Free-living birds face various health risks due to viruses, such as avian influenza, West Nile Virus, and paramyxoviruses (Callinan et al., 1979, Choi et al., 2012, De Thoisy et al., 2009, Dierauf et al., 2006, Gilchrist, 2005, Kaleta and Docherty, 2007, Mendelson et al., 1995, Niu et al., 2018, Senne, 2007). Herpesviruses have also been detected in more than 100 domestic and wild avian species (De Thoisy et al., 2009, Kaleta and Docherty, 2007, Pfaff et al., 2017, Tuxbury et al., 2020). Herpesviruses are large, double-stranded DNA viruses in which infection severity typically depends on virus strain and host susceptibility (MacLachlan and Dubovi, 2017). These viruses usually have low pathogenicity, as herpesviruses typically co-evolve with their host species; however, some herpesviruses can cause severe disease and mortality in aberrant, young, naïve, or immunosuppressed hosts (Kaleta and Docherty, 2007, Ritchie, 1995).

There are currently ten avian herpesviruses (order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae) in the genera Iltovirus and Mardivirus that are officially recognized by the International Committee on Taxonomy of Viruses (ICTV) (Gatherer et al., 2021). Several of these herpesviruses are associated with outbreaks and mortality among free-ranging marine and aquatic birds, and therefore, these viruses may serve as a potential threat to avian conservation (De Thoisy et al., 2009, Goldberg et al., 1990, Niemeyer et al., 2017, Pfaff et al., 2017, Tuxbury et al., 2020). Specifically, Mardivirus anatidalpha1 (Anatid alphaherpesvirus, duck enteritis virus) causes considerable mortality in multiple species of waterfowl (Goldberg et al., 1990), and Fregata magnificens herpesvirus 1 has been associated with massive annual mortality episodes in Magnificent Frigatebird (Fregata magnificens) chicks in French Guiana (De Thoisy et al., 2009). Currently, four genetically distinct herpesviruses have been identified in penguins (order Sphenisciformes, family Spheniscidae): spheniscid alphaherpesvirus 1 (Mardivirus spheniscidalpha1; Tuxbury et al., 2020), sphenicid alphaherpesvirus 2 (Seimon et al., 2025), Magellanic penguin herpesvirus 1, and Magellanic penguin herpesvirus 2 (Niemeyer et al., 2017).

The ICTV does not currently recognize Magellanic penguin herpesvirus 1 (MagHV1), but it is most closely related to Gallid herpesvirus 1 (order Herpesvirales, family Orthoherpesviridae, subfamily Alphaherpesvirinae, genus Iltovirus, species gallidalpha1). MagHV1 was first identified during a 2011 outbreak of severe respiratory disease in oiled Magellanic penguins (Spheniscus magellanicus) from Brazil. The penguins were housed at a rehabilitation center with albatross (Thalassarche chlororhynchos) and juvenile petrels (Macronectes giganteus) after stranding along the Rio Grande do Sul State beaches. Of the 168 penguins at the rehabilitation center, 98 individuals developed acute respiratory disease characterized by anorexia, gasping, coughing, expectoration of bloody mucus, and marked dyspnea. Eighty-five of them died; fourteen of these animals died within 24 h of arrival at the rehabilitation center, and the rest around two weeks after the onset of clinical signs, either naturally or via euthanasia due to severe respiratory distress (Niemeyer et al., 2017). Syncytial giant cells in the tracheal epithelium contained amphophilic intranuclear inclusions characteristic of herpesvirus infection, and the presence of a viral infection was confirmed using electron microscopy. The DNA from a novel herpesvirus (MagHV1) was then detected using conventional consensus PCR in affected tissues (Niemeyer et al., 2017).

While conventional consensus PCR was used to detect the presence of MagHV1, this assay is not specific for MagHV1, and DNA sequencing is required to confirm the identity of positive PCR products, which is a costly and time-consuming process (VanDevanter et al., 1996). Quantitative PCR (qPCR) could improve speed, sensitivity, specificity, and efficiency compared to conventional consensus PCR. A validated qPCR assay for MagHV1 could allow for rapid, early detection of birds shedding the virus and facilitate monitoring of clinically affected penguins. Because latency is a clinical feature of herpesvirus infections, timely detection of reoccurrence and viral shedding is crucial for making informed decisions regarding disease and flock management decisions, especially regarding translocation events or situations where multiple seabird species are kept together (Callinan et al., 1979, De Thoisy et al., 2009, Niemeyer et al., 2017).

The purpose of this study was to develop and partially validate a TaqMan qPCR assay for detecting MagHV1. We hypothesized that a qPCR hydrolysis probe-based assay would have high analytical sensitivity and specificity for detecting Magellanic penguin herpesvirus 1. This assay will enable the rapid, sensitive, and specific detection of MagHV1 in penguins, thereby supporting the epidemiological characterization of this virus through prevalence estimation and outbreak investigation during disease surveillance.