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The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe, frequently fatal, lymphoproliferative disease that affects a wide variety of ruminants and is caused by ovine gammaherpesvirus 2 (OvHV-2), a member of the MCF virus (MCFV) complex. The typical clinical manifestations of SA-MCF are well known and easily recognized by veterinarians, resulting in clinical diagnosis of MCF when characteristic clinical signs are present. This article describes the findings observed in cattle infected with OvHV-2 but without typical clinical manifestations of SA-MCF. Three calves with episodes of diarrhea before death and a yearling that died suddenly were investigated. Gross alterations were not suggestive of SA-MCF. Histopathology revealed a combination of proliferating vascular lesions (PVLs) and necrotizing vasculitis in three animals (two calves and the yearling); with PVLs being identified only at the carotid rete mirabile of two calves infected with OvHV-2. Additional significant histopathologic lesions included atrophic enteritis, portal lymphocytic hepatitis, interstitial pneumonia, suppurative bacterial bronchopneumonia, and pulmonary hemorrhage. An immunohistochemical assay designed to identify only antigens of MCFV revealed, positive, intralesional, intracytoplasmic immunoreactivity within epithelial cells of multiple tissues of all animals with PVLs. PCR assays amplified OvHV-2 DNA from multiple tissues of the animals that contained MCFV proteins, confirming the MCFV identified as OvHV-2. Additionally, bovine coronavirus (BCoV) nucleic acids were amplified from tissues of all animals, including the animal not infected by OvHV-2. Collectively, these findings confirmed the participation of OvHV-2 in the development of the disease patterns observed in these animals that were concomitantly infected by BCoV and provide additional confirmation that cattle can be subclinically infected with OvHV-2. Consequently, the real occurrence of OvHV-2-related disease may be more elevated than reported, since asymptomatic or subclinically infected animals are not likely to be investigated for OvHV-2. Furthermore, PVLs should be included as possible histologic indicators of OvHV-2-related diseases in ruminants.

This study investigated the possible occurrence of pulmonary disease in beef cattle from 13 municipalities within the State of Mato Grosso (MT), Brazil. The state of MT is a leading player in beef cattle production in Brazil, but with comparatively few data relative to the occurrence of pulmonary disease or ovine gammaherpesvirus 2 (OvGHV2)-related infections in cattle. Pulmonary samples from 44 beef cattle, with ages ranging between 18 and 28 months, were collected during slaughter and processed to determine the patterns of pulmonary lesions. Additionally, duplicate fragments were used in immunohistochemical (IHC) assays designed to detect malignant catarrhal fever (MCFV) antigens and in molecular assays to amplify 10 agents associated with the development of bovine respiratory disease (BRD). Interstitial pneumonia (IP) was diagnosed in most of the lungs (98%; 43/44) evaluated from all municipalities. MCFV antigens were detected in 37% (16/43) of the animals with IP. Only four pathogens were amplified by molecular assays within the lungs of cattle with IP: OvGHV2 (23%; 10/43), bovine viral diarrhea virus (12%; 5/43), bovine coronavirus (7%; 3/43), and Mannheimia haemolytica (2%; 1/43). The nucleic acids of bovine respiratory syncytial virus, bovine alphaherpesvirus 1, bovine parainfluenza virus 3, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis (Mycoplasma) bovis were not amplified. Singular infections in cattle from municipalities were associated with MCFV (n = 3) and OvGHV2 (n = 2), while OvGHV2 occurred in all dual and triple infections. However, in four animals with IP, none of the disease pathogens identified were detected. Statistically, MCFV antigens were more frequently (p = 0.048) detected in the lungs of female (75%; 12/16) cattle with IP relative to males (25%; 4/16). Additionally, there was a positive correlation (p < 0.001) between the IHC detection of MCFV antigens within the bronchial epithelium and the epithelium of peribronchial glands of cattle with IP. This is the first study to statistically demonstrate that female cattle are at greater risk of developing MCFV-related infections as compared to male animals. The detection of OvGHV2 in singular and multiple infections during this investigation supports earlier studies that associate this pathogen with the development of pulmonary disease in cattle, indicating that OvGHV2 can contribute to the etiology of IP. Additionally, the detection of OvGHV2-induced infections in asymptomatic cattle suggests that all animals were subclinically infected, confirming that subclinically OvGHV2-induced infections may be widespread in ruminants from Brazil. Furthermore, the occurrence of atypical interstitial pneumonia cannot be discarded, particularly in animals with IP but without any associated pathogen. These initial findings suggest the need for a more elaborate investigation to understand the dynamics of pulmonary disease within this state.

Bovine gammaherpesvirus 6 (BoGHV6), previously known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae. Other members of the genus Macavirus include viruses that produce malignant catarrhal fever (MCF) in mammalian hosts, collectively referred to as the MCF virus (MCFV) complex, and the porcine lymphotropic herpesvirus (PLHV). However, the current role of BoGHV6 in the development of diseases and/or disease syndromes remains uncertain and controversial. This paper investigated the participation of BoGHV6 in the development of pulmonary disease in a cow with interstitial pneumonia by histopathology and molecular testing. Tissue antigens of common viral agents of respiratory diseases and Mycoplasma bovis were not identified by immunohistochemistry. Additionally, molecular assays designed to amplify common bacterial and viral pathogens of pulmonary disease did not amplify the nucleic acids of these agents. However, a pan-PCR assay amplified the DNA of the herpesvirus polymerase gene, while the specific BoGHV6 nested-PCR assay amplified the partial fragment of the BoGHV6 polymerase gene derived from the pulmonary tissue with interstitial pneumonia. Phylogenetic analysis revealed that the BoGHV6 strain herein identified had 99.8% nucleotide (nt) sequence identity with reference strains of BoGHV6, but only 72.2–73.5% and 67.9–68.6% nt identity with reference strains of MCFV and PLHV, respectively. Consequently, these results suggest that BoGHV6 was associated with the pulmonary disease observed in this cow.

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AlHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SAMCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AlHV-1 genes and with annealing temperatures>11 °C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AlHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SAMCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AlHV-1 genes and with annealing temperatures > 11 °C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

The Macavirus , ovine gammaherpesvirus 2 (OvGHV2), is the cause of sheep-associated malignant catarrhal fever (SA-MCF). Although SA-MCF occurs in a wide range of mammalian hosts, there are few descriptions of this disease and/or infection in goats. This report describes the findings observed in a goat that was infected by OvGHV2 and adds to the rare description of this infection in this animal species. A 6.5-year-old, female, Anglo Nubian goat, with a neurological syndrome, that was euthanized after severe esophageal obstruction was investigated to determine the cause of the brain disease. Histopathology revealed cerebral cortical edema, hemorrhagic rhombencephalitis, severe hepatic necrosis, and atrophic enteritis. An immunohistochemical (IHC) assay identified intracytoplasmic antigens of a malignant catarrhal fever virus (MCFV) within epithelial cells of the intestine, liver, lungs, and kidneys. A semi-nested PCR assay amplified the partial fragment of the OvGHV2 tegument protein gene from the intestine, confirming that the MCFV identified by IHC was OvGHV2. A qPCR assay that targeted the OvGHV2 polymerase gene revealed an elevated quantification cycle ( Cq ), while nanoplate-based digital PCR (dPCR) detected low viral copy load within the OvGHV2 DNA. Furthermore, the nucleic acids of several disease pathogens associated with diseases in ruminants were not amplified. However, the exact cause of the neurological syndrome remained obscure since nucleic acids of neurological disease pathogens such as bovine viral diarrhea virus, bovine alphaherpesvirus 1 and 5, Histophilus somni , and OvGHV2 were not detected from the brain. Collectively, the results of the Cq and dPCR confirmed that this goat was infected with a low viral load of OvGHV2, which probably was insufficient to induce the typical histopathological alterations and subsequent clinical manifestations associated with SA-MCF and/or infections by OvGHV2. Therefore, elevated viral loads of OvGHV2 would have been required for the development of histological lesions and/or clinical manifestations of SA-MCF in this goat. Furthermore, the dPCR methodology can be used for the efficient detection and quantification of OvGHV2 DNA in animals with or without clinical and/or histopathological evidence of SA-MCF. Additionally, since previous cases of OvGHV2 infections in goats did not have the typical clinical manifestations of SA-MCF, one wonders if this Macavirus can induce SA-MCF in goats.

Sheep-associated malignant catarrhal fever (SA-MCF), the form of MCF that occurs in Brazil, is a severe, frequently fatal, infectious disease caused by ovine gammaherpesvirus-2 (OvHV-2), in which sheep are the asymptomatic hosts and cattle and other cloven-hoofed animals are the accidental hosts. This review provides a critical analysis of the historical, epidemiological aspects and the estimated economic impacts associated with SA-MCF in Brazil. Moreover, the clinical manifestations and pathological lesions associated with SA-MCF in cattle are reviewed and discussed and the phylogenetic distribution of OvHV-2 in Brazil is presented. OvHV-2 is the only MCF virus identified in animals from Brazil. It is recommended that a histopathologic diagnosis of SA-MCF be based on all aspects of vascular disease in the affected animal and not only lymphocytic/necrotizing vasculitis and/or fibrinoid change. Conformation of the intralesional participation of OvHV-2 in these alterations can be achieved by immunohistochemistry and/or in situ hybridization assays. Additionally, it is proposed that OvHV-2 should be considered as a possible infectious disease agent associated with the development of bovine respiratory disease in cattle. Furthermore, the possible role of the small intestine in the dissemination of OvHV-2 is discussed.

Malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AIHV-1) transmitted from wildebeest, is a lethal cattle disease with significant impacts on East African pastoralists. Development of a live attenuated MCF vaccine has prompted research into its use in communities at risk. This study reports results from the first utilisation of the MCF vaccine in locally-owned cattle under field conditions. The study involved a primary two-dose course vaccination of 1634 cattle, followed a year later, by boost vaccination of 385 of these cattle. It aimed to: (a) evaluate the antibody response to a two-dose AlHV-1 primary vaccination course, including initial response, antibody levels after one year, and clinical events post-vaccination; (b) assess how factors like age, reproductive status, body condition, and breed influence the initial response; and (c) compare antibody responses to single- and two-dose booster protocols one year after primary vaccination. Analyses were carried out using linear mixed-effects models and paired t-tests. Clinical incidents were reported in 11/1634 cattle vaccinated during the primary course and in 0/385 cattle during the boost regimens. The primary vaccination resulted in a 9-fold increase in comparison to pre-vaccination antibody levels and the response was consistent across animals of different ages, reproductive statuses and body conditions. While antibody levels declined 11 months after primary vaccination, they remained high, and a single-dose booster vaccination was sufficient to elicit a strong immune response, with only marginal increases after a second booster. The study provides evidence of high immunogenicity and low incidences of clinical events of the vaccine in cattle across individual host factors and immunologically vulnerable groups, under prevailing environmental conditions. It also indicates the utility of a single-dose booster regimen. These findings will support progress towards commercial production and larger-scale adoption which could generate important benefits for the livelihoods, and sustainability of pastoral livestock systems. [Display omitted] •Zebu cattle were vaccinated against malignant catarrhal fever using an attenuated live virus in remote northern Tanzania.•Strong antibody responses followed a two-dose vaccination and single booster, unaffected by age, reproductive status, or condition.•No major adverse effects were reported following primary or boost-vaccination.