Rabies in a Dog Imported from Azerbaijan - Pennsylvania, 2021.

On June 16, 2021, rabies virus infection was confirmed in a dog included in a shipment of rescue animals imported into the United States from Azerbaijan. A multistate investigation was conducted to prevent secondary rabies cases, avoid reintroduction of a dog-maintained rabies virus variant (DMRVV), identify persons who might have been exposed and would be recommended to receive rabies postexposure prophylaxis, and investigate the cause of importation control failures. Results of a prospective serologic monitoring (PSM) protocol suggested that seven of 32 (22%) animals from the same shipment as the dog with confirmed rabies virus infection and who had available titer results after rabies vaccine booster had not been adequately vaccinated against rabies before importation. A requirement for rabies vaccination certificates alone will not adequately identify improper vaccination practices or fraudulent paperwork and are insufficient as a stand-alone rabies importation prevention measure. Serologic titers before importation would mitigate the risk for importing DMRVV.

Phylogenetic and epidemiologic evidence of multiyear incubation in human rabies.

Eight years after emigrating from Brazil, an otherwise healthy man developed rabies. An exposure prior to immigration was reported. Genetic analysis revealed a canine rabies virus variant found only in the patient's home country, and the patient had not traveled internationally since immigrating to the United States. We describe how epidemiological, phylogenetic, and viral sequencing data provided confirmation that rabies encephalomyelitis may present after a long, multiyear incubation period, a consideration that previously has been hypothesized without the ability to exclude a more recent exposure. Accordingly, rabies should be considered in the diagnosis of any acute encephalitis, myelitis, or encephalomyelitis.

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.

Further Evidence of Inadequate Quality in Lateral Flow Devices Commercially Offered for the Diagnosis of Rabies.

As a neglected zoonotic disease, rabies causes approximately 5.9 × 104 human deaths annually, primarily affecting low- and middle-income countries in Asia and Africa. In those regions, insufficient surveillance is hampering adequate medical intervention and is driving the vicious cycle of neglect. Where resources to provide laboratory disease confirmation are limited, there is a need for user-friendly and low-cost reliable diagnostic tools that do not rely on specialized laboratory facilities. Lateral flow devices (LFD) offer an alternative to conventional diagnostic methods and may strengthen control efforts in low-resource settings. Five different commercially available LFDs were compared in a multi-centered study with respect to their diagnostic sensitivity and their agreement with standard rabies diagnostic techniques. Our evaluation was conducted by several international reference laboratories using a broad panel of samples. The overall sensitivities ranged from 0% up to 62%, depending on the LFD manufacturer, with substantial variation between the different laboratories. Samples with high antigen content and high relative viral load tended to test positive more often in the Anigen/Bionote test, the latter being the one with the best performance. Still, the overall unsatisfactory findings corroborate a previous study and indicate a persistent lack of appropriate test validation and quality control. At present, the tested kits are not suitable for in-field use for rabies diagnosis, especially not for suspect animals where human contact has been identified, as an incorrect negative diagnosis may result in human casualties. This study points out the discrepancy between the enormous need for such a diagnostic tool on the one hand, and on the other hand, a number of already existing tests that are not yet ready for use.

Enzootic rabies elimination from dogs and reemergence in wild terrestrial carnivores, United States.

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.

An ELISA-based method for detection of rabies virus nucleoprotein-specific antibodies in human antemortem samples.

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.

Novel mass spectrometry based detection and identification of variants of rabies virus nucleoprotein in infected brain tissues.

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.

An inter- laboratory proficiency testing exercise for rabies diagnosis in Latin America and the Caribbean.

The direct fluorescent antibody test (DFA), is performed in all rabies reference laboratories across Latin America and the Caribbean (LAC). Despite DFA being a critical capacity in the control of rabies, there is not a standardized protocol in the region. We describe the results of the first inter-laboratory proficiency exercise of national rabies laboratories in LAC countries as part of the regional efforts towards dog-maintained rabies elimination in the American region. Twenty three laboratories affiliated to the Ministries of Health and Ministries of Agriculture participated in this exercise. In addition, the laboratories completed an online questionnaire to assess laboratory practices. Answers to the online questionnaire indicated large variability in the laboratories throughput, equipment used, protocols availability, quality control standards and biosafety requirements. Our results will inform actions to improve and harmonize laboratory rabies capacities across LAC in support for the regional efforts towards elimination of dog-maintained rabies.

A need for standardized rabies-virus diagnostic procedures: effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test.

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: .

Virology, immunology and pathology of human rabies during treatment.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of an indirect rapid immunohistochemistry test for the differentiation of rabies virus variants.

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.

Emerging pattern of rabies deaths and increased viral infectivity.

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.