Mpox outbreak in Rio de Janeiro, Brazil: A translational approach.

Mpox is a zoonotic disease historically reported in Africa. Since 2003, limited outbreaks have occurred outside Africa. In 2022, the global spread of cases with sustained interhuman transmission and unusual disease features raised public health concerns. We explore the mpox outbreak in Rio de Janeiro (RJ) state, Brazil, in an observational study of mpox-suspected cases from June to December 2022. Data collection relied on a public healthcare notification form. Diagnosis was determined by MPXV-PCR. In 46 confirmed cases, anti-OPXV IgG was determined by ELISA, and seven MPXV genomes were sequenced. A total of 3095 cases were included, 816 (26.3%) with positive MPXV-PCR results. Most positive cases were men in their 30 s and MSM. A total of 285 (34.9%) MPXV-PCR+ patients live with HIV. Eight were coinfected with varicella-zoster virus. Anogenital lesions and adenomegaly were associated with the diagnosis of mpox. Females and individuals under 18 represented 9.4% and 5.4% of all confirmed cases, respectively, showing higher PCR cycle threshold (Ct) values and fewer anogenital lesions compared to adult men. Anti-OPXV IgG was detected in 29/46 (63.0%) patients. All analyzed sequences belonged to clade IIb. In RJ state, mpox presented a diverse clinical picture, represented mainly by mild cases with low complication rates and prominent genital involvement. The incidence in females and children was higher than usually reported. The observation of a bimodal distribution of Ct values, with few positive results, may suggest the need to review the diagnostic criteria in these groups.

ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

In vitro susceptibility of eighteen clinical isolates of human monkeypox virus to tecovirimat.

BACKGROUND: In 2022, an outbreak of mpox that started in European countries spread worldwide through human-to-human transmission. Cases have been mostly mild, but severe clinical presentations have been reported. In these cases, tecovirimat has been the drug of choice to treat patients with aggravated disease. OBJECTIVES: Here we investigated the tecovirimat susceptibility of 18 clinical isolates of monkeypox virus (MPXV) obtained from different regions of Brazil. METHODS: Different concentrations of tecovirimat were added to cell monolayers infected with each MPXV isolate. After 72 hours, cells were fixed and stained for plaque visualization, counting, and measurement. The ortholog of F13L gene from each MPXV isolate was polymerase chain reaction (PCR)-amplified, sequenced, and the predicted protein sequences were analyzed. FINDINGS: The eighteen MPXV isolates generated plaques of different sizes. Although all isolates were highly sensitive to the drug, two showed different response curves and IC50 values. However, the target protein of tecovirimat, F13 (VP37), was 100% conserved in all MPXV isolates and therefore does not explain the difference in sensitivity. MAIN CONCLUSIONS: Our results support screening different MPXV isolates for tecovirimat susceptibility as an important tool to better use of the restricted number of tecovirimat doses available in low-income countries to treat patients with mpox.

Genomic diversity of vaccinia virus strain Cantagalo isolated in southeastern Brazil during the early years of the outbreak, 1999-2006.

Outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported in Brazil since 1999 when the vaccinia virus strain Cantagalo was first isolated in the State of Rio de Janeiro. However, the genomic diversity of the viral isolates associated with these outbreaks is not well known, particularly in the southeastern states that represent the focal point of virus spread to other regions. Here, we report the genomic sequences and an analysis of the polymorphic site profiles and genotypic diversity of four clinical isolates of vaccinia virus strain Cantagalo collected from 1999 to 2006 in southeastern Brazil.

Validation of a real-time PCR assay for detection of swinepox virus.

Swine are the only known hosts of swinepox virus (SWPV), the sole member of the genus Suipoxvirus, family Poxviridae. Rapid diagnosis is recommended for appropriate interventions because of the high morbidity associated with this virus. This study describes a real-time quantitative PCR (qPCR) assay for rapid detection and quantification of SWPV. The detection limit, repeatability, reproducibility, and specificity of this assay were determined. The efficiency was 96%, and the R2 value was 0.996. The detection limit was 1 fg or 10-0.5 TCID50/50 µL. Tests showed that the greatest source of error in the SWPV qPCR assay was variation between analysts rather than different qPCR kits or equipment. All nucleic acids from other viruses or samples collected from swine were negative in the specificity test. qPCR for SWPV is a new method with tested variables that allows main sources of error in laboratory diagnosis and viral quantification to be identified.

Autochthonous Transmission of East/Central/South African Genotype Chikungunya Virus, Brazil.

We isolated East/Central/South African genotype chikungunya virus during the 2016 epidemic in Rio de Janeiro, Brazil. Genome sequencing revealed unique mutations in the nonstructural protein 4 (NSP4-A481D) and envelope protein 1 (E1-K211T). Moreover, all Brazil East/Central/South isolates shared the exclusive mutations E1-M407L and E2-A103T.

Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus.

UNLABELLED: Smallpox was declared eradicated in 1980 after an intensive vaccination program using different strains of vaccinia virus (VACV; Poxviridae). VACV strain IOC (VACV-IOC) was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication program. However, little is known about the biological and immunological features as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. Both clones had low virulence in infected mice and induced a protective immune response against a lethal infection comparable to the response of the licensed vaccine ACAM2000 and the parental strain VACV-IOC. Full-genome sequencing revealed the presence of several fragmented virulence genes that probably are nonfunctional, e.g., F1L, B13R, C10L, K3L, and C3L. Most notably, phylogenetic inference supported by the structural analysis of the genome ends provides evidence of a novel, independent cluster in VACV phylogeny formed by VACV-IOC, the Brazilian field strains Cantagalo (CTGV) and Serro 2 viruses, and horsepox virus, a VACV-like virus supposedly related to an ancestor of the VACV lineage. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved in parallel with VACV-IOC after splitting from a most recent common ancestor, probably an ancient smallpox vaccine strain related to horsepox virus. Our data, together with an interesting historical investigation, revisit the origins of VACV and propose new evolutionary relationships between ancient and extant VACV strains, mainly horsepox virus, VACV-IOC/CTGV-like viruses, and Dryvax strain. IMPORTANCE: First-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current health care standards. Moreover, these vaccines are genetically heterogeneous and consist of a pool of quasispecies of VACV. Therefore, the search for new-generation smallpox vaccines that combine low pathogenicity, immune protection, and genetic homogeneity is extremely important. In addition, the phylogenetic relationships and origins of VACV strains are quite nebulous. We show the characterization of two clones of VACV-IOC, a unique smallpox vaccine strain that contributed to smallpox eradication in Brazil. The immunogenicity and reduced virulence make the IOC clones good options for alternative second-generation smallpox vaccines. More importantly, this study reveals the phylogenetic relationship between VACV-IOC, feral VACV established in nature, and the ancestor-like horsepox virus. Our data expand the discussion on the origins and evolutionary connections of VACV lineages.

Biological characterization and next-generation genome sequencing of the unclassified Cotia virus SPAn232 (Poxviridae).

Cotia virus (COTV) SPAn232 was isolated in 1961 from sentinel mice at Cotia field station, São Paulo, Brazil. Attempts to classify COTV within a recognized genus of the Poxviridae have generated contradictory findings. Studies by different researchers suggested some similarity to myxoma virus and swinepox virus, whereas another investigation characterized COTV SPAn232 as a vaccinia virus strain. Because of the lack of consensus, we have conducted an independent biological and molecular characterization of COTV. Virus growth curves reached maximum yields at approximately 24 to 48 h and were accompanied by virus DNA replication and a characteristic early/late pattern of viral protein synthesis. Interestingly, COTV did not induce detectable cytopathic effects in BSC-40 cells until 4 days postinfection and generated viral plaques only after 8 days. We determined the complete genomic sequence of COTV by using a combination of the next-generation DNA sequencing technologies 454 and Illumina. A unique contiguous sequence of 185,139 bp containing 185 genes, including the 90 genes conserved in all chordopoxviruses, was obtained. COTV has an interesting panel of open reading frames (ORFs) related to the evasion of host defense, including two novel genes encoding C-C chemokine-like proteins, each present in duplicate copies. Phylogenetic analysis revealed the highest amino acid identity scores with Cervidpoxvirus, Capripoxvirus, Suipoxvirus, Leporipoxvirus, and Yatapoxvirus. However, COTV grouped as an independent branch within this clade, which clearly excluded its classification as an Orthopoxvirus. Therefore, our data suggest that COTV could represent a new poxvirus genus.

Gene duplication, gene loss, and recombination events with variola virus shaped the complex evolutionary path of historical American horsepox-based smallpox vaccines.

IMPORTANCE: Modern smallpox vaccines, such as those used against mpox, are made from vaccinia viruses, but it is still unknown whether cowpox, horsepox, or vaccinia viruses were used in the early 20th century or earlier. The mystery began to be solved when the genomes of six historical smallpox vaccines used in the United States from 1850 to 1902 were determined. Our work analyzed in detail the genomes of these six historical vaccines, revealing a complex genomic structure. Historical vaccines are highly similar to horsepox in the core of their genomes, but some are closer to the structure of vaccinia virus at the ends of the genome. One of the vaccines is a recombinant virus with parts of variola virus recombined into its genome. Our data add valuable information for understanding the evolutionary path of current smallpox vaccines and the genetic makeup of the potentially extinct group of horsepox viruses.

Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.

Emerging Monkeypox virus and neuroinflammatory disorders.

Alarming situation: Monkeypox is a zoonosis caused by a double-stranded DNA virus. The virus was isolated in monkeys in 1958. The first human case was detected in Africa in 1970. It is endemic in western and central Africa. The infection has worried public health authorities around the world since May 2022 with the emergence of thousands of cases in non-African countries. Objective: We discuss the neurological manifestations associated with monkeypox infection. Rare and Severe Complications: Although in the current outbreak, the disease appears to be self-limiting, with predominance of focal skin lesions, complications may occur, mainly in children and immunosuppressed patients. Neurological manifestations such as encephalitis, convulsion, dizziness, pain, fatigue, visual alteration, photophobia, headache and myalgia were previously reported. Encephalitis may be due to viral invasion of the central nervous system or an immune-mediated process. In both situations, rapid recognition is extremely important with the investigation of the cerebrospinal fluid exam, which can demonstrate the local inflammatory reaction, specific IgM and, possibly viral detection, in addition to the imaging study. Conclusions: We emphasize that health professionals should be alert to the emergence of neurological disorders associated with monkeypox infection in order to avoid sequelae and better characterize the current disease.

Genomic Characterization of the Historical Smallpox Vaccine Strain Wyeth Isolated from a 1971 Seed Vial.

The Wyeth strain of vaccinia virus (VACV) produced by Wyeth Pharmaceuticals was supposedly used to manufacture the old freeze-dried American smallpox vaccine, Dryvax, until its discontinuation in 2008. Although the genomic sequences of numerous Dryvax clones have been reported, data on VACV-Wyeth genomes are still lacking. Genomic analysis of old VACV strains is relevant to understand the evolutionary relationships of smallpox vaccines, particularly with the recent resumption of smallpox vaccination in certain population groups as an attempt to control the worldwide monkeypox outbreak. Here we analyzed the complete genome sequences of three VACV-Wyeth clonal isolates obtained from a single seed vial donated to the Brazilian eradication program in the 1970s. Wyeth clones show >99.3% similarity to each other and >95.3% similarity with Dryvax clones, mapping together in clade I of the vaccinia group. Although the patterns of SNPs and INDELs comparing Dryvax and Wyeth clones are overall uniform, important differences were detected particularly at the ends of the genome. In addition, we detected recombinant events of clone Wyeth A111 and the Dryvax clone Acam2000, suggesting that other regions of the genomes may have similar patchy patterns of recombination. A small-scale serological survey using VACV-Wyeth as antigen in ELISA assays revealed that 63 of the 65 individuals born before the end of smallpox vaccination in Brazil still have anti-VACV IgG antibodies, demonstrating the usefulness of the VACV-Wyeth strain in future extended serological studies of the Brazilian population.

Animal movement and establishment of vaccinia virus Cantagalo strain in Amazon biome, Brazil.

To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008-2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondonia State, leading to the establishment and spread of this pathogen.

clpB, a class III heat-shock gene regulated by CtsR, is involved in thermotolerance and virulence of Enterococcus faecalis.

Here, we transcriptionally and phenotypically characterized the clpB gene from Enterococcus faecalis. Northern blot analysis identified a monocistronic mRNA strongly induced at 48 and 50 °C. In silico analysis identified that the clpB gene encodes a protein of 868 aa with a predicted molecular mass of approximately 98 kDa, presenting two conserved ATP-binding domains. Sequence analysis also identified a CtsR-binding box upstream of the putative -10 sequence, and inactivation of the ctsR gene resulted in an approximately 2-log increase in clpB mRNA expression, confirming ClpB as a member of the CtsR regulon. While expression of clpB was induced by heat stress, a ΔclpB strain grew relatively well under many different stressful conditions, including elevated temperatures. However, expression of ClpB appears to play a major role in induced thermotolerance and in pathogenesis, as assessed by using the Galleria mellonella virulence model.

Cidofovir inhibits genome encapsidation and affects morphogenesis during the replication of vaccinia virus.

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Early smallpox vaccine manufacturing in the United States: Introduction of the "animal vaccine" in 1870, establishment of "vaccine farms", and the beginnings of the vaccine industry.

For the first 80-90 years after Jenner's discovery of vaccination in 1796, the main strategy used to disseminate and maintain the smallpox vaccine was arm-to-arm vaccination, also known as Jennerian or humanized vaccination. A major advance occurred after 1860 with the development of what was known as "animal vaccine", which referred to growing vaccine material from serial propagation in calves before use in humans. The use of "animal vaccine" had several advantages over arm-to-arm vaccination: it would not transmit syphilis or other human diseases, it ensured a supply of vaccine even in the absence of the spontaneous occurrence of cases of cowpox or horsepox, and it allowed the production of large amounts of vaccine. The "animal vaccine" concept was introduced in the United States in 1870 by Henry Austin Martin. Very rapidly a number of "vaccine farms" were established in the U.S. and produced large quantities of "animal vaccine". These "vaccine farms" were mostly established by medical doctors who saw an opportunity to respond to an increasing demand of smallpox vaccine from individuals and from health authorities, and to make a profit. The "vaccine farms" evolved from producing only smallpox "animal vaccine" to manufacturing several other biologics, including diphtheria- and other antitoxins. Two major incidents of tetanus contamination happened in 1901, which led to the promulgation of the Biologics Control Act of 1902. The US Secretary of the Treasury issued licenses to produce and sell biologicals, mainly vaccines and antitoxins. Through several mergers and acquisitions, the initial biologics licensees eventually evolved into some of the current major American industrial vaccine companies. An important aspect that was never clarified was the source of the vaccine stocks used to manufacture the smallpox "animal vaccines". Most likely, different smallpox vaccine stocks were repeatedly introduced from Europe, resulting in polyclonal vaccines that are now recognized as "variants" more appropriately than "strains". Further, clonal analysis of modern "animal vaccines" indicate that they are probably derived from complex recombinational events between different strains of vaccinia and horsepox. Modern sequencing technologies are now been used by us to study old smallpox vaccine specimens in an effort to better understand the origin and evolution of the vaccines that were used to eradicate the smallpox.

Re-assembly of nineteenth-century smallpox vaccine genomes reveals the contemporaneous use of horsepox and horsepox-related viruses in the USA.

According to a recent article published in Genome Biology, Duggan and coworkers sequenced and partially assembled five genomes of smallpox vaccines from the nineteenth century. No information regarding the ends of genomes was presented, and they are important to understand the evolutionary relationship of the different smallpox vaccine genomes during the centuries. We re-assembled the genomes, which include the largest genomes in the vaccinia lineage and one true horsepox strain. Moreover, the assemblies reveal a diverse genetic structure in the genome ends. Our data emphasize the concurrent use of horsepox and horsepox-related viruses as the smallpox vaccine in the nineteenth century.