Isolation and Characterization of Akhmeta Virus from Wild-Caught Rodents (Apodemus spp.) in Georgia.

In 2013, a novel orthopoxvirus was detected in skin lesions of two cattle herders from the Kakheti region of Georgia (country); this virus was named Akhmeta virus. Subsequent investigation of these cases revealed that small mammals in the area had serological evidence of orthopoxvirus infections, suggesting their involvement in the maintenance of these viruses in nature. In October 2015, we began a longitudinal study assessing the natural history of orthopoxviruses in Georgia. As part of this effort, we trapped small mammals near Akhmeta (n = 176) and Gudauri (n = 110). Here, we describe the isolation and molecular characterization of Akhmeta virus from lesion material and pooled heart and lung samples collected from five wood mice (Apodemus uralensis and Apodemus flavicollis) in these two locations. The genomes of Akhmeta virus obtained from rodents group into 2 clades: one clade represented by viruses isolated from A. uralensis samples, and one clade represented by viruses isolated from A. flavicollis samples. These genomes also display several presumptive recombination events for which gene truncation and identity have been examined.IMPORTANCE Akhmeta virus is a unique Orthopoxvirus that was described in 2013 from the country of Georgia. This paper presents the first isolation of this virus from small mammal (Rodentia; Apodemus spp.) samples and the molecular characterization of those isolates. The identification of the virus in small mammals is an essential component to understanding the natural history of this virus and its transmission to human populations and could guide public health interventions in Georgia. Akhmeta virus genomes harbor evidence suggestive of recombination with a variety of other orthopoxviruses; this has implications for the evolution of orthopoxviruses, their ability to infect mammalian hosts, and their ability to adapt to novel host species.

Human Monkeypox in Sierra Leone after 44-Year Absence of Reported Cases.

We note the reemergence of human monkeypox in Sierra Leone following a 44-year absence of reported disease. The persons affected were an 11-month-old boy and, several years later, a 35-year-old man. The reappearance of monkeypox in this country suggests a need for renewed vigilance and awareness of the disease and its manifestations.

Human infection with a zoonotic orthopoxvirus in the country of Georgia.

During 2013, cutaneous lesions developed in two men in the country of Georgia after they were exposed to ill cows. The men had never received vaccination against smallpox. Tests of lesion material with the use of a quantitative real-time polymerase-chain-reaction assay for non-variola virus orthopoxviruses were positive, and DNA sequence analysis implicated a novel orthopoxvirus species. During the ensuing epidemiologic investigation, no additional human cases were identified. However, serologic evidence of exposure to an orthopoxvirus was detected in cows in the patients' herd and in captured rodents and shrews. A third case of human infection that occurred in 2010 was diagnosed retrospectively during testing of archived specimens that were originally submitted for tests to detect anthrax. Orthopoxvirus infection should be considered in persons in whom cutaneous lesions develop after contact with animals.

Development of a relative potency test using ELISA for human rabies vaccines.

The NIH potency test for human rabies vaccines has disadvantages for use, especially in developing countries where rabies is endemic and prophylaxis needs ample, rapid, and reliable vaccine supplies. In China, 60-75 million doses of human rabies vaccines are administered each year. Vaccine quality control is of paramount importance, as is the release of potency-validated vaccines. We intended to design an alternative to the NIH in vivo method, and developed a relative potency test using an ELISA. Using Pearson's correlation analysis, we found a close relationship between the rabies vaccine glycoprotein content in vitro and the potency values in vivo. We suggest the relative potency test developed here as a simplified method for human rabies vaccine quality control in China and a possible alternative to the NIH method.

Retrospective Proteomic Analysis of Serum After Akhmeta Virus Infection: New Suspect Case Identification and Insights Into Poxvirus Humoral Immunity.

Serologic cross-reactivity, a hallmark of orthopoxvirus (OPXV) infection, makes species-specific diagnosis of infection difficult. In this study, we used a variola virus proteome microarray to characterize and differentiate antibody responses to nonvaccinia OPXV infections from smallpox vaccination. The profile of 2 case patients infected with newly discovered OPXV, Akhmeta virus, exhibited antibody responses of greater intensity and broader recognition of viral proteins and includes the B21/22 family glycoproteins not encoded by vaccinia virus strains used as vaccines. An additional case of Akhmeta virus, or nonvaccinia OPXV infection, was identified through community surveillance of individuals with no or uncertain history of vaccination and no recent infection. The results demonstrate the utility of microarrays for high-resolution mapping of antibody response to determine the nature of OPXV exposure.

Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus Exposure - United States (Including U.S. Territories), July 2017.

CDC has updated the interim guidance for U.S. health care providers caring for pregnant women with possible Zika virus exposure in response to 1) declining prevalence of Zika virus disease in the World Health Organization's Region of the Americas (Americas) and 2) emerging evidence indicating prolonged detection of Zika virus immunoglobulin M (IgM) antibodies. Zika virus cases were first reported in the Americas during 2015-2016; however, the incidence of Zika virus disease has since declined. As the prevalence of Zika virus disease declines, the likelihood of false-positive test results increases. In addition, emerging epidemiologic and laboratory data indicate that, as is the case with other flaviviruses, Zika virus IgM antibodies can persist beyond 12 weeks after infection. Therefore, IgM test results cannot always reliably distinguish between an infection that occurred during the current pregnancy and one that occurred before the current pregnancy, particularly for women with possible Zika virus exposure before the current pregnancy. These limitations should be considered when counseling pregnant women about the risks and benefits of testing for Zika virus infection during pregnancy. This updated guidance emphasizes a shared decision-making model for testing and screening pregnant women, one in which patients and providers work together to make decisions about testing and care plans based on patient preferences and values, clinical judgment, and a balanced assessment of risks and expected outcomes.

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat.

The genome of Eptesipoxvirus (EPTV) is the first poxvirus genome isolated from a microbat. The 176,688 nt sequence, which is believed to encompass the complete coding region of the virus, is 67% A+T and is predicted to encode 191 genes. 11 of these genes have no counterpart in GenBank and are therefore unique to EPTV. The presence of a distantly related ortholog of Vaccinia virus F5L in EPTV uncovered a link with fragmented F5L orthologs in Molluscum contagiosum virus/squirrelpox and clade II viruses. Consistent with the unique position of EPTV approximately mid-point between the orthopoxviruses and the clade II viruses, EPTV has 11 genes that are specific to the orthopoxviruses and 13 genes that are typical, if not exclusive, to the clade II poxviruses. This mosaic nature of EPTV blurs the distinction between the old description of the orthopoxvirus and clade II groups. Genome annotation and characterization failed to find any common virulence genes shared with the other poxvirus isolated from bat (pteropoxvirus); however, EPTV encodes 3 genes that may have been transferred to or from deerpox and squirrelpox viruses; 2 of these, a putative endothelin-like protein and a MHC class I-like protein are likely to have immunomodulatory roles.

The genomes of three North American orthopoxviruses.

The complete genomes of a skunkpox, volepox, and raccoonpox virus were sequenced and annotated. Phylogenetic analysis of these genomes indicates that although these viruses are all orthopoxviruses, they form a distinct clade to the other known species. This supports the ancient divergence of the North American orthopoxviruses from other members of the orthopoxviruses. Only two open reading frames appear to be unique to this group of viruses, but a relatively small number of insertions/deletions contribute to the varied gene content of this clade. The availability of these genomes will help determine whether skunkpox and volepox viruses share the characteristics that make raccoonpox a useful vaccine vector.

Novel poxvirus infection in 2 patients from the United States.

BACKGROUND: Some human poxvirus infections can be acquired through zoonotic transmission. We report a previously unknown poxvirus infection in 2 patients, 1 of whom was immunocompromised; both patients had known equine contact. METHODS: The patients were interviewed and clinical information was abstracted from the patients' medical files. Biopsies of the skin lesions were collected from both patients for histopathology, immunohistochemistry, and transmission electron microscopy analysis. Oral and skin swabs were collected from animals with frequent contact with the patients, and environmental sampling including rodent trapping was performed on the farm where the immunosuppressed patient was employed. "Pan-pox and high Guanine-cytosine" polymerase chain reaction assays were performed on patient, animal, and environmental isolates. Amplicon sequences of the viral DNA were used for agent identification and phylogenetic analysis. RESULTS: Specimens from both human cases revealed a novel poxvirus. The agent shares 88% similarity to viruses in the Parapoxvirus genus and 78% to those in the Molluscipoxvirus genus but is sufficiently divergent to resist classification as either. All animal and environmental specimens were negative for poxvirus and both patients had complete resolution of lesions. CONCLUSIONS: This report serves as a reminder that poxviruses should be considered in cutaneous human infections, especially in individuals with known barnyard exposures. The clinical course of the patients was similar to that of parapoxvirus infections, and the source of this virus is currently unknown but is presumed to be zoonotic. This report also demonstrates the importance of a comprehensive approach to diagnosis of human infections caused by previously unknown pathogens.

Rabies in a Dog Imported from Egypt with a Falsified Rabies Vaccination Certificate--Virginia, 2015.

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.

Effects of anthropogenic and demographic factors on patterns of parasitism in African small mammal communities.

Habitat disturbance often results in alterations in community structure of small mammals. Additionally, the parasites harboured by these small mammals may be impacted by environmental changes or indirectly affected by changes in available hosts. To improve our understanding of this interplay, we examined the patterns of parasitism in small mammal communities from a variety of habitats in forested Uganda. Small mammals were collected from areas experiencing variable habitat disturbance, host density and species richness. The analysis focused on 3 most abundant rodent species, Lophuromys aquilus, Praomys jacksoni and Hylomyscus stella, and a diverse group of parasites they harbour. The impact of various habitat and host community factors on parasite prevalence was examined using linear regression and Spearman's rank-order correlation. We further investigated the parasite communities associated with each individual using correspondence analysis. We determined that, parasite prevalence and richness may be occasionally influenced by community and habitat factors, but taxonomy is a driving force in influencing the parasite community harboured by an individual host. Ultimately, applying general principles across a broad range of disturbance levels and diverse host communities needs to be approached with caution in complex communities.

Estimating the geographic distribution of human Tanapox and potential reservoirs using ecological niche modeling.

BACKGROUND: Tanapox virus is a zoonotic infection that causes mild febrile illness and one to several nodular skin lesions. The disease is endemic in parts of Africa. The principal reservoir for the virus that causes Tanapox is unknown, but has been hypothesized to be a non-human primate. This study employs ecological niche modeling (ENM) to determine areas of tropical Africa suitable for the occurrence of human Tanapox and a list of hypothetical reservoirs. The resultant niche model will be a useful tool to guide medical surveillance activities in the region. METHODS: This study uses the Desktop GARP software to predict regions where human Tanapox might be expected to occur based on historical human case locations and environmental data. Additional modeling of primate species, using occurrence data from museum records was performed to determine suitable disease reservoirs. RESULTS: The final ENM predicts a potential distribution of Tanapox over much of equatorial Africa, exceeding the borders of Kenya and Democratic Republic of Congo (DRC) where it has been historically reported. Five genera of non-human primates were found to be potential reservoir taxa. CONCLUSIONS: Validity testing suggests the model created here is robust (p < 0.04). Several genera of primates were identified as having ENMs overlapping with that of Tanapox and are suggested as potential reservoirs, mainly members of the Genus Cercopithecus. The ENM modeling technique has several limitations and results should be interpreted with caution. This study may increase knowledge and engage further research in this neglected disease.

Novel poxvirus in big brown bats, northwestern United States.

A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of Poxviridae.

Phylogenetic and ecologic perspectives of a monkeypox outbreak, southern Sudan, 2005.

Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans.

Use of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Tests by US Infectious Disease Physicians: Results of an Emerging Infections Network Survey, March 2022.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests have had limited recommended clinical application during the coronavirus disease 2019 (COVID-19) pandemic. To inform clinical practice, an understanding is needed of current perspectives of United States-based infectious disease (ID) physicians on the use, interpretation, and need for SARS-CoV-2 antibody tests. Methods: In March 2022, members of the Emerging Infections Network (EIN), a national network of practicing ID physicians, were surveyed on types of SARS-CoV-2 antibody assays ordered, interpretation of test results, and clinical scenarios for which antibody tests were considered. Results: Of 1867 active EIN members, 747 (40%) responded. Among the 583 who managed or consulted on COVID-19 patients, a majority (434/583 [75%]) had ordered SARS-CoV-2 antibody tests and were comfortable interpreting positive (452/578 [78%]) and negative (405/562 [72%]) results. Antibody tests were used for diagnosing post-COVID-19 conditions (61%), identifying prior SARS-CoV-2 infection (60%), and differentiating prior infection and response to COVID-19 vaccination (37%). Less than a third of respondents had used antibody tests to assess need for additional vaccines or risk stratification. Lack of sufficient evidence for use and nonstandardized assays were among the most common barriers for ordering tests. Respondents indicated that statements from professional societies and government agencies would influence their decision to order SARS-CoV-2 antibody tests for clinical decision making. Conclusions: Practicing ID physicians are using SARS-CoV-2 antibody tests, and there is an unmet need for clarifying the appropriate use of these tests in clinical practice. Professional societies and US government agencies can support clinicians in the community through the creation of appropriate guidance.

Effects of georeferencing effort on mapping monkeypox case distributions and transmission risk.

BACKGROUND: Maps of disease occurrences and GIS-based models of disease transmission risk are increasingly common, and both rely on georeferenced diseases data. Automated methods for georeferencing disease data have been widely studied for developed countries with rich sources of geographic referenced data. However, the transferability of these methods to countries without comparable geographic reference data, particularly when working with historical disease data, has not been as widely studied. Historically, precise geographic information about where individual cases occur has been collected and stored verbally, identifying specific locations using place names. Georeferencing historic data is challenging however, because it is difficult to find appropriate geographic reference data to match the place names to. Here, we assess the degree of care and research invested in converting textual descriptions of disease occurrence locations to numerical grid coordinates (latitude and longitude). Specifically, we develop three datasets from the same, original monkeypox disease occurrence data, with varying levels of care and effort: the first based on an automated web-service, the second improving on the first by reference to additional maps and digital gazetteers, and the third improving still more based on extensive consultation of legacy surveillance records that provided considerable additional information about each case. To illustrate the implications of these seemingly subtle improvements in data quality, we develop ecological niche models and predictive maps of monkeypox transmission risk based on each of the three occurrence data sets. RESULTS: We found macrogeographic variations in ecological niche models depending on the type of georeferencing method used. Less-careful georeferencing identified much smaller areas as having potential for monkeypox transmission in the Sahel region, as well as around the rim of the Congo Basin. These results have implications for mapping efforts, as each higher level of georeferencing precision required considerably greater time investment. CONCLUSIONS: The importance of careful georeferencing cannot be overlooked, despite it being a time- and labor-intensive process. Investment in archival storage of primary disease-occurrence data is merited, and improved digital gazetteers are needed to support public health mapping activities, particularly in developing countries, where maps and geographic information may be sparse.

Sealpox virus in marine mammal rehabilitation facilities, North America, 2007-2009.

Sealpox, a zoonotic disease affecting pinnipeds (seals and sea lions), can occur among captive and convalescing animals. We surveyed 1 worker each from 11 marine mammal centers and interviewed 31 other marine mammal workers to ascertain their knowledge of and experience with sealpox virus and to identify factors associated with sealpox virus outbreaks among pinnipeds in marine rehabilitation facilities. Demographic and health data were obtained for 1,423 pinnipeds at the 11 facilities. Among the 23 animals in which sealpox was clinically diagnosed, 4 arrived at the facility ill, 11 became ill <5 weeks after arrival, and 2 became ill ≥5 weeks after arrival; the timing of illness onset was unknown for 6 animals. Most infections occurred in pinnipeds <1 year of age. Nine affected animals were malnourished; 4 had additional illnesses. Sealpox had also occurred among workers at 2 facilities. Sealpox is a noteworthy zoonosis of rehabilitating convalescing pinnipeds; workplace education can help to minimize risks for human infection.

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.

Pharmacokinetics and Efficacy of a Potential Smallpox Therapeutic, Brincidofovir, in a Lethal Monkeypox Virus Animal Model.

Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 105 plaque-forming units (PFU; 90% lethal dose [LD90]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID-1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID-1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [Cmax]) and the time of the last quantifiable concentration (AUClast) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival.IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today's current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel.

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.