Lessons Learned from Active Clinical and Laboratory Surveillance during the Sheep Pox Virus Outbreak in Spain, 2022-2023.

In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.

Poxviruses in Children.

The family Poxviridae is a large family of viruses with a ubiquitous distribution, subdivided into two subfamilies: Chordopoxvirinae (poxviruses of vertebrates) and Entomopoxvirinae (poxviruses of insects). Only three species from the first subfamily, Orthopoxvirus (OPV), Molluscipoxvirus and Parapoxvirus, can infect the human being. In the paediatric population, viruses belonging to the first two subfamilies have the greatest importance. Following the eradication of smallpox in 1980, vaccination of the general population was discontinued after careful consideration of the risks and benefits. However, nearly all children and most of the world's population had little to no protection against OPV. The aim of this chapter is to review the current evidence on the aetiology, clinical manifestations, diagnosis and management of Poxviridae infections in children.

Role of the Laboratory in the Diagnosis of Poxvirus Infections.

Although WHO-led global efforts led to eradication of smallpox over four decades ago, other poxviruses, especially monkeypox, have re-emerged to occupy the ecological niche vacated by smallpox. Many of these viruses produce similar lesions thus mandating a prompt laboratory confirmation. There has been considerable evolution in the techniques available to diagnose these infections and differentiate between them. With the 2022 multi-country outbreak of monkeypox, significant efforts were made to apprise the laboratory diagnosis of the virus and numerous real-time-PCR-based assays were made commercially available. This chapter discusses the sample collection and biosafety aspects along with the repertoire of diagnostic modalities, both traditional and emerging, for poxviruses which a special focus on monkeypox. The advantages and disadvantages of each technique have been illustrated. We have also reflected upon the newer advances and the existing lacunae.

Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections.

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

Poxvirus infections in dermatology - the neglected, the notable, and the notorious.

The family Poxviridae currently comprises 22 genera that infect vertebrates. Of these, members of the Ortho-, Para-, Mollusci- and Yatapoxvirus genera have been associated with human diseases of high clinical relevance in dermatology. Historically, smallpox had been a notorious health threat until it was declared eradicated by the World Health Organization in 1979. Today, dermatologists are confronted with a variety of poxviral infections, such as farmyard pox, which occurs as a zoonotic infection after contact with animals. In the tropics, tanapox or vaccinia may be in the differential diagnosis as neglected tropical dermatoses. Molluscum contagiosum virus infection accounts for significant disease burden worldwide and is classified as a sexually transmitted infection in certain scenarios. Recently, mpox (monkeypox) has emerged as a public health emergency of international concern, requiring rapid recognition and appropriate management by dermatologists and infectious disease specialists. Advances and new insights into the epidemiology, diagnosis, clinical manifestations and complications, treatment, and prevention of poxviral infections require a high level of expertise and interdisciplinary skills from healthcare professionals linking virology, infectious diseases, and dermatology. This CME article provides a systematic overview and update to assist the practicing dermatologist in the identification, differential diagnosis, and management of poxviral infections.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Development and application of a real-time polymerase chain reaction assay to detect lumpy skin disease virus belonging to the Kenyan sheep and goat pox group.

Lumpy skin disease (LSD) outbreaks in Southeast and South Asia are attributed to different lineages of LSD virus (LSDV). Variants belonging to the novel recombinant cluster 2.5 circulate in China and Thailand, while a Kenyan sheep and goat pox (KSGP) strain from cluster 1.1 circulates in India, Pakistan, and Bangladesh. The clusters representing these circulating strains are vastly different. However, if their distribution encroaches into each other's ranges, it will be impossible to differentiate between them due to the lack of suitable molecular tools. Thus, fit-for-purpose molecular tools are in demand to effectively and timeously diagnose and investigate the epidemiology of LSDVs in a region. These could significantly contribute to the phylogenetic delineation of LSDVs and the development of preventive measures against transboundary spillovers. This work aimed to develop a real-time polymerase chain reaction assay targeting open reading frame LW032, capable of specifically detecting KSGP-related isolates and recombinant LSDV strains containing the KSGP backbone. The analytical specificity was proven against the widest possible panel of recombinant vaccine-like LSDV strains known to date. The amplification efficiency was 91.08%, and the assay repeatability had a cycle threshold variation of 0.56-1.1 over five repetitions across three runs. This KSGP-specific assay is reliable and fast and is recommended for use in LSDV epidemiological studies where the accurate detection of KSGP genetic signatures is a priority, particularly in regions where KSGP-like and other lineages are circulating.

Poxvirus Infections in Dairy Farms and Transhumance Cattle Herds in Nigeria.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria.

Multiple accurate and sensitive arrays for Capripoxvirus (CaPV) differentiation.

Capripoxvirus (CaPV) contains three viruses that have caused massive losses in the livestock and dairy industries. Accurate CaPV differentiation has far-reaching implications for effectively controlling outbreaks. However, it has a great challenge to distinguishing three viruses due to high homology of 97%. Here, we established a sensitive CRISPR/Cas12a array based on Multiple-recombinase polymerase amplification (M-RPA) for CaPV differentiation, which provided a more comprehensive and accurate differentiation mode targeting VARV B22R and RPO30 genes. By sensitive CRISPR/Cas12a and M-RPA, the actual detection limits of three viruses were as low as 50, 40 and 60 copies, respectively. Moreover, Lateral flow dipstick (LFD) array based on CRISPR/Cas12a achieved portable and intuitive detection, making it suitable for point-of-care testing. Therefore, CRISPR/Cas12a array and LFD array paved the way for CaPV differentiation in practice. Additionally, we constructed a real-time quantitative PCR (qPCR) array to fill the qPCR technical gap in differentiation and to facilitate the quarantine departments.

Tanapox, South Africa, 2022.

Tanapox is a rarely diagnosed zoonosis known to be endemic to equatorial Africa. All previously reported human cases were acquired within 10° north or south of the Equator, most recently 19 years ago. We describe a human case of tanapox in South Africa (24° south of the Equator). Expanded surveillance for this pathogen is warranted.

Molecular epidemiology of swinepox viruses circulating in India.

Swinepox is a sporadic virus disease of domestic and wild pigs that mainly occurs during the rainy season. Though the disease is known for a century, research on swinepox especially genetic characterization is scanty. Self-limiting nature of the disease, the non-availability of specific diagnostics as well as the resemblance of clinical signs with other pathogens are some of the issues in the slow progress in swinepox-related research. Recent whole genome sequencing data from the USA, India, and Germany enhanced our understanding of the biology of swinepox virus (SWPV). The objective of the present study is to investigate the molecular epidemiology of two swinepox outbreaks that occurred in 2015 and 2016 one each in Uttar Pradesh, and the Haryana states of India. The appearance of clinical signs in different swine breeds was recorded. The scab samples from infected pigs were collected, DNA extracted, host range genes of SWPV were PCR amplified, sequenced and analyzed for genetic and phylogenetic characterization. Desi (nondescript breed), Yorkshire White pigs, and Landrace cross were found to be infected with SWPV. Host range genes of SWPV analyzed from clinical samples showed very high nucleotide identity with each other. Phylogenetic analyses revealed that SWPVs circulating in India are distinct (Indian lineage) from the SWPV of the USA, Germany, and Russia (European-North American lineage). Our study affirms the existence of two distinct lineages of SWPV globally with differences in clinical lesions between breeds.

Validation of molecular diagnostics for the detection of pseudocowpox virus.

The pseudocowpox virus (PCPV) is recognized for causing exanthematic lesions in cattle and humans. The diagnosis is important because it is a zoonosis and its clinical signs can be confused with foot-and-mouth disease, a high-impact bovine disease in livestock. The objective of this work is to validate a SYBR Green qPCR and a conventional PCR for virus detection in bovine samples. Detection limit tests, repeatability, reproducibility, sensitivity, and specificity were compared. When two analysts were compared, results demonstrated that training and pipetting influence the repeatability. The qPCR was more sensitive than conventional PCR but showed nonspecific reactions distinguishable by the melting curve. Both showed high repeatability and reproducibility.

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus and foot-and-mouth disease virus.

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV) and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included ß-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV and FMDV) with no cross-reactivity against other viruses that cause similar clinical signs. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 CaPV, 7 PaPV and 15 FMDV copies per assay. The amplification efficiency (AE) and correlation coefficient (R2 ), estimated from the standard curves (Ct vs. log10 template dilution), were 94%-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96%-116% (AE) and >0.98 (R2 ), respectively, for CaP/FMD rule-out assays and 91%-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 CaPV and 39 FMDV clinical specimens from experimentally infected (CS-E) animals, and 29 CaPV (LSDV), 28 FMDV and 36 PaPV clinical specimens from naturally infected (CS-N) animals; all tested positive (DSe 100%) except two CS-E FMDV specimens that were tested negative by FMD rule-out and the corresponding singleplex (FMDV) assays (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV and FMDV in suspected animals and animals with mixed infections.

Retrospective study of poxviruses diagnosed in cattle from Goiás State, Brazil (2010-2018) / Estudo retrospectivo de poxviroses diagnosticadas em bovinos no estado de Goiás (2010-2018)

A retrospective study of poxvirus infections diagnosed in cattle from Goiás state (GO), Brazil, from 2010 to 2018, was performed. All cases have been investigated by the GO Official Veterinary Service (Agrodefesa), from which technical forms and protocols of veterinary diagnosis laboratories were reviewed. In most cases, samples of oral or cutaneous tissues and/or swabs were submitted for virological diagnosis by polymerase chain reaction (PCR) and/or virus isolation. Thirty seven outbreaks/cases of vesicular disease were notified in cattle of 25 counties; in 33 cases the animals presented lesions clinically compatible with poxviruses. The etiology of 25 out of 33 outbreaks/cases was confirmed as poxviruses by PCR and/or viral isolation: 13 as bovine vaccinia virus (VACV), six as pseudocowpox virus (PCPV), five as bovine papular stomatitis virus (BPSV) and one coinfection (VACV and an Orf virus-like parapoxvirus). The laboratory confirmed that cases occurred mainly in dairy cattle (19/25) and during the dry season (22/25). In adult cattle, gross changes were observed mainly in the teats and udder and included vesicles, ulcers, crusts, papules and scars and varied of type, severity and affected region, depending on the poxvirus species. In calves, the main lesions were ulcers in the mouth and muzzle. Zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, affecting especially the hands of milkers and other farm workers. Our data demonstrate the sanitary and economic relevance of these diseases and the wide circulation of different poxviruses in cattle from GO.(AU)

Foi realizado um estudo retrospectivo das infecções por poxvírus diagnosticadas em bovinos do estado de Goiás (GO), entre 2010 e 2018. Todos os casos foram investigados pela Agência Goiana de Defesa Agropecuária (Agrodefesa). Foram revisados formulários técnicos e protocolos de laboratórios de diagnóstico veterinário. Na maioria dos casos, amostras de tecidos orais ou cutâneos e/ou swabs foram encaminhadas para diagnóstico virológico. Foram notificados 37 surtos/casos de doença vesicular em bovinos em 25 municípios; em 33 casos os animais apresentavam lesões clinicamente compatíveis com poxvírus. A etiologia de 25 de 33 surtos/casos foi confirmada como poxvírus por PCR e/ou isolamento viral: 13 como vírus vaccínia (VACV), seis como vírus pseudocowpox (PCPV), cinco como vírus da estomatite papular bovina (BPSV) e um caso de coinfecção (VACV e um parapoxvírus semelhante ao Orf vírus). Os casos confirmados laboratorialmente ocorreram principalmente em bovinos leiteiros (19/25) e durante a estação seca (22/25). Em bovinos adultos, alterações macroscópicas foram observadas principalmente nas tetas e úbere e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes e variaram quanto ao tipo, gravidade e região afetada, dependendo da espécie do poxvírus. Em bezerros, as principais lesões foram úlceras na boca e focinho. Lesões zoonóticas compatíveis com infecção por poxvírus foram observadas em todas as poxviroses diagnosticadas, afetando principalmente as mãos dos ordenhadores e outros trabalhadores rurais. Nossos dados demonstram a relevância sanitária e econômica dessas doenças e a ampla circulação de diferentes poxvírus em bovinos de GO.(AU)