Bat and Lyssavirus Exposure among Humans in Area that Celebrates Bat Festival, Nigeria, 2010 and 2013.

Using questionnaires and serologic testing, we evaluated bat and lyssavirus exposure among persons in an area of Nigeria that celebrates a bat festival. Bats from festival caves underwent serologic testing for phylogroup II lyssaviruses (Lagos bat virus, Shimoni bat virus, Mokola virus). The enrolled households consisted of 2,112 persons, among whom 213 (10%) were reported to have ever had bat contact (having touched a bat, having been bitten by a bat, or having been scratched by a bat) and 52 (2%) to have ever been bitten by a bat. Of 203 participants with bat contact, 3 (1%) had received rabies vaccination. No participant had neutralizing antibodies to phylogroup II lyssaviruses, but >50% of bats had neutralizing antibodies to these lyssaviruses. Even though we found no evidence of phylogroup II lyssavirus exposure among humans, persons interacting with bats in the area could benefit from practicing bat-related health precautions.

Seroprevalence and Risk Factors Possibly Associated with Emerging Zoonotic Vaccinia Virus in a Farming Community, Colombia.

In 2014, vaccinia virus (VACV) infections were identified among farmworkers in Caquetá Department, Colombia; additional cases were identified in Cundinamarca Department in 2015. VACV, an orthopoxvirus (OPXV) used in the smallpox vaccine, has caused sporadic bovine and human outbreaks in countries such as Brazil and India. In response to the emergence of this disease in Colombia, we surveyed and collected blood from 134 farmworkers and household members from 56 farms in Cundinamarca Department. We tested serum samples for OPXV antibodies and correlated risk factors with seropositivity by using multivariate analyses. Fifty-two percent of farmworkers had OPXV antibodies; this percentage decreased to 31% when we excluded persons who would have been eligible for smallpox vaccination. The major risk factors for seropositivity were municipality, age, smallpox vaccination scar, duration of time working on a farm, and animals having vaccinia-like lesions. This investigation provides evidence for possible emergence of VACV as a zoonosis in South America.

Potential Confounding of Diagnosis of Rabies in Patients with Recent Receipt of Intravenous Immune Globulin.

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.

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.

Detection and Molecular Characterization of Zoonotic Poxviruses Circulating in the Amazon Region of Colombia, 2014.

During 2014, cutaneous lesions were reported in dairy cattle and farmworkers in the Amazon Region of western Colombia. Samples from 6 patients were analyzed by serologic and PCR testing, and results demonstrated the presence of vaccinia virus and pseudocowpox virus. These findings highlight the need for increased poxvirus surveillance in Colombia.

A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories.

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron®. Genetron® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield.

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.

Vaccinia virus A19 protein participates in the transformation of spherical immature particles to barrel-shaped infectious virions.

The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.

Interactions of the vaccinia virus A19 protein.

The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.

Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain.

The vaccinia virus O3 protein, a component of the entry-fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry-fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry-fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry.

Orthopoxvirus species and strain differences in cell entry.

Vaccinia virus (VACV) enters cells by a low pH endosomal route or by direct fusion with the plasma membrane. We previously found differences in entry properties of several VACV strains: entry of WR was enhanced by low pH, reduced by bafilomycin A1 and relatively unaffected by heparin, whereas entry of IHD-J, Copenhagen and Elstree were oppositely affected. Since binding and entry modes may have been selected by specific conditions of in vitro propagation, we now examined the properties of three distinct, recently isolated cowpox viruses and a monkeypox virus as well as additional VACV and cowpox virus strains. The recent isolates were more similar to WR than to other VACV strains, underscoring the biological importance of endosomal entry by orthopoxviruses. Sequence comparisons, gene deletions and gene swapping experiments indicated that viral determinants, other than or in addition to the A26 and A25 "fusion-suppressor" proteins, impact entry properties.

Sequence-divergent chordopoxvirus homologs of the o3 protein maintain functional interactions with components of the vaccinia virus entry-fusion complex.

Composed of 35 amino acids, O3 is the smallest characterized protein encoded by vaccinia virus (VACV) and is an integral component of the entry-fusion complex (EFC). O3 is conserved with 100% identity in all orthopoxviruses except for monkeypox viruses, whose O3 homologs have 2 to 3 amino acid substitutions. Since O3 is part of the EFC, high conservation could suggest an immutable requirement for interaction with multiple proteins. Chordopoxviruses of other genera also encode small proteins with a characteristic predicted N-terminal α-helical hydrophobic domain followed by basic amino acids and proline in the same relative genome location as that of VACV O3. However, the statistical significance of their similarity to VACV O3 is low due to the large contribution of the transmembrane domain, their small size, and their sequence diversity. Nevertheless, trans-complementation experiments demonstrated the ability of a representative O3-like protein from each chordopoxvirus genus to rescue the infectivity of a VACV mutant that was unable to express endogenous O3. Moreover, recombinant viruses expressing O3 homologs in place of O3 replicated and formed plaques as well or nearly as well as wild-type VACV. The O3 homologs expressed by the recombinant VACVs were incorporated into the membranes of mature virions and, with one exception, remained stably associated with the detergent-extracted and affinity-purified EFC. The ability of the sequence-divergent O3 homologs to coordinate function with VACV entry proteins suggests the conservation of structural motifs. Analysis of chimeras formed by swapping domains of O3 with those of other proteins indicated that the N-terminal transmembrane segment was responsible for EFC interactions and for the complementation of infectivity.

Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.

Characterization of a newly identified 35-amino-acid component of the vaccinia virus entry/fusion complex conserved in all chordopoxviruses.

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function.

A single point mutation disrupts the capsid assembly in Sesbania Mosaic Virus resulting in a stable isolated dimer.

Protein-protein interactions play a crucial role in virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable soluble dimers of the capsid protein. The X-ray crystal structure of one of the isolated dimer mutants - rCPDeltaN65W170K was determined to a resolution of 2.65 A. Detailed analysis of the dimeric mutant protein structure revealed that a number of structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated dimer was "more relaxed" than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca(2+) ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the virus, has different conformations in the isolated dimer and the intact virus suggesting its flexible nature and the conformational changes that accompany assembly. The isolated dimer mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca(2+) mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.

Vaccinia virus H7 protein contributes to the formation of crescent membrane precursors of immature virions.

Crescent membranes are the first viral structures that can be discerned during poxvirus morphogenesis. The crescents consist of a lipoprotein membrane and an outer lattice scaffold, which provides uniform curvature. Relatively little is known regarding the composition of the crescent membrane or its mode of formation. Here, we show that the H7 protein, which is conserved in all vertebrate poxviruses but has no discernible functional motifs or nonpoxvirus homologs, contributes to the formation of crescents and immature virions. Synthesis of the 17-kDa H7 protein was dependent on DNA replication and occurred late during vaccinia virus infection. Unlike many late proteins, however, H7 was not incorporated into mature virions or localized in cellular organelles. To gain insight into the role of H7, an inducible mutant was constructed and shown to have a conditional lethal phenotype: H7 expression and infectious virus formation were dependent on isopropyl-beta-D-thiogalactopyranoside. In the absence of inducer, viral late proteins were made, but membrane and core proteins were not processed by the I7 protease. A block in morphogenesis was demonstrated by transmission electron microscopy: neither typical crescents nor immature virions were detected in the absence of inducer. Instead, factory areas of the cytoplasm contained large, electron-dense inclusions, some of which had partially coated membrane segments at their surfaces. Separate, lower-density inclusions containing the D13 scaffold protein and endoplasmic reticulum membranes were also present. These features are most similar to those previously seen when expression of A11, another conserved nonvirion protein, is repressed.

Inhibition of the ubiquitin-proteasome system prevents vaccinia virus DNA replication and expression of intermediate and late genes.

The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.

Structure of recombinant capsids formed by the beta-annulus deletion mutant -- rCP (Delta48-59) of Sesbania mosaic virus.

A unique feature of several T=3 icosahedral viruses is the presence of a structure called the beta-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T=3 capsid assembly. In order to examine the importance of the beta-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48-59 involved in the formation of the beta-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Delta48-59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 A resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the beta-annulus. Therefore, the beta-annulus is not essential for T=3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the beta-annulus could be closely similar.

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket.

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.