Epidemiological and genetic analysis of Cetacean Morbillivirus circulating on the Italian coast between 2018 and 2021

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea.

Epidemiological surveillance and genetic analysis of the detected sequences revealed that, from 2012, a new strain with a northeast (NE) Atlantic origin caused deaths of cetaceans along the Spanish Mediterranean coast (20) and subsequently in Italy: both an UME (18) and isolated cases (21,22). The systematic sanitary surveillance of cetaceans is a priority for understanding potential infectious disease outbreaks (23) and elucidating the dynamics of the CeMV infection (24). In this study, our primary objectives were to document the prevalence of CeMV in the cetacean populations from the Italian waters and to provide detailed epidemiological data in terms of species, age, and stranding locations. In addition, through the phylogenetic analysis performed, we characterized the CeMV strain circulating.

Methods Animals
All animals were stranded cetaceans examined during routine pathological analysis and cause-of-death assessment by the Italian Cetacean Stranding Network of the Istituti Zooprofilattici Sperimentali, the veterinary public health institutions under the Italian Ministry of Health, and coordinated by the National Reference Center for Diagnostic Investigations in Stranded Marine Mammals (C.Re.Di.Ma). The animals were submitted for a complete post-mortem examination according to standard protocols (25).
Between 2018 and 2021, 829 cetacean strandings were reported along the Italian coastlines (26)(27)(28)(29). A complete necropsy was performed, when possible (n = 376/829; 45.36%), based on the decomposition condition code (DCC), according to standardized protocols (25,30). Age classes (neonates-calves, juveniles-subadults, and adults) were estimated based on total body length (25,31). During necropsy, tissue samples from major organs were collected and divided into three aliquots for subsequent analysis: one was kept frozen at −20 • C for microbiological analysis, one at −80 • C for biomolecular analysis, and the third was preserved in neutral buffered formalin for histological and immunohistochemical (IHC) analysis.
Annual stranding reports elaborated by C.Re.Di.Ma (26)(27)(28)(29), referring to the period 2018-2021, were retrieved and further analyzed to get (i) the CeMV prevalence during the study period, (ii) the signaling (species, sex, and age) and epidemiological information (stranding date and location), (iii) the descriptions of the histopathological examination, with a focus on CeMV suggestive lesions (2), and (iv) the results of the ancillary investigations of selected pathogens in CeMV-positive animals.

CeMV analysis: confirmation and characterization
A total of 134 CeMV target tissues (brain, cerebrospinal fluid, pharyngeal tonsils, lung, mesenteric, pre-scapular and pulmonary lymph nodes, spleen, liver, kidney, urinary bladder, placenta, tongue, and blood plasma, when possible) from 33 cetaceans out of a total of 113 CeMV molecularly positive cetaceans stranded along the Italian coastlines in the period under study were submitted to confirmatory and characterization analysis following Verna et al. (32), and Bellière et al. (33) methodologies, respectively.
Briefly, tissues were physically disrupted using a TissueLyser II homogenizer (Qiagen, Hilden, Germany) by high-speed shaking in plastic tubes with stainless-steel beads (5 mm diameter), and RNA was extracted using an All-Prep DNA/RNA Mini kit (Qiagen, Hilden, Germany), based on the manufacturer's instructions. A molecular diagnosis following Verna et al. (32) and targeting the nucleocapsid (N) gene with the use of degenerate primers. Characterization based on the phosphoprotein (P), N, and fusion protein (F) genes was conducted according to Bellière et al. (33) through various conventional reverse transcription-PCR assays. The N gene was amplified using NgeneF and NgeneR primers, resulting in a 158-bp amplicon. The F gene was amplified with DMVFu-F and DMVFu-R primers, generating a 152-bp amplicon. The P gene was amplified using DMV-C and DMV-P2 primers, producing a 358-bp amplicon. Positive products from the characterization analysis were subsequently sequenced by the Sanger sequencing method.

Phylogenetic study
Nucleotide identities (1 -p-distance) for the sequences of P and N genes were calculated using the MEGA X software (34).
The CeMV nucleotide sequences were analyzed phylogenetically using the maximum likelihood method in the MEGA X software (34). To generate a reliable phylogenetic tree, we confirmed the accuracy of sequence alignments, since the average amino acid p-distances were 0.08 and 0.03 for P and N genes, respectively. These values were smaller than the acceptance threshold value (<0.8) for average p-distance (35, 36). The Kimura 2-parameter model was used with 2,000 bootstrap replicates, and the resulting trees were rooted and edited with the iTOL editor (37).
The phylogeny constructed based on the sequences of the P and N genes showed two main clades in the DMV sequences that separate the Mediterranean and NE-Atlantic strains (Figure 2), and the sequences of this study clustered together with the latter. Accordingly, our sequences showed the highest identity with GenBank sequences MF472881, MG000861, MF472882, KP835987, KP835991, KP836003, and KT878660 (identities: 1.0000 -0.9908) for the P gene and with GenBank sequences MG000862, KP835984, KP836000, KT878655, and KP836004 (identities: 1.0000 -0.9943)

Discussion
Our study assessed the prevalence of CeMV in cetaceans stranded along the Italian coasts between 2018 and 2021 and successfully molecularly typed the detected isolates. These insights enhance our knowledge on the current epidemiological status of CeMV in the Mediterranean Sea.
Molecular positivity for CeMV was detected in cetaceans stranded in all the marine sectors of the Italian coastline, with the only exception being the northern Adriatic coast (n = 0 in the regions of Emilia-Romagna, Veneto, and Friuli-Venezia Giulia) ( Table 1 and Figure 1). This could be related to the fact that most of the cetaceans stranded during this period in the aforementioned regions (56/70, 80.0%) had a DCC of 4 or 5 (26)(27)(28)(29), which might cause the degradation of the RNA virus. However, the area where the highest number of CeMV-positive cetaceans stranded during the study period was the southwest of the Italian peninsula (Calabria, n = 26; Campania, n = 25) ( Table 1 and Figure 1). It has been suggested that chemical contaminants have the potential to impact susceptibility to CeMV infection (48). A recent investigation has revealed a correlation between the presence of pathological evidence of infection and the elevated levels of chemical pollutants in the Pelagos Sanctuary, located in northern Italy (49). Considering the existing body of research indicating higher concentrations of pollutants in southern Italy (41, 50), it is plausible to attribute the higher number of CeMV-positive animals in this geographical region to such environmental factors. However, further studies are necessary to elucidate the origins of these observed differences in prevalence between the different regions. Continued monitoring of strandings and their causes will provide valuable insights into whether these geographical patterns are repeated and help identify potential underlying causes.
Regarding the DMV strains present in the Mediterranean Sea, recent evidence indicates that the previous DMV Mediterranean strain has been replaced by the NE -Atlantic strain (18, 21, 22), first reported in the Mediterranean Sea in 2012 (20). P and N gene sequences described in the present study clustered together with NE-Atlantic DMV strain sequences (Figure 2). These sequences were identical or showed the highest identity with the NE-Atlantic strain, detected on the Portuguese and Spanish Atlantic coasts and in Italy (see Supplementary material). Taken together, these results provide evidence of an active circulation of the NE-Atlantic strain along the Italian coastline during the studied period, as previously reported (18, 21,22,40).
It was hypothesized that the epidemiology of the DMV strain that circulated in 2011 along the Spanish Mediterranean coast was changing from an epizootic to an enzootic infection (14). Similarly, chronic forms of DMV infection were predominant in 2011 (51) and 2013 (16) in Spain and Italy, respectively. However, during the 2016 Italian UME, acute and subacute forms of DMV infection were mainly reported (18, 52). This could be explained by the fact that the cetacean population of the Mediterranean Sea was naïve to this NE-Atlantic strain (18, 20, 22) and the greater neurovirulence described in this strain, which prevented affected cetaceans from surviving the acute and subacute stages of the infection, as previously proposed (40). In line with this, non-suppurative meningoencephalitis was the main lesion detected in this series of cases (Table 1). However, the present findings have demonstrated that, in more than a third (n = 31/87, 35.63%) of the 87 CeMV-positive individuals that received a histopathological examination, no lesions consistent with DMV infection were detected, suggesting a chronic form of the infection (no lesions directly attributable to CeMV, but viral antigen is detectable) (2). In addition, almost half of these animals (n = 14/31) presented coinfections with important viral, bacterial, or protozoan agents, and/or severe nematode infestations (26-29, 39, 53). These coinfections are typical of the chronic phase of DMV infection and are related to host immunosuppression caused by the virus (2). However, a complete and exhaustive histopathological study was not always performable due to the autolysis status of the animals involved: only 12 of these 31 animals were fresh carcasses (DCC = 2), 11 were in moderate decomposition (DCC = 3), and eight in advanced decomposition (DCC = 4) (data not shown). Therefore, it is possible that our ratio of CeMV-positive cetaceans lacking CeMV-associated lesions was overestimated due to the conservation status of the animals, which in some cases precluded a reliable examination. This could explain the difference in terms of the predominant stage of infection reported in a recent study (40) involving some of the animals described here (acute/subacute vs. chronic). In addition, in the aforementioned study, the stage of infection was determined by a multiparametric assessment from the cerebral and cerebellar cortex samples (40), while in our study, a systemic determination was considered.
The infection with the NE-Atlantic strain was considered endemic in the striped dolphin populations from Portugal and Galicia since no outbreaks were detected and positive samples were detected annually, indicating that the virus was actively circulating (38). In addition, the prevalence obtained in that study was 24% (38), which is very similar to the prevalence obtained in the last 2 years of our study: 23.91% (2020) and 26.88% (2021). Thus, our results support the possibility of an endemic circulation of this strain in the Mediterranean Sea, as previously theorized (21).
This NE-Atlantic strain most likely entered the immunologically naïve striped dolphin population with a lack of specific antiviral immunity, resulting in a UME in 2016 (18) and several associated strandings since then (21,40). However, it is possible that some individuals outgrow the acute/subacute phase of the infection, resulting in chronic forms with detectable DMV antigen but with no associated lesions. This specific immunity would explain the absence of UME since 2016. These observations are in good agreement with previous findings that support an endemic DMV circulation in the Mediterranean Sea and with the occurrence of cyclic outbreaks when herd immunity decreases (14). Nevertheless, further research is needed to clarify the epidemiology of this new DMV strain in the Mediterranean Sea.
It has been suggested that the NE-Atlantic strain entered the Mediterranean Sea through the Strait of Gibraltar, where cetacean populations from both seas come into contact, and subsequently spread into the Mediterranean Sea (18, 21,22), where it has continued to circulate until 2021, as this study confirms. As previously hypothesized, for the possibility of an endemic circulation of CeMV in the Mediterranean Sea, the transmission and maintenance of CeMV strains between different species should be considered (21). In the Mediterranean Sea, the striped dolphin is the species in which CeMV has been detected most frequently (11,13,14,16,18,(20)(21)(22). Accordingly, 70.80% (80/113) of the CeMV-positive cetaceans stranded in Italy during the study period were striped dolphins (Table 1). However, in the literature, it has .
/fvets. . also been reported in the Mediterranean Sea in other species such as short-beaked common dolphins (18), bottlenose dolphins (15,22), long-finned pilot whales (54), Cuvier's beaked whales (55), sperm whales (17,22), and fin whales (19,22). In line with this, between 2018 and 2021 in Italy, CeMV was detected in bottlenose dolphins, fin whales, long-finned pilot whales, Cuvier's beaked whales, and sperm whales ( Table 1). Some of these species, such as long-finned pilot whales, Cuvier's beaked whales, and sperm whales, have been suggested to spread the virus between geographically distant populations (42,54,56). Specifically, sperm whales are known to be susceptible to infection by the NE-Atlantic CeMV strain (22), and they have been suggested to be a spillover host with a role in disseminating CeMV to cetaceans from widely dispersed geographical regions since they travel long distances during seasonal migration (56). During the period spanning from 2018 to 2021, a total of six sperm whales tested positive for CeMV, as indicated in Table 1. Among these cases, the presence of the NE-Atlantic strain of CeMV was confirmed in one individual (Table 2). However, the NE-Atlantic CeMV strain was also previously reported in bottlenose dolphins in the Mediterranean Sea (40). During the study period, this strain was reported in 20 bottlenose dolphins (Table 1), with at least four of these individuals found to be infected with the NE-Atlantic strain ( Table 2). However, the epidemiological role of these species in the transmission pattern of this strain in the Mediterranean Sea is still unknown, and further studies would be necessary to shed light on this field.

Conclusion
In this study, we report a CeMV prevalence of 31.9% in cetaceans stranded along the Italian coasts between 2018 and 2021. The greater proportion of CeMV-positive cetaceans during the study period was found in the south-west of the Italian peninsula ( Figure 1). A total of 57 sequences from novel strains were generated and submitted to GenBank. Phylogenetic analysis revealed that these sequences belonged to the NE-Atlantic DMV strain. The species affected by the detected strains were striped dolphins, bottlenose dolphins, and sperm whales, and the individuals were mainly adults and, to a lesser extent, juvenilessubadults and neonates-calves. The evidence from this research points toward the idea of an endemic circulation of the NE-Atlantic strain in the Italian Sea based on the epidemiological, phylogenetic, and histopathological information.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethics statement
Ethical review and approval was not required for the animal study because in the collection of post-mortem tissues for research purposes, the approval of the corresponding Ethics Committee is not required.  . /fvets. .