Insect-specific Alphamesonivirus-1 (Mesoniviridae) in lymph node and lung tissues from two horses with acute respiratory syndrome

25 As members of the RNA virus order Nidovirales include those that infect hosts ranging from 26 marine invertebrates to terrestrial mammals, understanding their emergence, host range and 27 disease potential is of clear importance. The Mesoniviridae are a recently documented family 28 of viruses within the Nidovirales. To date, mesoniviruses have only been associated with the 29 infection of arthropods, particularly mosquitoes. Herein, we report the first detection of a 30 mesonivirus – Alphamesonivirus-1 – in mammals. Specifically, w e utilized genomic and 31 histological techniques to identify the presence of Alphamesonivirus -1 in lung and lymph 32 node tissues of two horses that succumbed to an acute respiratory syndrome . Notably, no 33 other pathogens typically associated with respiratory disease in horses were detected in 34 these samples. Counter to the previous contention that mesoniviruses only infect insects, our 35 findings suggest a potential ly broader host range and cross -species transmission of these 36 viruses. The genome sequences of Alphamesonivirus-1 obtained from the two horses were 37 closely related to those from a local Culex mosquito pool a s well as an Alphamesonivirus-1 38 previously in identified Italy, suggestive of ongoing local transmission. Th e discovery of 39 Alphamesonivirus-1 in tissue s from diseased horses not only challenges current 40 understandings of mesonivirus host range, but prompts further investigation into the role of 41 insect-specific viruses in mammalian disease processes. Our results emphasize the 42 importance of considering atypical pathogens in cases of unexplained animal deaths and 43 suggest a potential zoonotic threat posed by previously overlooked viral families. 44 45 IMPORTANCE 46 Alphamesoniviruses, members of the Mesoniviridae family, have long been considered insect-47 specific viruses with no known association with vertebrate hosts. Herein, we describe the first 48 detection of Alphamesonivirus-1 in mammals, marking a significant expansion of the known 49 host range for this newly described virus family. Using detailed molecular and histological 50 analyses we identified Alphamesonivirus-1 in lung and lymph node tissues of two horses that 51 presented with an acute respiratory syndrome. Our findings indicate that Alphamesoniviruses 52 may possess a broader host range than previously believed and could potentially induce 53 severe disease in mammals. This unexpected host jump not only challenges existing 54 knowledge on the ecology of mesoniviruses , but suggests that insect -specific viruses may 55 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 3 pose a previously unrecognized health risk to vertebrates, including domesticated animals. 56 These insights prompt the need for increased surveillance of atypical pathogens, especially in 57 cases of unexplained respiratory illness, and may have implications for zoonotic disease 58 emergence. 59 60

Alphamesonivirus-1, Mesoniviridae, horses, acute respiratory syndrome, lymph 61 node, lung. 62 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 4

63 The emergence and global spread of SARS-CoV-2 has highlighted the capacity of viruses from 64 the RNA virus order Nidovirales (i.e., coronaviruses) to jump species boundaries, occasionally 65 resulting in outbreaks of infectious disease 1. In most cases, such virus host jumps occur 66 between closely related host taxa, such as different mammalian species, in part reflecting 67 conserved virus-cell receptor relationships. SARS-CoV-2 has also been characterized by multi-68 species host jumping , with the virus passing from humans to a diverse array of mammalian 69 species2. As a consequence, understanding the exact nature of host-pathogen interactions, 70 including the barriers to successful cross -species transmission, is of considerable research 71 interest3. Fortunately, o ur knowledge of the host range of many viruses has been greatly 72 enhanced by the increasing use of metagenomic sequencing, which enables the entire 73 viromes of species to rapidly documented. In addition, metagenomic sequencing has 74 advanced diagnostics, enabling the identification and characterization of poorly described 75 animal pathogens. However, despite our expanded knowledge of the virosphere, there has 76 been a general neglect of the Nidovirales aside from the mammalian coronaviruses, including 77 their prevalence, host range and frequency of host jumps, and their capacity to cause disease. 78 Respiratory problems are common in horses and are often diagnosed as a cause of 79 poor athletic performance. However, the basic diagnostic techniques of the equine 80 respiratory tract examination are not always sufficient for a complete diagnosis of the disease, 81 its exacerbation, remission, or response to treatment. Of the different causes that might lead 82 to respiratory system problems, infections are the most common disorders. This is particularly 83 so with racehorses, in which respiratory system infections are often cited as the second most 84 common reason for horses failing to train (Ainsworth and Hackett, 2004). Pathogens of the 85 greatest concern in horses are influenza A viruses (AIV) , Equine herpesvirus 1 and 4 (EHV1, 86 EHV4), Streptococcus zooepidemicus, Streptococcus pneumoniae, Streptococcus equi s ubsp. 87 equi, Rhodococcus equi , and Pasteurella spp .4–6. In addition, h orses are susceptible to a 88 plethora of viruses transmitted by biting arthropods, including mosquitoes, midgets, flies, 89 ticks, as well as parasites7. 90 The Mesoniviridae are a newly assigned family of viruses within the Nidovirales. Unlike 91 other nidoviruses, mesoniviruses are considered insect-specific viruses (ISVs) mainly found in 92 mosquitoes and are not known to infect vertebrate cells. This family contains only one 93 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 5 subfamily, Hexponivirinae, that currently contains a single genus – Alphamesonivirus – and 94 nine subgenera (https://ictv.global/taxonomy). 95 The genome of Alphamesonivirus-1 (Alphamesonivirus cavallyense , subgenus 96 Namcalivirus) contains seven open reading frames (ORFs), with ORF1a and -1b located at the 97 5′ end, encompassing two-thirds of the genome, and the smaller ORF2a, -2b, -3a, -3b, and -4 98 occupying the 3′-proximal end of the genome. The five major 3′-ORFs are predicted to encode 99 a spike (S) glycoprotein (in ORF2a) , a nucleocapsid (N) protein ( in ORF2b), two proteins with 100 membrane-spanning regions (in ORF3a and -3b), and a small protein with unknown function 101 (in ORF4)8,9. 102 Alphamesonivirus-1 still comprises most of the mesoniviruses identified and was 103 initially considered the prototype specie s of the family (https://ictv.global/taxonomy). The 104 family was first represented by two closely related viruses : Cavally virus (CavV), isolated in 105 Ivory Coast and initially named as a first insect -associated nidovirus 8, and Nam Dinh virus 106 (NDiV) isolated in Vietnam 10, with the new family Mesoniviridae proposed the following 107 year11. Mesoniviruses have only been identified from naturally infected mosquitoes 12,13 and 108 hence are considered to be ISVs14,15 in a similar manner to insect-specific flaviviruses16 and 109 mosquito-associated bunyaviruses 17. Indeed, t o date, mesoniviruses have only been 110 associated with invertebrates, with no reports in vertebrates16,18. Of note, a growing number 111 of mesonivirus species have been identified from mosquitos collected in the Americas19, 112 Asia20, Africa 12, and Australia 21, suggesting a near global distribution. Additionally, a 113 mesonivirus was identified from Aphis citricidus aphids collected in China in 201222, while a 114 mesoni-like virus has been detected in an obligate fungal pathogen – Leveillula Taurica – in 115 Italy23. Hence, the host range of mesoniviruses is likely to be far broader than currently 116 known. 117 Here, using a combination of genomic and histological t echniques, we identified 118 Alphamesonivirus-1 in two horses that succumbed to an acute respiratory syndrome . This 119 represents the first detection of a mesonivirus in vertebrates. 120 121 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 6

122 Bronchopneumonia and unspecific-viral infection detected in lungs and lymph nodes of 123 two horses that succumbed to an acute respiratory syndrome 124 The body condition score of the horse carcasses were in the physiological range, with no signs 125 of external trauma observed. The mare was in foal, and the intra-uterus foetus was of normal 126 physiological size and shape. Advanced putrefaction and decomposition of internal digestive 127 organs as late post -mortem changes were apparent. Post-mortem analysis revealed foamy 128 nasal discharge, severe g elatinous subcutaneous oedema of the neck region, and 129 hemorrhagic pleural exudate, while severe pulmonary oedema and thickness of interlobular 130 septa were observed in the lungs of both horses. Splenomegaly, enlargement of bronchial, 131 submandibular, and retropharyngeal lymph nodes (LN), the necrosis of submandibular LN, 132 and petechial hemorrhages of the large intestine mucosa were evident as well (Figure 1A). 133 Histopathology analyses revealed severe alveolar oedema, massive and severe thickness of 134 visceral pleura, areas of bronchopneumonia and hemorrhagic foci in bronchial, 135 submandibular and retropharyngeal lymph nodes (Figure 1B). Both the mare and the foal 136 showed gross and microscopic similar lesions in terms of localization, type , and extension 137 (Figure 1C and 1D). 138 All s amples tested negative for the common agents responsible for respiratory 139 syndrome in horses: EHV1, EHV4, WNV, USUV, EAV, IAV, AHSV, Babesia caballi, Theileria equi, 140 Trichinella spiralis (Table 1 ). Bacterial growth was not observed when the brain , lung and 141 lymph nodes homogenates w ere cultured with standard protocol s. Anaerobic bacterial 142 growth was observed in spleen, kidney, and liver tissue homogenates likely as result of post-143 mortem proliferation. 144 145 Identification and confirmation of Alphamesonivirus-1 in horse samples 146 Metatranscriptomic sequencing produced a total number of 28,231,914 and 26,934,576 raw 147 reads from the foal and mare lung tissue samples, respectively. After quality check s and 148 trimming, the remaining 26,944,930 and 25,958,036 reads were analyzed using the CZID tool: 149 this assigned 14,683 reads (0.73% of all reads , foal) and 746 ( 0.43%, mare) reads to 150 Alphamesonivirus-1 strain pool 11/2008 (GenBank: MF281710.1) identified from a C. pipiens 151 pool in north Ital y in 2008. Similar results were obtained by sequential Blast analyses , with 152 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 7 RPKM values for Alphamesonivirus-1 ranging from 28.47-54.48 for the foal, and 0.96-1.63 for 153 the mare. Hence, the virus was at considerably higher abundance in the foal than the mare. 154 A subsequent reference-based assembly of Alphamesonivirus-1 by iVar (1.3.1) 155 produced two consensus sequences with a horizontal coverage (Hcov) of 99% for the foal 156 sample and 57% for the mare sample. Hcov for the mare sample was improved to 87% after 157 re-mapping of reads using the Alphamesonivirus -1 consensus sequence obtained from the 158 foal sample as reference. Mean vertical coverage (Vcov) was 37.22 (min = 1, max = 554) for 159 the foal sample and 5 (min = 0, max= 65) for the mare sample. Pan-mesonivirus real time RT-160 PCR confirmed the presence of RNA belonging to Alphamesonivirus -1 in both samples and 161 threshold cycles (CT) were 33 and 34 for foal and mare lungs, respectively. 162 In addition to the equine samples, 10 pools of Culex sp. were analyzed with this 163 metatranscriptomic protocol. The taxonomic classification of reads by CZID revealed the 164 presence of reads assigned to Alphamesonivirus-1 species in one pooled sample collected in 165 Sant’Omero municipality (Teramo province, Abruzzo region, a neighboring region of Molise). 166 Deep sequencing of this sample resulted in 30,775,924 raw reads. After quality check s and 167 trimming, the remaining 13,125,482 reads were again analyzed using CZID. As before, the 168 most abundant species was Alphamesonivirus-1 (15.07%, with 1,977,945 reads), followed by 169 arthropod-specific microbial species including Culex-associated Tombus -like virus (8.7%), 170 Wolbachia bacteria (1.53%), and a member of the Negevirus genus of positive-sense RNA 171 viruses (0.23%; see below). 172 In situ hybridization for Alphamesonivirus-1 in both animals demonstrated 173 intracytoplasmic occurrence of viral RNA in macrophages residing the sub-capsular sinus in a 174 bronchial lymph node and in lung alveoli (Figure 1E and F). 175 176 Presence of additional microbial species 177 As noted above, besides Alphamesonivirus-1 a variety of other microbial species were 178 identified in the horse samples following metatranscriptomics analysis. Perhaps of most note 179 was the presence of transcripts\ for Biggie virus (Negevirus, unclassified positive-sense RNA 180 virus) in the foal (RPKM = 1.63), matching its identification in the Culex samples. 181 In addition, almost 2 million reads from the foal lung sample and 250,000 reads from 182 the mare lung samples were assigned to different bacterial species known to be normally non-183 pathogenic or environmental contaminants (Table 1). Reads assigned to the Clostridium genus 184 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 8 represented the highest relative sequence abundance in both the mare (91.09%) and the foal 185 (87.27%). Although the vast majority of clostridial species are non-pathogenic commensal or 186 soil bacteria, Clostridium perfringens and C. botulinum are responsible for severe diseases of 187 horses41. Therefore, mapping analysis was performed using the reference sequence for C. 188 perfringens (Genbank: CP009557.1) and for C. botulinum (CP063816.1) as indicated by the 189 CZID tool. Mapping analysis for both samples failed, resulting in Hcov of 0.01% (foal) and 190 0.04% (mare) for C. perfringens and Hcov of 0.04% (foal) and 0.03% (mare) for C. botulinum. 191 192 Characterization of horse Alphamesonivirus-1 193 The Alphamesonivirus-1 sequences obtained from the foal and mare samples were identical 194 with the exception of ambiguities due to low coverage (resulting in an overall pairwise nt 195 identity of 98.5%). A total of 21 amino acid substitutions were found between the 196 Alphamesonivirus-1 sequences obtained from the horses and from the Culex mosquito pool 197 (Figure 2). Overall, eight non-synonymous mutations were found in ORF1a, and ORF1b each, 198 and six in ORF2a. A phylogenetic tree of Alphamesonivirus-1 was estimated using publicly 199 available complete genome sequences combined with those generated here (Figure 3). This 200 revealed some geographical clustering , with monophyletic groups for virus sequences 201 sampled from South Kor ea, North America, Australia, and Asia. Notably, the sequences 202 generated in this study form ed a well-supported monophyletic group (99% bootstrap 203 support) within a European clade (90% bootstrap support) that includes an Alphamesonivirus-204 1 sequence identified in Italy in 2008 which falls as the sister-group to the horse and Culex 205 pool sequences. Hence, this topological pattern is indicative of the ongoing transmission of 206 Alphamesonivirus-1 in Italy. 207 There was no evidence for molecular clock structure in these data (negative 208 correlation coefficient -0.29, R squared 0.08), suggesting that Alphamesonivirus-1 is evolving 209 relatively slowly over the time course of sampling and precluding more detailed molecular 210 clock analysis. This is in marked contrast to other Nidovirales such as SARS-CoV-1 and SARS-211 CoV-2 that have experienced measurable evolutionary rates of ~10-3 nucleotide substitutions 212 per site, per year during their spread through human populations42. Although some sporadic 213 in silico evidence for limited positive selection across the Alphamesonivirus-1 phylogeny was 214 observed, there was no evidence for any adaptive evolution associated with the Italian viruses 215 (results not shown). 216 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 9 217

218 Respiratory infections are important causes of morbidity and mortality in horses. However, 219 the causative agents are frequently unidentified, often misdiagnosed or overlooked. 220 Infections of the upper respiratory tract, both viral and bacterial, are usually diagnosed in 221 weanling and yearling horses, while airway disorders, pleuropneumonia or epistaxis 222 conditions caused by inflammation or exercise are found in horses older than two years. In 223 contrast, recurrent airway disease or neoplasia of the respiratory tract are diagnosed 224 primarily in the middle -aged and older horses 43. Conditions such as inflammatory airway 225 disease, chronic obstructive pulmonary disease or exercise -induced pulmonary hemorrhage 226 are examples of the pathological conditions affecting respiratory system of horses, whose 227 complex etiology remain uncertain. Nevertheless, viral respiratory infections are the most 228 important causes of respiratory disease in horses worldwide. 229 Mesoniviruses have no known association with human or animal disease. To the best 230 of our knowledge, this is the first report describing Alphamesonivirus-1 infection in a 231 vertebrate. The presence of this virus in two horses located in the same barn was suggested 232 by metatranscriptomic analysis and confirmed by an Alphamesonivirus-1-specific-PCR and in 233 situ hybridisation on infected pulmonary tissues. Phylogenetic analysis revealed that the 234 Alphamesonivirus-1 identified from the mare and the foal from the Molise region of Italy were 235 closely related to a sequence derived from a mosquito pool collected in the neighboring 236 Abruzzo region in 2022. The high genetic similarity between these sequences, and to 237 sequence from a virus identified in north Italy in 2008 , suggests that Alphamesonivirus-1 is 238 continuously circulating in Italy. 239 Microbiological analyses of kidneys, liver, and spleen tested positive for the presence 240 of anaerobic bacteria, yet culture isolation, including the pathogenic Clostridium perfrigens, 241 yielded negative results. Observed anaerobic bacterial growth was, therefore, likely present 242 due to post-mortem changes of decomposition and putrefaction of the internal organs. 243 As no other well-recognized horse pathogens were identified in our analysis , it is 244 tempting to speculate that the Alphamesonivirus-1 infection had a pathogenic role, alone or 245 in association with an undiagnosed pathogen, in the acute respiratory syndromes observed 246 in the horses. However, this will need to be confirmed , and the mechanisms triggering the 247 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 10 infection remain unknown. Similarly, it is uncertain whether the virus was maternally 248 transmitted from mare to foal, or whether these animals were simultaneously infected by 249 local Culex mosquitoes. 250 The presence of the Biggie virus (Negevirus) in the foal and in the Culex mosquito pool 251 is also intriguing. Biggie virus is associated with Culex mosquitoes and was first identified at 252 relatively high abundance in C. pipiens and C. torrentium from Sweden44. Although, in a similar 253 manner to Alphamesonivirus-1, there is no prior evidence for negeviruses in mammalian 254 species, these two viruses were recently found to interact in vitro with several co-infecting 255 arboviruses (Flaviviridae, Togaviridae, Peribunyaviridae) inhibiting USUV and Bunyamwera 256 orthobunyavirus infection45. The co -infection of Alphamesonivirus-1 and Biggie virus 257 therefore merits additional attention. 258 Similarly, we cannot exclude a major contributing role of a cytokine storm following 259 Alphamesonivirus-1 infection for the observed respiratory syndrome, particularly as this is 260 well known in related Nidovirales including feline infectious peritonitis virus and SARS -CoV-261 246–49. COVID-19 pathology was generally characterized as biphasic with an acute phase 262 dominated by active SARS -CoV-2 infection and a post -viral clearance phase dominated by 263 host reparative and immunologic processes 50. Hence, w e cannot exclude macrophage 264 hyperactivation in the two horses since macrophages in the sub-capsular sinus in a bronchial 265 lymph node and in lung alveoli were shown to be positive for Alphamesonivirus-1 by in situ 266 hybridization. This scenario is also described in SARS -CoV-2 infected individuals in which 267 CD169+ macrophages were detected in lymph node subcapsular spaces 51. Macrophages 268 disorders such as secondary hemophagocytic lymphohistiocytosis have been well described 269 in COVID-19 and in other coronavirus infections such as SARS and MERS 52. Hemophagocytic 270 lymphohistiocytosis is a hyperinflammatory syndrome characterized by a fulminant and fatal 271 hypercytokinaemia with multiorgan failure in humans. In adults, this phenomenon is mostly 272 triggered by viral infections, autoimmune diseases, and neoplasms53. 273 The low viral load found in our study indicated by high ct values and low number of 274 Alphamesonivirus-1 reads calls into the question whether this virus was the causative agent 275 of the fatal respiratory disease in the horses. However, similar circumstances were observed 276 when Schmallenberg virus was first detected in blood samples from infected cows54. This virus 277 is transmitted by the female biting midges of the Culicoides obsoletus complex and is 278 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 11 associated with disease ruminants includ ing fever, fetal malformation, drop in milk 279 production, diarrhoea and stillbirths, becoming a burden for small and large farms55. 280 Despite all attempts with different mammalian and mosquito cell lines (VeroE6 and 281 C6/36), we were unable to isolate the virus. As the experiments undertaken in by Diagne et 282 al.12 demonstrated the inability of Alphamesonivirus-1 to replicate in the mosquito C6/ 36 283 cells at 37 °C but at only 28 °C, we performed all isolation attempt (also with mammalian cell 284 lines) at 28 °C. Another limitation of the study is that we were unable to screen a healthy 285 population of horses from the same farm to investigate the presence of Alphamesonivirus-1 286 RNA genome in the rest of the herd. Furthermore, it would have been beneficial to collect 287 mosquitoes from that farm. Due to the lack of specific serological tools for Alphamesonivirus-288 1, the local virus circulation and potential seroconversion in horses could not be addressed. 289 Due to the presentation as an acute respiratory syndrome, the gross lesions described, and 290 the advanced state of putrefaction observed in the carcasses, we initially focused only on the 291 lungs and did not perform the Alphamesonivirus -1 PCR and metatranscriptomics on other 292 organ samples. 293 Overall, the presence of Alphamesonivirus-1 in two horses may provide new insights 294 into the pathogenesis of respiratory disease s of horses, and enhances our understanding of 295 the diversity and evolution of mesoniviruses. The correlation between the pathological 296 condition observed in these horses and the presence of Alphamesonivirus-1 in their lungs 297 represents an important first step in understanding mesonivirus evolution, host range , and 298 its potential to infect and cause disease in other animals than insects . Since the presence 299 and/or replication of Alphamesonivirus-1 in mammalian hosts has not been reported to date, 300 our identification of a supposedly insect-specific virus in a mammalian host clearly 301 necessitates the further in vivo investigation and broader surveillance of this virus. 302 303

304 Sample collection and diagnostic approach 305 In October 2021, an 18 -month-old foal and a 7 -years-old mare Haflinger horse died 306 unexpectedly due to acute respiratory syndrome in the same farm located in Miranda, 307 province of Isernia, Molise region, Italy. The two carcasses were sent to the Istituto 308 Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) for necropsy. Lung and 309 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 12 bronchial lymph nodes were sampled from each animal, fixed in 10% neutral buffered 310 formalin, routinely processed for histology and stained with Haematoxylin and Eosin (HE). 311 Samples from the retropharyngeal, submandibular and bronchial lymph nodes, lungs and 312 spleens were collected and homogenized in a sterile phosphate -buffered saline (PBS), and 313 then centrifuged. Nucleic acid was extracted from 200 µl of supernatants using the MagMAX 314 CORE Nucleic Acid Purification Kit (Applied Biosystems) on an automatic extractor KingFisher 315 Flex (ThermoFisher Scientific), with an elution volume of 100 μl, following the manufacturer’s 316 instructions. The samples were tested by molecular assays for the presence of RNA/DNA of 317 several respiratory viruses, including EHV1 and EHV4 24, West Nile virus (WNV)25, Usutu virus 318 (USUV)26, Alphaarterivirus equid (EAV) (VetMax EAV Kit, Applied Biosystems), IAV27,28, African 319 horse sickness virus (AHSV) 29. Whole blood samples were analyzed for the detection of 320 Babesia caballi and Theileria equi by conventional PCR 30,31, while diaphragmatic muscle was 321 tested for the presence of Trichinella spiralis by means of magnetic stirrer method 32. All 322 samples were also tested by standard procedures for aerobic and anaerobic bacterial 323 isolations. 324 325 Sample preparation, library construction and metatranscriptomic sequencing 326 To assist with pathogen identification, RNA purified from lung samples of both horses was 327 processed for metatranscriptomic analysis. After Turbo DNAse (Thermo Fisher Scientific, 328 Waltham, MA, USA) treatment at 37°C for 20 min, total RNA was purified by an RNA Clean & 329 Concentrator™-5 Kit (Zymo Research, Irvine, CA, USA). The RNA obtained was processed using 330 sequence-independent single -primer amplification protocol (SISPA) with some 331 modifications33. The amplicons were purified by ExpinTM PCR SV (GeneAll Biotechnology CO., 332 LTD Seoul, Korea), and quantified by Qubit dsDNA HS assay (Thermo Fisher Scientific, 333 Waltham, MA, USA). The samples were diluted to obtain a concentration of 100 –500 ng and 334 used for library preparation with the Illumina DNA Prep kit (Illumina Inc., San Diego, CA, USA) 335 according to the manufacturer’s protocol. Deep sequencing was performed on the NextSeq 336 500 (Illumina Inc., San Diego, CA, USA) using the NextSeq 500/550 Mid Output Reagent 337 Cartridge v2, performing 300 cycles and generating 150 bp paired end reads. Raw sequencing 338 reads underwent quality trimming before adapter removal using Trimmomatic v0.38. Quality 339 control of raw and trimmed reads was performed with FASTQC v0.11.8. 340 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 13 Fastq files were initially analyzed using the Chan Zuckerberg ID (CZID) software 341 (https://czid.org/), an open -source software platform that helps identify pathogens in 342 metatranscriptomic sequencing data after host sequence removal. Following indications on 343 microbial composition provided by CZID, fastq data of both horses were mapped by BWA 344 software package (v.0.7.17) 34 to Alphamesonivirus-1 reference accession number 345 NC_015668. Alphamesonivirus-1 consensus sequences were obtained by iVar (v1.3.1). Paired-346 end reads were de novo assembled into contigs using MEGAHIT v1.2.9 with default settings. 347 To confirm these initial observations, the assembled contigs were compared to the NCBI non-348 redundant database (NCBI -nr) using DIAMOND v2.1.6 with an e -value cut -off ≥1E -435. To 349 provide further validation of hits, contigs were screened against the nucleotide database 350 (NCBI-nt) with an e-value cut-off ≥1E-10. Virus abundance was quantified and normalized by 351 contig length using TPM (transcripts per million) and RPKM (reads per kilobase million) 352 metrics as implemented in RSEM v1.3.0. 353 Within genomic surveillance activities performed at IZSAM in 2022 within the National 354 surveillance system of arboviral diseases, 10 pools of Culex sp. were collected in different 355 parts of Teramo province, Italy (in the Abruzzo region, a neighboring region to Molise). These 356 samples also underwent metatranscriptomic analysis using the protocol described above. 357 358 Specific Pan-Mesonivirus real time RT-PCR 359 The presence of Alphamesonivirus-1 was confirmed by real-time RT-PCR assay using primers 360 and probes as described in Diagne et al. (2020). PCR reactions were prepared with 10x GoTaq 361 Probe qPCRMaster Mix (Promega) containing final concentrations of 0.5 mM forward primer, 362 0.5 mM reverse primer, 0.25 mM TaqMan probe, 5 µl of double -strand cDNA obtained after 363 SISPA protocol, and nuclease -free water up to 20 µl reaction volume. Real -time RT -PCR 364 reactions were performed on a QuantStudio 7 Flex Real -Time PCR System (Applied 365 Biosystems) in fast mode with the following settings: initial denaturation at 95°C for 20 sec, 366 followed by 40 cycles of denaturation at 95°C for 1 sec and annealing/extension at 55°C for 367 20 sec. 368 369 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 14 RNA in situ hybridization 370 Lung and bronchial lymph nodes derived from the foal and mare were subjected to an RNA in 371 situ hybridization performed by RNA scope analysis platform (RNAscope 2.5 HD Assay – 372 BROWN kit, Biotechne) following the manufacturer’s instructions. To detect viral RNA, 373 sections were incubated with an ad hoc probe designed and manufactured commercially 374 (ACDbio, Bio -Techne, USA). The probe was designed to detect Alphamesonivirus ORF1a 375 (GenBank: MT096515.1) targeting nt 1084 -2098. The endogenous housekeeping gene 376 Ubiquitin C (UBC) was used as positive control to assess both tissue RNA integrity and assay 377 procedure. Slides were counter -stained with Mayer’s Hematoxylin (Bio -Optica, Italy) and 378 mounted with Eukitt® mounting media (Bio -Optica, Italy) before analyzing on a Zeiss Axio 379 Scope.A1 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). 380 381 Phylogenetic and evolutionary analysis 382 Nucleotide sequences representing the full -genome of Alphamesonivirus -1 were obtained 383 from NCBI (accessed 5 June 2024; n=52) and combined with two reference sequences 384 (NC_015874.1 and NC_015668.1) and the two sequences isolated from both horses and from 385 the mosquito pool. A multiple sequence alignment was performed in Mafft v7.450 using the 386 FFT-NS-i x1000 algorithm (Katoh and Standley, 2013). The alignment was manually inspected 387 in Geneious Prime 2021.1.1 ( https://www.geneious.com) for accuracy. Sequences with 388 extensive genetic diversity were removed. A maximum likelihood (ML) phylogenetic tree was 389 then estimated for the final data set of 43 full -genome sequences using RAxML v 8.2.11 390 (Stamatakis, 2014) implementing a gamma time reversible + Γ model of among suite rate 391 heterogeneity (GTR+ Γ) nucleotide substitution model and 200 bootstrap replicates. 392 To assess the extent of temporal (i.e., clock-like) structure in the data we performed a 393 regression of root-to-tip genetic distance on the ML tree against date (year) of sampling using 394 the TempEST method (Rambaut et al., 2016). The lack of temporal structure (see Results 395 section) precluded additional analyses of evolutionary dynamics. 396 To test for the presence of positive selection (i.e., adaptive evolution), especially on 397 the amino acid substitutions associated with the viruses in the horses, we utili zed the 398 FUBAR36, MEME 37, BUSTED 38 methods in HyPhy/Datamonkey 39,40 that explore various 399 distributions of the numbers of nonsynonymous (d N) and synonymous (d S) substitutions per 400 site. 401 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 15 402 Data availability 403 The two Alphamesonivirus-1 consensus sequences obtained in this study from the Haflinger 404 mare and foal, as well as the sequence from the Culex mosquito pool, were deposited in the 405 NCBI/GenBank database under accession numbers PP961236, PP961235, and PP961237, 406 respectively. Metatranscritptomic data are available on the Sequence Read Archive (SRA) 407 under bioproject number PRJNA1126112. 408 409

410 We acknowledge Dr. Addolorato Ruberto (Istituto Zooprofilattico Sperimentale dell’Abruzzo 411 e del Molise) for horse necropsy. 412 413 Funding 414 This work was funded by the Ministry of Health (Ricerca Corrente 2022 "OneCoV: coronavirus 415 animali emergenti e impatto nella Salute Pubblica " recipient Alessio Lorusso , and Ricerca 416 Corrente 2023 “CARBO: biological characterization and virulence factors of old and emerging 417 arboviruses” recipient Alessio Lorusso). This research was partially supported by EU funding 418 within the NextGenerationEU -MUR PNRR Extended Partnership initiative on Emerging 419 Infectious Diseases (Project no. PE00000007, INF -ACT). The work undertaken in this paper 420 was supported by a National Health & Medical Research Council (NHMRC) grant to E.C.H. 421 (GNT2017197). Mention of trade names or commercial products in this article is solely for the 422 purpose of providing specific information and does not imply recommendation or 423 endorsement by the IZSAM. 424 425 Authorship contributions 426 Lucija Jurisic: Conceptualization, Data curation, Writing – original draft 427 Heidi Auerswald: Writing - review & editing 428 Maurilia Marcacci: Conceptualization, Investigation, Writing – original draft 429 Francesca Di Giallonardo: Formal analysis, Visualization 430 Laureen M. Coetzee: Investigation, Methodology, Formal analysis 431 Valentina Curini: Formal analysis 432 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 16 Daniela Averaimo: Formal analysis 433 Giovanni Di Teodoro: Methodology, Software, Validation, Formal analysis, Investigation, 434 Ayda Susana Ortiz-Baez: Methodology, Software, Validation, Formal analysis, Data curation 435 Cesare Cammà: Writing – review & editing 436 Juergen A. Richt: Conceptualization, Writing – review & editing 437 Edward C. Holmes: Conceptualization, Writing – original draft, Writing – review & editing 438 Alessio Lorusso : Conceptualization, Writing – original draft, Writing – review & editing , 439 Funding acquisition, Supervision, Project administration 440 441 Declaration of competing interest 442 The authors declare that they have no known competing financial interests or personal 443 relationships that could have appeared to influence the work reported in this paper. 444 445 Preprint to bioRxiv 446 All authors agree on posting this article to bioRxiv 447 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 17 Table 1. Pathogen detection in horses (mare and foal) and a Culex mosquito pool* 448 PATHOGEN METHOD HORSE SAMPLES#

MARE FOAL CULEX MOSQUITO POOL& Equine Herpesvirus 1 and 4 (EHV1 & EHV4) EHV1/4-specific real-time RT- PCR Retropharyngeal, submandibular, and bronchial lymph nodes, lungs, spleen Negative Negative Not tested West Nile virus (WNV) WNV-specific real-time RT-PCR Negative Negative Not tested Usutu virus (USUV) USUV-specific real-time RT-PCR Negative Negative Not tested Equine Arteritis virus (EAV) EAV-specific real-time RT-PCR Negative Negative Not tested Influenza A virus (IAV) IAV-specific real-time RT-PCR Negative Negative Not tested African horse sickness virus (AHSV) AHSV-specific real-time RT-PCR Negative Negative Not tested Babesia caballi Babesia caballi conventionel PCR Whole blood Negative Negative Not tested Theileria equi Theileria equi conventionel PCR Whole blood Negative Negative Not tested Trichinella spiralis Magnetic stirrer method Diaphragmatic muscle Negative Negative Not tested Alphamesonivirus-1 Pan-Mesonivirus real-time RT- PCR Lungs Positive (CT=34) Positive (CT=33) Not tested Alphamesonivirus-1 In situ hybridization Lungs Positive Positive Not tested Non-targeted Metatranscriptomic screening Lungs Alphamesonivirus-1, Massilia sp., Bradyrhizobium sp., Bacillus sp., Filimonas sp., Chitinophaga sp., Providencia sp. Alphamesonivirus-1, Biggie virus Bradyrhizobium sp., Panacibacter sp., Filimonas sp., Chitinophaga sp., Escherichia sp. Alphamesonivirus-1, Tombus-like virus, Wolbachia ap., Biggie virus * Results are only shown for the one pool found positive for Alphamesonivirus-1 using a metatranscriptomic approach. 449 # The supernatant of homogenized organ samples or whole blood was used for the horse samples 450 & The supernatant of the homogenized mosquito pool was used for the metatranscriptomic analysis of the Culex pool 451 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 18 FIGURE LEGENDS 452 453 Figure 1. Organ photographs and histological staining of tissues from the horses that 454 succumbed to an acute respiratory syndrome. 455 At gross examination of the lung, the mare showed pleural effusion (A), and on cut section, 456 pulmonary oedema and marked enlargement of interlobular septa (B). Histological analyses 457 (hematoxylin and eosin stain) in the lung demonstrated (C; 50x magnification) thickness of 458 visceral pleura (black asterisk) and diffuse alveolar oedema (white asterisk), and in a bronchial 459 lymph node (D; 100x magnification) sub -cortical multifocal hemorrhages (black arrows). 460 Alphamesonivirus-1 in situ hybridization (400x magnification) detected intracytoplasmic 461 signals visual as brown spots in scattered macrophages residing the sub -capsular sinus in a 462 bronchial lymph node (E) and in lung alveoli (F). 463 464 Figure 2: Amino acid substitutions that distinguish the Alphamesonivirus -1 sequences 465 obtained from the horses and from the mosquito pool. 466 A total of 22 amino acid mutations were identified (red arrows) between the 467 Alphmesonivirus-1 sequence from the Culex mosquito pool collected in 2022 from Abruzzo 468 region of Italy, and the sequence obtained from the horses with acute respiratory syndrome 469 sampled in 2021. A schematic of the Alphmesonivirus-1 genome is shown. 470 471 Figure 3. Phylogenetic analysis of the alphamesoniviruses showing the position of the 472 Italian sequences. 473 Maximum likelihood tree of the full genome sequences of alphamesoniviruses with the 474 sequences generated in this study marked in bold (E. f. caballus = horse sequences; Cx pool = 475 mosquito sequences). The European clade of sequences is also marked. The tree is rooted on 476 the first detected Nam Dinh virus sequence (from 2009) and all horizontal branches are drawn 477 to a scale of nucleotide substitutions per site. Bootstrap support values >70% are shown at 478 main internal nodes. 479 480 481 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint 19

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At gross examination of the lung, the mare showed pleural effusion (A), and on cut section, pulmonary oedema and marked enlargement of interlobular septa (B). Histological analyses (hematoxylin and eosin stain) in the lung demonstrated (C; 50x magnification) thickness of visceral pleura (black asterisk) and diffuse alveolar oedema (white asterisk), and in a bronchial lymph node (D; 100x magnification) sub-cortical multifocal hemorrhages (black arrows). Alphamesonivirus-1 in situ hybridization (400x magnification) detected intracytoplasmic signals visual as brown spots in scattered macrophages residing the sub-capsular sinus in a bronchial lymph node (E) and in lung alveoli (F). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint Figu re 2. Amino acid substitutions that distinguish the Alphamesonivirus-1 sequences obtained from the horses and from the mosquito pool. A total of 22 amino acid mutations were identified (red arrows) between the Alphmesonivirus-1 sequence from the Culex mosquito pool collected in 2022 from Abruzzo region of Italy, and the sequence obtained from the horses with acute respiratory syndrome sampled in 2021. A schematic of the Alphmesonivirus-1 genome is shown. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint Figu re 3. Phylogenetic analysis of the alphamesoniviruses showing the position of the Italian sequences. Maximum likelihood tree of the full genome sequences of alphamesoniviruses with the sequences generated in this study marked in bold (E. f. caballus = horse sequences; Cx pool = mosquito sequences). The European clade of sequences is also marked. The tree is rooted on the first detected Nam Dinh virus sequence (from 2009) and all horizontal branches are drawn to a scale of nucleotide substitutions per site. Bootstrap support values >70% are shown at main internal nodes. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.10.622896doi: bioRxiv preprint