Exploring Viral Genome Profile in Mpox Patients during the 2022 Outbreak, in a North-Eastern Centre of Italy.

In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.

Molecular Virology of Orthopoxviruses with Special Reference to Monkeypox Virus.

Poxviruses are large (200-450 nm) and enveloped viruses carrying double-stranded DNA genome with an epidermal cell-specific adaptation. The genus Orthopoxvirus within Poxviridae family constitutes several medically and veterinary important viruses including variola (smallpox), vaccinia, monkeypox virus (MPXV), and cowpox. The monkeypox disease (mpox) has recently emerged as a public health emergency caused by MPXV. An increasing number of human cases of MPXV have been documented in non-endemic nations without any known history of contact with animals brought in from endemic and enzootic regions, nor have they involved travel to an area where the virus was typically prevalent. Here, we review the MPXV replication, virus pathobiology, mechanism of viral infection transmission, virus evasion the host innate immunity and antiviral therapies against Mpox. Moreover, preventive measures including vaccination were discussed and concluded that cross-protection against MPXV may be possible using antibodies that are directed against an Orthopoxvirus. Despite the lack of a specialised antiviral medication, several compounds such as Cidofovir and Ribavirin warrant consideration against mpox.

An Update of Orthopoxvirus Molecular Evolution.

Although variola virus (VARV) has been eradicated through widespread vaccination, other orthopoxviruses pathogenic for humans circulate in nature. Recently, new orthopoxviruses, including some able to infect humans, have been found and their complete genomes have been sequenced. Questions about the orthopoxvirus mutation rate and the emergence of new threats to humankind as a result of the evolution of circulating orthopoxviruses remain open. Based on contemporary data on ancient VARV DNA and DNA of new orthopoxvirus species, an analysis of the molecular evolution of orthopoxviruses was carried out and the timescale of their emergence was estimated. It was calculated that the orthopoxviruses of the Old and New Worlds separated approximately 40,000 years ago; the recently discovered Akhmeta virus and Alaskapox virus separated from other orthopoxviruses approximately 10,000-20,000 years ago; the rest of modern orthopoxvirus species originated from 1700 to 6000 years ago, with the exception of VARV, which emerged in approximately 300 AD. Later, there was a separation of genetic variants of some orthopoxvirus species, so the monkeypox virus West African subtype originated approximately 600 years ago, and the VARV minor alastrim subtype emerged approximately 300 years ago.

Outbreak of a Systemic Form of Camelpox in a Dromedary Herd (Camelus dromedarius) in the United Arab Emirates.

Camelpox virus (CMLV) is the causative agent of camelpox, which frequently occurs in the Old World camelids-rearing countries except for Australia. It has also been described in experimentally inoculated New World camelids. Camelpox outbreaks are often experienced shortly after the rainy season, which occurs twice a year on the Arabian Peninsula because of the increased density of the insect population, particularly mosquitos. A systemic form of camelpox outbreak in seven dromedary camels was diagnosed by histology, virus isolation, and PCR. A phylogenetic analysis using full length CMLV genomes of the isolated CMLV strains showed a single phylogenetic unit without any distinctive differences between them. The United Arab Emirates (UAE) isolate sequences showed phylogenetical relatedness with CMLV isolates from Israel with only minor sequence differences. Although the sequences of viruses from both countries were closely related, the disease manifestation was vastly different. Our study shows that the virulence is not only determined by genetic features of CMLV alone but may also depend on other factors such as unknown aspects of the host (e.g., age, overall fitness), management, and the environment.

New methylene blue derivatives suggest novel anti-orthopoxviral strategies.

Decades after the eradication of smallpox and the discontinuation of routine smallpox vaccination, over half of the world's population is immunologically naïve to variola virus and other orthopoxviruses (OPXVs). Even in those previously vaccinated against smallpox, protective immunity wanes over time. As such, there is a concomitant increase in the incidence of human OPXV infections worldwide. To identify novel antiviral compounds with potent anti-OPXV potential, we characterized the inhibitory activity of PAV-866 and other methylene blue derivatives against the prototypic poxvirus, vaccinia virus (VACV). These compounds inactivated virions prior to infection and consequently inhibited viral binding, fusion and entry. The compounds exhibited strong virucidal activity at non-cytotoxic concentrations, and inhibited VACV infection when added before, during or after viral adsorption. The compounds were effective against other OPXVs including monkeypox virus, cowpox virus and the newly identified Akhmeta virus. Altogether, these findings reveal a novel mode of inhibition that has not previously been demonstrated for small molecule compounds against VACV. Additional studies are in progress to determine the in vivo efficacy of these compounds against OPXVs and further characterize the anti-viral effects of these derivatives.

(+)-Camphor and (-)-borneol derivatives as potential anti-orthopoxvirus agents.

Although the World Health Organisation had announced that smallpox was eradicated over 40 years ago, the disease and other related pathogenic poxviruses such as monkeypox remain potential bioterrorist weapons and could also re-emerge as natural infections. We have previously reported (+)-camphor and (-)-borneol derivatives with an antiviral activity against the vaccinia virus. This virus is similar to the variola virus (VARV), the causative agent of smallpox, but can be studied at BSL-2 facilities. In the present study, we evaluated the antiviral activity of the most potent compounds against VARV, cowpox virus, and ectromelia virus (ECTV). Among the compounds tested, 4-bromo-N'-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide 18 is the most effective compound against various orthopoxviruses, including VARV, with an EC50 value of 13.9 µM and a selectivity index of 206. Also, (+)-camphor thiosemicarbazone 9 was found to be active against VARV and ECTV.

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR.

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.

Rapid protocol of dot-immunnoassay for orthopoxviruses detection.

The aim of the work was to create a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the "point of care" format. This work presents the results of the comparative evaluation of a single-stage (rapid version) and two-stage protocol of dot-immunoassay based on plane protein array for detection of vaccinia virus (VACV), cowpoxvirus (CPXV) and ectromelia virus (ECTV) in viral culture materials with different degrees of purification. It has been established that rabbit polyclonal VACV-antibodies can be used in a one-stage dot-analysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the sensitivity of detection of OPXV is inversely related to the degree of purification of viruses. The one-stage variant of the dot-immunoassay allows reducing the analysis time to 39 min and increasing the detection sensitivity of all the studied orthopoxviruses in crude viral samples to a range of 104-103 PFU/mL. The increase in sensitivity in the rapid version of the analysis, presumably, occurs due to binding of capture antibodies to subviral structures that form large aggregates of gold particles. Ultrasonic treatment of culture virus reduces the detection sensitivity, presumably due to both the destruction of conformational epitopes located on the surface of subvirus structures, as well as the increase in the dispersion of cell debris, which limits diffusion and contacts of viral antigens with capture antibodies on the substrate. Both versions of the analysis are specific and do not detect interactions both with preparations of non-infected cell culture and with heterogeneous controls of the causative agents of erythematous infections. The rapid protocol of dot-immunnoassay described above can be used to detect, or help to exclude, the presence of threat viruses in samples and could be useful in a variety of biodefense applications. Ready-to-use setup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.

Here, There, and Everywhere: The Wide Host Range and Geographic Distribution of Zoonotic Orthopoxviruses.

The global emergence of zoonotic viruses, including poxviruses, poses one of the greatest threats to human and animal health. Forty years after the eradication of smallpox, emerging zoonotic orthopoxviruses, such as monkeypox, cowpox, and vaccinia viruses continue to infect humans as well as wild and domestic animals. Currently, the geographical distribution of poxviruses in a broad range of hosts worldwide raises concerns regarding the possibility of outbreaks or viral dissemination to new geographical regions. Here, we review the global host ranges and current epidemiological understanding of zoonotic orthopoxviruses while focusing on orthopoxviruses with epidemic potential, including monkeypox, cowpox, and vaccinia viruses.

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.

Genome of Alaskapox Virus, A Novel Orthopoxvirus Isolated from Alaska.

Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.

Whole Genome Characterization of Orthopoxvirus (OPV) Abatino, a Zoonotic Virus Representing a Putative Novel Clade of Old World Orthopoxviruses.

Orthopoxviruses (OPVs) are diffused over the complete Eurasian continent, but previously described strains are mostly from northern Europe, and few infections have been reported from Italy. Here we present the extended genomic characterization of OPV Abatino, a novel OPV isolated in Italy from an infected Tonkean macaque, with zoonotic potential. Phylogenetic analysis based on 102 conserved OPV genes (core gene set) showed that OPV Abatino is most closely related to the Ectromelia virus species (ECTV), although placed on a separate branch of the phylogenetic tree, bringing substantial support to the hypothesis that this strain may be part of a novel OPV clade. Extending the analysis to the entire set of genes (coding sequences, CDS) further substantiated this hypothesis. In fact the genome of OPV Abatino included more CDS than ECTV; most of the extra genes (mainly located in the terminal genome regions), showed the highest similarity with cowpox virus (CPXV); however vaccinia virus (VACV) and monkeypox virus (MPXV) were the closest OPV for certain CDS. These findings suggest that OPV Abatino could be the result of complex evolutionary events, diverging from any other previously described OPV, and may indicate that previously reported cases in Italy could represent the tip of the iceberg yet to be explored.

The Virology of Taterapox Virus In Vitro.

Taterapox virus (TATV) is phylogenetically the closest related virus to variola-the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

Genome Sequences of Akhmeta Virus, an Early Divergent Old World Orthopoxvirus.

Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure to that of the cowpox virus (CPXV), but a lower sequence identity was found between AKMV and Old World OPXVs than between other known species of Old World OPXVs. Phylogenetic analysis showed that AKMV diverged prior to other Old World OPXV. AKMV isolates formed a monophyletic clade in the OPXV phylogeny, yet the sequence variability between AKMV isolates was higher than between the monkeypox virus strains in the Congo basin and West Africa. An AKMV isolate from Vani contained approximately six kb sequence in the left terminal region that shared a higher similarity with CPXV than with other AKMV isolates, whereas the rest of the genome was most similar to AKMV, suggesting recombination between AKMV and CPXV in a region containing several host range and virulence genes.

Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?

Diagnostic electron microscopy (DEM) was an essential component of viral diagnosis until the development of highly sensitive nucleic acid amplification techniques (NAT). The simple negative staining technique of DEM was applied widely to smallpox diagnosis until the world-wide eradication of the human-specific pathogen in 1980. Since then, the threat of smallpox re-emerging through laboratory escape, molecular manipulation, synthetic biology or bioterrorism has not totally disappeared and would be a major problem in an unvaccinated population. Other animal poxviruses may also emerge as human pathogens. With its rapid results (only a few minutes after arrival of the specimen), no requirement for specific reagents and its "open view", DEM remains an important component of virus diagnosis, particularly because it can easily and reliably distinguish smallpox virus or any other member of the orthopoxvirus (OPV) genus from parapoxviruses (PPV) and the far more common and less serious herpesviruses (herpes simplex and varicella zoster). Preparation, enrichment, examination, internal standards and suitable organisations are discussed to make clear its continuing value as a diagnostic technique.

First Diagnosed Case of Camelpox Virus in Israel.

An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype.

Understanding orthopoxvirus host range and evolution: from the enigmatic to the usual suspects.

In general, orthopoxviruses can be considered as falling into one of three host-utilization categories: highly specialized, single-host; broad host range; or 'cryptic', the last encompassing those viruses about which very little is known. Single-host viruses tend to exploit abundant hosts that have consistent patterns of interaction. For these viruses, observed genome reduction and loss of presumptive host-range genes is thought to be a consequence of relaxed selection. In contrast, the large genome size retained among broad host range orthopoxviruses suggests these viruses may depend on multiple host species for persistence in nature. Our understanding of the ecologic requirements of orthopoxviruses is strongly influenced by geographic biases in data collection. This hinders our ability to predict potential sources for emergence of orthopoxvirus-associated infections.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1.

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Novel Orthopoxvirus Infection in an Alaska Resident.

BACKGROUND.: Human infection by orthopoxviruses is being reported with increasing frequency, attributed in part to the cessation of smallpox vaccination and concomitant waning of population-level immunity. In July 2015, a female resident of interior Alaska presented to an urgent care clinic with a dermal lesion consistent with poxvirus infection. Laboratory testing of a virus isolated from the lesion confirmed infection by an Orthopoxvirus. METHODS.: The virus isolate was characterized by using electron microscopy and nucleic acid sequencing. An epidemiologic investigation that included patient interviews, contact tracing, and serum testing, as well as environmental and small-mammal sampling, was conducted to identify the infection source and possible additional cases. RESULTS.: Neither signs of active infection nor evidence of recent prior infection were observed in any of the 4 patient contacts identified. The patient's infection source was not definitively identified. Potential routes of exposure included imported fomites from Azerbaijan via the patient's cohabiting partner or wild small mammals in or around the patient's residence. Phylogenetic analyses demonstrated that the virus represents a distinct and previously undescribed genetic lineage of Orthopoxvirus, which is most closely related to the Old World orthopoxviruses. CONCLUSIONS.: Investigation findings point to infection of the patient after exposure in or near Fairbanks. This conclusion raises questions about the geographic origins (Old World vs North American) of the genus Orthopoxvirus. Clinicians should remain vigilant for signs of poxvirus infection and alert public health officials when cases are suspected.

The French Armed Forces Virology Unit: A Chronological Record of Ongoing Research on Orthopoxvirus.

Since the official declaration of smallpox eradication in 1980, the general population vaccination has ceased worldwide. Therefore, people under 40 year old are generally not vaccinated against smallpox and have no cross protection against orthopoxvirus infections. This naïve population may be exposed to natural or intentional orthopoxvirus emergences. The virology unit of the Institut de Recherche Biomédicale des Armées (France) has developed research programs on orthopoxviruses since 2000. Its missions were conceived to improve the diagnosis capabilities, to foster vaccine development, and to develop antivirals targeting specific viral proteins. The role of the virology unit was asserted in 2012 when the responsibility of the National Reference Center for the Orthopoxviruses was given to the unit. This article presents the evolution of the unit activity since 2000, and the past and current research focusing on orthopoxviruses.