Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

YTHDF2 Is Downregulated in Response to Host Shutoff Induced by DNA Virus Infection and Regulates Interferon-Stimulated Gene Expression.

Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

Varíola dos macacos: o que é a doença, seus sintomas e por que ela afeta humanos

Surgimento de casos em vários países da Europa e nos Estados Unidos deixam autoridades de saúde em alerta Publicado em: 20/05/2022

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.

Protocol for analyzing and visualizing antiviral immune responses after acute infection of the murine oral mucosa.

The oral mucosa is an important site for virus infection and transmission, yet few animal models exist to examine the virology, pathology, and immunology of acute oral mucosal viral infection. Here, we provide a protocol for infecting and imaging the inner lip (labial mucosa) of mice with the poxvirus vaccinia virus (VACV). Inoculation of the labial mucosa with a bifurcated needle results in viral replication and priming of an adaptive antiviral response that can be imaged using intravital microscopy. For complete details on the use and execution of this protocol, please refer to Shannon et al. (2021).

Swinepox Virus Strains Isolated from Domestic Pigs and Wild Boar in Germany Display Altered Coding Capacity in the Terminal Genome Region Encoding for Species-Specific Genes.

Swinepox virus (SWPV) is a globally distributed swine pathogen that causes sporadic cases of an acute poxvirus infection in domesticated pigs, characterized by the development of a pathognomonic proliferative dermatitis and secondary ulcerations. More severe disease with higher levels of morbidity and mortality is observed in congenitally SWPV-infected neonatal piglets. In this study, we investigated the evolutionary origins of SWPV strains isolated from domestic pigs and wild boar. Analysis of whole genome sequences of SWPV showed that at least two different virus strains are currently circulating in Germany. These were more closely related to a previously characterized North American SWPV strain than to a more recent Indian SWPV strain and showed a variation in the SWPV-specific genome region. A single nucleotide deletion in the wild boar (wb) SWPV strain leads to the fusion of the SPV019 and SPV020 open reading frames (ORFs) and encodes a new hypothetical 113 aa protein (SPVwb020-019). In addition, the domestic pig (dp) SWPV genome contained a novel ORF downstream of SPVdp020, which encodes a new hypothetical 71aa protein (SPVdp020a). In summary, we show that SWPV strains with altered coding capacity in the SWPV specific genome region are circulating in domestic pig and wild boar populations in Germany.

Sero-Epidemiological Survey of Orthopoxvirus in Stray Cats and in Different Domestic, Wild and Exotic Animal Species of Central Italy.

Orthpoxvirus infection can spread more easily in a population with a waning immunity with the subsequent emergence/re-emergence of the viruses pertaining to this genus. In the last two decades, several cases of Orthopoxvirus, and in particular Cowpoxvirus infections in humans were reported in different parts of the world, possibly due to the suspension of smallpox vaccinations. To date, in Italy, few investigations were conducted on the presence of these infections, and because of this a serosurvey was carried out to evaluate Cowpoxvirus infection in feline colonies situated in the province of Rome, since these are also susceptible to other zoonotic viruses belonging to Orthopoxvirus, and from which humans may contract the infection. The sample design was set at an expected minimum seroprevalence of 7.5%, a 5% standard error and 95% confidence level. In parallel, a serological investigation was conducted using convenience sampling in domestic, exotic and wild susceptible animals of the Latium and Tuscany Regions, which are areas in the jurisdiction of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, coordinating this study. The serological methods employed were indirect immunofluorescence for 36 sera of nonhuman primate and virus neutralization for 1198 sera of different species. All the 1234 sera examined were negative for the presence of antibodies against Cowpoxvirus, indicating its limited circulation in the areas of investigation. The methodology applied for the serosurveillance could be adopted in the case of outbreaks of this infection and for the evaluation of the spread of this infection in the area of interest, to obtain essential information crucial for animal and public health policies according to the One Health concept.

Genetic diversity of the carp oedema virus in France.

The koi sleepy disease of carp caused by the carp oedema virus (CEV) was observed on farms and in ponds in France since the 2010s. Samples of CEV collected in France over a period of eight years were characterized at the molecular level by sequencing the partial p4a gene. All the sequences, except one, fell into two well-defined genogroups. Sequences obtained from CEV detected in common carp generally clustered in genogroup I and sequences from CEV detected in the koi were assigned to genogroup II. A particular sample was different to the others and represented a putative new genogroup possibly arose from a recombination event between a genogroup II sequence and one from an unknown genogroup. Compared with sequences from CEV of other countries, most of the French sequences exhibited high degree of DNA identities with those published previously, indicating identical sources of viruses. The sequence diversity suggests multiple introductions of the viruses in France. Among the French sequences, two genogroup-specific molecular markers were identified. One was an insertion/deletion identified within a microsatellite and other was a group of single nucleotide polymorphisms. CEV seems to generate genetic diversity via diverse mechanisms: substitutions, indels and recombination events.

Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV) Infection in Juvenile Egyptian Fruit Bat (Rousettus aegyptiacus): Clinical Findings and Molecular Detection.

During 2019, five carcasses of juvenile Egyptian fruit bats (Rousettus aegyptiacus) were submitted to the Kimron Veterinary Institute. These bats exhibited typical poxvirus like lesion plaques of different sizes on the skin, abdomen and the ventral side of the wings. Clinical and histopathological findings suggested a poxvirus infection. Infectious virus was isolated from skin swabs, skin tissue and tongue of the dead bats and was further confirmed to be a Poxvirus by molecular diagnosis using PCR with pan-chordopoxviruses primers. All the dead bats were found positive for two Poxvirus genes encoding a metalloproteinase and DNA dependent DNA polymerase. In this study, a novel real time quantitative PCR (qPCR) assay was established to further confirmed the presence of specific poxvirus viral DNA in all pathologically tested tissues. Moreover, according to sequence analysis, the virus was found to be highly similar to the recently discovered Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV).

Evidence of Transmission of Goatpox between Domestic Goats and Wild Himalayan Goral (Naemorhedus goral) in Arunachal Pradesh, India.

Examination of carcasses of Himalayan goral (Naemorhedus goral) revealed nodular, pox-like eruptions in the skin. Similar disease was also seen in domestic goats (Capra aegagrus hircus) in the same area. Goatpox virus was identified as the etiology of the disease in both cases, with probable transmission between the species.

Geographic distribution and genetic characterization of poxviruses from human infections in Georgia, 2009-2014.

Anthrax is endemic in Georgia, as are multiple zoonotic poxviruses. Poxvirus-associated infections share some clinical manifestations and exposure risks with anthrax, and so it is important to distinguish between the two. With this in mind, an archived collection of anthrax-negative DNA samples was retrospectively screened for poxviruses, and of the 148 human samples tested, 64 were positive. Sequence analysis confirmed the presence of orf virus, bovine papular stomatitis virus, and pseudocowpox virus. This study provides evidence of previously unrecognized poxvirus infections in Georgia and highlights the benefit of the timely identification of such infections by improving laboratory capacity.

Experimental infection of indigenous North African goats with goatpox virus.

BACKGROUND: Goatpox is a viral disease caused by infection with goatpox virus (GTPV) of the genus Capripoxvirus, Poxviridae family. Capripoxviruses cause serious disease to livestock and contribute to huge economic losses. Goatpox and sheeppox are endemic to Africa, particularly north of the Equator, the Middle East and many parts of Asia. GTPV and sheeppox virus are considered host-specific; however, both strains can cause clinical disease in either goats or sheep with more severe disease in the homologous species and mild or sub-clinical infection in the other. Goatpox has never been reported in Morocco, Algeria or Tunisia despite the huge population of goats living in proximity with sheep in those countries. To evaluate the susceptibility and pathogenicity of indigenous North African goats to GTPV infection, we experimentally inoculated eight locally bred goats with a virulent Vietnamese isolate of GTPV. Two uninfected goats were kept as controls. Clinical examination was carried out daily and blood was sampled for virology and for investigating the antibody response. After necropsy, tissues were collected and assessed for viral DNA using real-time PCR. RESULTS: Following the experimental infection, all inoculated goats displayed clinical signs characteristic of goatpox including varying degrees of hyperthermia, loss of appetite, inactivity and cutaneous lesions. The infection severely affected three of the infected animals while moderate to mild disease was noticed in the remaining goats. A high antibody response was developed. High viral DNA loads were detected in skin crusts and nodules, and subcutaneous tissue at the injection site with cycle threshold (Ct) values ranging from 14.6 to 22.9, while lower viral loads were found in liver and lung (Ct = 35.7 and 35.1). The results confirmed subcutaneous tropism of the virus. CONCLUSION: Clinical signs of goatpox were reproduced in indigenous North African goats and confirmed a high susceptibility of the North African goat breed to GTPV infection. A clinical scoring system is proposed that can be applied in GTPV vaccine efficacy studies.

Molecular phylogenetics of a recently isolated goat pox virus from Vietnam.

BACKGROUND: After a decade of silence, an outbreak of the contagious and Asian endemic disease, goat pox re-emerged in North Vietnam affecting more than 1800 heads with a mortality rate of 6.5%. The inevitable impact of goat pox on hide quality, breeding, chevon and milk production has resulted in a significant economic losses to the developing goat industry of Vietnam. In the act of establishing an effective control of this devastating disease, tracing the source of re-emergence via a phylogenetic study was carried out to reveal their genetic relatedness. Either skin scab or papule from the six affected provinces were collected, cultured into Vero cells followed by restricted enzyme digestion of targeted P32 gene DNA encoding. The P32 gene was then cloned and transformed into E.coli competent cells for further sequencing. RESULTS: The isolated sequence is deposited into GenBank under Accession No. MN317561/VNUAGTP1. The phylogenetic tree revealed high similarity of nucleotide and amino acid sequences to references goat pox strains accounting for 99.6 and 99.3, respectively. The Vietnamese strain is clustered together with currently circulating goat pox virus in China, India and Pakistan which suggested the origin of South China. CONCLUSIONS: This Vietnam isolate is clustered together with other Asian goat pox strains indicating the dissemination of a common goat pox virus within this continent.

(+)-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.

Emergence of a Novel Pathogenic Poxvirus Infection in the Endangered Green Sea Turtle (Chelonia mydas) Highlights a Key Threatening Process.

Emerging viral disease is a significant concern, with potential consequences for human, animal and environmental health. Over the past several decades, multiple novel viruses have been found in wildlife species, including reptiles, and often pose a major threat to vulnerable species. However, whilst a large number of viruses have been described in turtles, information on poxvirus in cheloniids remains scarce, with no molecular sequence data available to date. This study characterizes, for the first time, a novel poxvirus, here tentatively designated cheloniid poxvirus 1 (ChePV-1). The affected cutaneous tissue, recovered from a green sea turtle (Chelonia mydas) captured off the Central Queensland coast of Australia, underwent histological examination, transmission electron microscopy (TEM), DNA extraction and genomic sequencing. The novel ChePV-1 was shown to be significantly divergent from other known poxviruses and showed the highest sequence similarity (89.3%) to avipoxviruses (shearwater poxvirus 2 (SWPV2)). This suggests the novel ChePV-1 may have originated from a common ancestor that diverged from an avipoxvirus-like progenitor. The genome contained three predicted unique genes and a further 15 genes being truncated/fragmented compared to SWPV2. This is the first comprehensive study that demonstrates evidence of poxvirus infection in a marine turtle species, as well as a rare example of an avipoxvirus crossing the avian-host barrier. This finding warrants further investigations into poxvirus infections between species in close physical proximity, as well as in vitro and in vivo studies of pathogenesis and disease.

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family Teiidae).

A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

Carp edema virus-related mortality in wild adult common carp (cyprinus carpio) in Italy.

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Yellow-Eyed Penguin (Megadyptes antipodes).

Emerging viral diseases have become a significant concern due to their potential consequences for animal and environmental health. Over the past few decades, it has become clear that viruses emerging in wildlife may pose a major threat to vulnerable or endangered species. Diphtheritic stomatitis, likely to be caused by an avipoxvirus, has been recognised as a significant cause of mortality for the endangered yellow-eyed penguin (Megadyptes antipodes) in New Zealand. However, the avipoxvirus that infects yellow-eyed penguins has remained uncharacterised. Here, we report the complete genome of a novel avipoxvirus, penguinpox virus 2 (PEPV2), which was derived from a virus isolate obtained from a skin lesion of a yellow-eyed penguin. The PEPV2 genome is 349.8 kbp in length and contains 327 predicted genes; five of these genes were found to be unique, while a further two genes were absent compared to shearwaterpox virus 2 (SWPV2). In comparison with penguinpox virus (PEPV) isolated from an African penguin, there was a lack of conservation within the central region of the genome. Subsequent phylogenetic analyses of the PEPV2 genome positioned it within a distinct subclade comprising the recently isolated avipoxvirus genome sequences from shearwater, canary, and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV2 (96.27%). This is the first reported genome sequence of PEPV2 from a yellow-eyed penguin and will help to track the evolution of avipoxvirus infections in this rare and endangered species.

First complete genome characterization of swinepox virus directly from a clinical sample indicates divergence of a Eurasian-lineage virus.

In this study, we report the complete genome sequence of swinepox virus from a clinical sample from a naturally occurring infection in India. The sequencing was done on a Nanopore MinION sequencer from Oxford Nanopore Technologies. Two new annotations were added to the genome. Three of the genes were found to have frameshifts, which might be of importance in relation to infection. When compared to the only other reported whole genome sequence of swinepox virus, which was obtained from an isolate from America in 1999, our sequence is only 98.19% identical at the nucleotide level. The average amino acid sequence identity of the viral proteins, based on the common 149 annotations, is also 98.19%, demonstrating that these viruses are distinctly divergent. Owing to the fact that swinepox virus infects only swine, it could not have entered America until the introduction of swine in the 16th century from Europe. The swinepox viruses in both continents have continued to evolve independently. The sequence divergence identified here indicates a Eurasian-lineage virus that is geographically distinct from the American-lineage swinepox virus.

Carp edema virus and immune response in carp (Cyprinus carpio): Current knowledge.

The importance of world aquaculture production grows annually together with the increasing need to feed the global human population. Common carp (Cyprinus carpio) is one of the most important freshwater fish in global aquaculture. Unfortunately, carp production is affected by numerous diseases of which viral diseases are the most serious. Koi herpesvirus disease (KHVD), spring viraemia of carp (SVC), and during the last decades also koi sleepy disease (KSD) are currently the most harmful viral diseases of common carp. This review summarizes current knowledge about carp edema virus (CEV), aetiological agent causing KSD, and about the disease itself. Furthermore, the article is focused on summarizing the available information about the antiviral immune response of common carp, like production of class I interferons (IFNs), activation of cytotoxic cells, and production of antibodies by B cells focusing on anti-CEV immunity.