Herpesviruses in migrating procellariforms, northeastern Brazil

Seabirds are one of the most threatened avian groups. Viruses, including herpesvirus, represent considerable threats to marine avifauna. Herein, our goal was to survey herpesvirus in Procellariiformes that stranded in Brazil between June and July 2021. We analyzed 12 Cory's shearwaters (Calonectris borealis), two Great Shearwaters (Ardenna gravis, syn. Puffinus gravis) and one Yellow-nosed Albatross (Thalassarche chlororynchos) found in an unusual mortality event in Bahía state, northeastern Brazil. After necropsy, selected tissue samples were tested for herpesvirus using a broad-range nested PCR. Overall, 20% (3/15) of the birds were herpesvirus-positive, i.e., two Cory's Shearwaters and one Great Shearwater. One alphaherpesvirus sequence type was identified in each shearwater species, classified into the genus Mardivirus. This study describes two likely novel herpesviruses in shearwaters, contributing to the currently very scarce data regarding infectious agents in Procellariiformes. Further studies are necessary to evaluate the presence and characteristics of herpesvirus in Procellariiformes, and the presence (or not) of related disease in order to understand the epidemiology of this infectious agent and eventually contribute to the conservation of this endangered seabird group.


Introduction
Seabirds are among the most threatened avian groups, also considered good indicators of marine ecosystems health (Croxall et al. 2012).Epidemiological surveillance provides crucial information regarding pathogens' hosts and geographic distribution, shedding light into the ecology, To this date, alphaherpesvirus within the genera Iltovirus and Mardivirus have been described in a variety of avian orders (Davison et al. 2009;Niemeyer et al. 2017;de Francisco et al. 2023;Ewbank et al. 2023).In seabirds, several alphaherpesviruses have been described in Charadriiformes, Gaviiformes, Procellariiformes, Phaethontiformes, Sphenisciformes, and Suliformes (Quesada et al. 2011;Niemeyer et al. 2017;Verdugo et al. 2019;Sebastiano et al. 2020); however, little is known about herpesviruses in Procellariiformes, limited to the description of a herpesvirus in a Yellow-nosed Albatross (Thalassarche chlororynchos) (Niemeyer et al. 2017).Stranding events involving pelagic seabirds provide an opportunity to assess population threats, infectious agents and related stressors.Thus, our goal was to molecularly survey and characterize herpesviruses in migrating Procellariiformes stranded alive in northeastern Brazilian coast and admitted into a rehabilitation center.
Rescue and postmortem examinations were performed by the Instituto Mamíferos Aquáticos (IMA), a rehabilitation center in the same state.All animals died, either during transport (n = 2) or while undergoing rehabilitation (n = 13), being necropsied following standard procedures (Hocken 2002).The body condition was classified into good, moderate, poor, or cachectic, based on pectoral muscle development and presence (or absence) of internal and subcutaneous fat deposits.Age class and sex were determined based on plumage pattern and upon visualization of the gonads.Tissue samples (i.e., lungs, brain, liver, spleen, and kidneys) were collected and fixed in 10% buffered formalin and frozen at -20/-80 °C until processed.

Molecular techniques
Total DNA of selected frozen tissue samples (brain, lung and kidney) was extracted using the DNeasy Blood and Tissue kit, according to the manufacturer's guidelines.No swabs were available.Subsequently, DNA was tested with a broad-range nested PCR protocol for herpesviruses, with an annealing temperature of 46 ºC for both PCRs, able to amplify a fragment of approximately 215-315 bp of the DNA polymerase gene of different alpha, beta-and gammaherpesviruses (VanDevanter et al. 1996).In herpesvirus-positive animals, other available frozen tissue samples (spleen and liver) were also tested following the same methodology described above.A previously confirmed cetacean gammaherpesvirus positive sample was used as positive and DPEC water was used as no template control.All amplicons of the expected size were purified with ExoSAP-IT and directionally sequenced.The obtained consensus sequences were analyzed for further phylogenetic classification using Mega 7.0.Subsequently, a DNA polymerase amino acid maximum likelihood phylogram was constructed including the obtained sequences, representative sequences of the genera Mardivirus and Iltovirus, and Human gammaherpesvirus 8 as an outgroup, using Mega 7.0.

Histopathology
Light microscopy histopathological evaluation was performed in the HV-PCR-positive individuals, on formalinfixed paraffin-embedded tissues sectioned at 5 μm and stained with hematoxylin and eosin.
In the Cory's Shearwaters, case IMA4473 was herpesvirus-positive in the spleen and the kidney, and negative in brain, lung and liver; while IMA4486 was only herpesviruspositive in the lung and brain, and negative in spleen, liver and kidney.Both Cory's Shearwater herpesviral sequences were identical between them, and presented the highest nucleotide (68.9%) and amino acid identities (67.8%) to different Columbid alphaherpesvirus 1 sequences (e.g., Gen-Bank accession nº NC_034266, MW625939, MW625940, detected in Rock Pigeons (Columba livia) in China in 2013, and in Slovenia in 2019).

Gross and histopathological findings
Overall, tissue samples of herpesvirus-positive individuals were compromised due to advanced autolysis.Nevertheless, in two of the positive birds (Cory's Shearwater 4473 and the herpesvirus-positive Great Shearwater) pulmonary edema was observed.Additionally, mononuclear adrenalitis and moderate mixed celomitis were present in the Great Shearwater (Table 1).

Discussion
Herein we found two likely novel alphaherpesvirus sequence types, reported for the first time in Cory's and Great Shearwaters, and in the family Procellariidae.To the authors' knowledge, these are the second and third descriptions of herpesviruses in Procellariiformes worldwide, following the report of Thalassarchid herpesvirus 1 in one of the 12 Yellow-nosed Albatross under rehabilitation in Rio Grande do Sul State, southern Brazil (Niemeyer et al.The gross and histopathological examinations of this individual were not conclusive to determine the causa mortis.
In our study, no associated lesions were observed in any of the herpesvirus-positive individuals, although the advanced autolysis precluded a detailed histopathological examination in some tissue samples.Herpesviruses generally show low virulence in their natural host, which could be the case here (Davison et al. 2009).The herpesviruspositive species -Cory's Shearwater and the Great Shearwater-breed, respectively, in Portugal and Canary Islands (Spain), and in the Tristan da Cunha archipelago (United Kingdom) and Malvinas/Falkland Islands, spending their non-breeding season in the Atlantic (Birdlife International 2023a, b).We hypothesize that the stress associated with the long migration of these species from their breeding territories to Brazil likely triggered the reactivation of the detected herpesviruses (Niemeyer et al. 2017), which were detected in several tissue samples.
Large interannual coastal strandings of seabirds (alive and/or dead) have been reported worldwide (Bugoni et of long-lived, pelagic species that spend most of their life cycle at sea (Stidworthy and Denk 2018).Due to these particular life history traits, procellariforms are amongst the most endangered threatened taxonomic groups (Croxall et al. 2012), considered highly sensitive to anthropogenic activities (e.g., fisheries interaction, habitat degradation, climate change, invasive and non-native species) (Tavares et al. 2019;Rodríguez et al. 2022).Therefore, data on the infectious agents affecting Procellariiformes is pivotal for the conservation of this group.
The obtained sequence types are highly divergent when compared to the closest from GenBank/DDBJ/EMBL database, with more than 30% of difference.This fact, along with their detection in novel host species, support their classification as novel herpesviral sequences.Nevertheless, further studies detecting more Cory's and the Great Shearwater individuals infected with these likely novel herpesviruses are necessary in order to establish if these birds are their natural hosts.According to our phylogenetic tree, both our sequence types are classified within the genus Mardivirus,

Fig. 1
Fig. 1 Maximum likelihood phylogram of the alignment of the herpesviral DNA polymerase sequences (1) obtained in this study (blue dots), and (2) previously detected in birds, including those classified into the genus Mardivirus (yellow square) and Iltovirus (blue square).Human gammaherpesvirus 8 was selected as outgroup.The phylogram was

Table 1
Species, age, sex, gross and histopathologic findings, and comments regarding the herpesvirus-PCR-positive Cory's Shearwater (Calonectris borealis) and Great Shearwater (Ardenna gravis) described in this study ID# Species Age Sex Gross Findings