primers to the rabies virus nucleoprotein gene, as well as in four of seven samples from the buccal mucosa and one of seven from the tongue. Rabies virus antigen was detected in all tonsils by an immunohistochemistry test, confirming the RT-PCR results. In addition, virus was isolated from one tonsil sample collected at 96 h, providing supportive evidence of viral replication. Ten of 12 (83%) of the vaccinated dogs demonstrated an anamnestic response, with viral neutralizing antibody titers (⩾0.5 IU/ml), after rabies virus challenge. These ten dogs survived, whereas all control dogs succumbed to rabies. Attenuated rabies viruses, such as SAG-2, replicate in local tissues of the oral cavity and can be cleared relatively quickly, without viral excretion, leading to protective immunity against the disease.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.

A novel rabies virus was identified after death in a man who had immigrated from Oaxaca, Mexico, to California, USA. Despite the patient’s history of exposure to domestic and wild carnivores, molecular and phylogenetic characterizations suggested that the virus originated from insectivorous bats. Enhanced surveillance is needed to elucidate likely reservoirs.

SUMMARY—During 2012, 49 states and Puerto Rico reported 6,162 rabid animals and 1 human rabies case to the CDC, representing a 2.1% increase from the 6,031 rabid animals and 6 human cases reported in 2011. Approximately 92% of reported rabid animals were wildlife. Relative contributions by the major animal groups were as follows: 1,953 raccoons (31.7%), 1,680 bats (27.3%), 1,539 skunks (25.0%), 340 foxes (5.5%), 257 cats (4.2%), 115 cattle (1.9%), and 84 dogs (1.4%). Compared with 2011, there was a substantial increase in the number of rabid cattle reported. One case of rabies involving a human was reported from California after the patient died abroad. The infection was determined to be a result of a rabies virus variant associated with Tadarida brasiliensis, with exposure occurring in California.

Changes in demographics, land use, recreation and hunting practices in the last 50 years dramatically increased the public health importance of reservoirs for rabies in wild species in the United States. This article focuses on attributes of host natural history to interpret the molecular phylogenies of the rabies variant transmitted within a particular animal population and the threat to human health presented by this reservoir.

The nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) genes of Irkut and West Caucasian bat viruses (WCBV) were sequenced and compared with those of other lyssaviruses. N gene nucleotide identities provided unequivocal separation of all lyssavirus genotypes with an identity threshold of 82%. On this basis, Irkut virus should be considered as a new genotype with particular relatedness to genotypes 4 and 5 (78.0-78.6% identity for N gene nucleotides and 90.4-92.6% for amino acids). Furthermore, genotypes 4-6, together with Aravan, Khujand and Irkut viruses, present a solid phylogroup of Old World bat lyssaviruses. This relationship is apparent using all three viral genes, and causes overlap between intragenotype and intergenotype identities for the P gene (Aravan, Khujand viruses and genotype 6) and for the G gene (Aravan, Khujand, genotypes 5 and 6). WCBV is the most divergent of known lyssaviruses with only limited relatedness to genotypes 2 and 3.

Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.

Rabies is a fatal viral disease transmitted from animals to humans. It causes more than 35,000 human deaths per year.1 Successful application of veterinary vaccines can eliminate canine rabies in an area, but control of rabies in free-ranging carnivores requires other strategies, such as oral vaccination.2 Live viral vaccines containing modified live rabies or recombinant vaccinia–rabies glycoprotein virus, placed in a bait, are used for disease control in Europe and North America.2–5 In the United States, more than 22 million doses of vaccinia–rabies glycoprotein vaccine were distributed from 1990 to 2000, mainly to control rabies in raccoons in the . . .

Twenty-eight samples from humans and domestic and wild animals collected in Mexico between 1990 and 1995 were characterized by using anti-nucleoprotein monoclonal antibodies and limited sequence analysis of the nucleoprotein gene. The variants of rabies viruses identified in these samples were compared with other isolates from Mexico and the rest of the Americas to establish epidemiologic links between cases and outbreaks and to increase the understanding of rabies epidemiology in the Western Hemisphere. Antigenic and genetic diversity was found in all samples from dogs and dog-related cases, suggesting a long-term endemic situation with multiple, independent cycles of virus transmission. Two isolates from bobcats were antigenically and genetically homologous to the rabies variant circulating in the Arizona gray fox population, indicating a wider distribution of this variant than previously reported. Rabies isolates from skunks were unrelated to any variant analyzed in this study and represent a previously unrecognized cycle of rabies transmission in skunks in Baja California Sur. Two antigenic and genetic variants co-circulating in southern and eastern Mexico were found in viruses obtained from cases epidemiologically related to vampire bats. These results serve as a baseline for the better understanding of the molecular epidemiology of rabies in Mexico.

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre ofor = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.

Background: Alternatives to antigenic typing are needed for epidemiologic surveys of the rabies virus associated with translocated coyotes and foxes, especially in areas where a closely related rabies virus is transmitted by striped skunks. Objectives: We developed and evaluated two enzyme based typing methods for rabies virus. The products of a reverse transcription-polymerase chain reaction (RT/PCR) of the nucleoprotein gene were hybridized to type specific probes and detected by enzyme assay after immobilization on microtiter plates. Study design: We tested RT/PCR products of 27 rabies isolates by two different DNA enzyme immunoassays (DEIA) and evaluated the quality of the results from the corresponding nucleotide sequence of the samples. Results: Using a set of two probes, one of the DEIAs correctly identified 26/27 samples as variants of rabies virus associated with either skunks, foxes, or coyotes. The identity of one fox rabies sample was unresolved by this assay. The second DEIA correctly identified 24/27 samples as variants of rabies virus associated with either skunks, foxes, or coyotes. This assay did not resolve the identity of two fox rabies samples, and misidentified one fox rabies sample as a skunk rabies sample. Conclusions: DEIA can be used for epidemiologic studies of variants of rabies virus associated with skunks, foxes, and coyotes. Both DEIA methods were effective when typing probes recognized changes at a minimum of two nucleotide positions between variants, but only one assay method was sufficiently stringent to detect a single base pair mismatch. The inherent mutability of RNA viruses must be considered when designing and evaluating typing methods.

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.

Nucleotide sequence analysis of a 200-bp region of the nucleoprotein (N) gene of rabies virus differentiated unique genetic groups of rabies virus from samples collected in areas where dog rabies is enzootic in Asia, Africa, Europe, and the Americas. Patterns of nucleotide sequence identified for an outbreak area were conserved in samples collected over three decades. Epidemiologic relationships among isolates were determined by patterns of conserved nucleotide sequence, and the degree of sequence divergence between samples from separate outbreak areas were measured. This approach suggested that a historical reconstruction of events leading to the introduction of rabies into an area would be possible. In this broader view of rabies epidemiology, the cultural legacy of European exploration and colonization may have also included zoonotic disease.

To summarize the epidemiologic, diagnostic, and clinical features of the 32 laboratory-confirmed cases of human rabies diagnosed in the United States from 1980 to 1996.Data were obtained from case reports of human rabies submitted to the Centers for Disease Control and Prevention by state or local health authorities.All cases of human rabies reported in the United States from 1980 to 1996 in which infection with rabies virus was confirmed by laboratory studies.Patients were reviewed for demographic characteristics, exposure history, rabies prophylaxis, clinical presentation, treatment, clinical course, diagnostic laboratory tests, identification of rabies virus variants, and the number of medical personnel or family members who required postexposure prophylaxis after coming in contact with an exposed person.32 cases of human rabies were reported from 20 states. Patients ranged in age from 4 to 82 years and were predominantly male (63%). Most patients (25 of 32) had no definite history of an animal bite or other event associated with rabies virus transmission. Of the 32 cases, 17 (53%) were associated with rabies virus variants found in insectivorous bats, 12 (38%) with variants found in domestic dogs outside the United States, 2 (6%) with variants found in indigenous domestic dogs, and 1 (3%) with a variant found in indigenous skunks. Among the 7 patients with a definite exposure history, 6 cases were attributable to dog bites received in foreign countries and 1 was attributable to a bat bite received in the United States. In 12 of the 32 patients (38%), rabies was not clinically suspected and was diagnosed after death. In the remaining 20 cases (63%), the diagnosis of rabies was considered before death and samples were obtained specifically for laboratory confirmation a median of 7 days (range, 3 to 17 days) after the onset of clinical signs. Of the clinical differences between patients in whom rabies was diagnosed before death and those in whom it was diagnosed after death, the presence of hydrophobia or aerophobia was significantly associated with antemortem diagnosis (odds ratio, 11.0 [95% CI, 1.05 to 273.34]). The median number of medical personnel or familial contacts of the patients who received postexposure prophylaxis was 54 per patient (range, 4 to 179). None of the 32 patients with rabies received postexposure prophylaxis before the onset of clinical disease.In the United States, human rabies is rare but probably underdiagnosed. Rabies should be included in the differential diagnosis of any case of acute, rapidly progressing encephalitis, even if the patient does not recall being bitten by an animal. In addition to situations involving an animal bite, a scratch from an animal, or contact of mucous membranes with infectious saliva, postexposure prophylaxis should be considered if the history indicates that a bat was physically present, even if the person is unable to reliably report contact that could have resulted in a bite. Such a situation may arise when a bat bite causes an insignificant wound or the circumstances do not allow recognition of contact, such as when a bat is found in the room of a sleeping person or near a previously unattended child.