First detection of Hedgehog coronavirus 1 in Poland

Hedgehogs are common in the majority of European countries and are known to host various pathogens, including viruses. The recent discovery of MERS-related coronaviruses (CoVs) in hedgehogs from Germany, France, the UK, China, and Italy suggests that hedgehogs may represent a wild reservoir of betacoronaviruses. This study reports the first detection and characterization of novel betacoronovirus, subgenus Merbecovirus in wild hedgehogs in Poland. The CoV RNA was detected in 10 out of 40 hedgehogs’ rectal swabs and in 1 out of 18 samples of the lung. No viral RNA was identified in the duodenum and kidney. There was no significant relationship between clinical status, gender, hedgehogs' age, and coronaviral RNA detection. Phylogenetic analysis showed that CoVs detected in our study grouped together with other representatives of Hedgehog coronavirus 1 species identified in Western Europe. Our findings provide further evidence that hedgehogs are a natural reservoir of Merbecovirus. Considering the high mutation rate of CoVs and their potential for crossing interspecies barriers, the proper management of hedgehogs admitted to wildlife rehabilitation centres is needed. It cannot be excluded that merbecovirus strains detected in hedgehogs may recombine with other CoVs leading to new viruses with potential for interspecies transmission.

www.nature.com/scientificreports/ significant genetic variation 22 . The epidemiological, biological, and virological characteristics of CoVs, mainly based on Spike-protein plasticity, suggest species barriers to infection may be easily crossed 24,25 . Thus, CoVs identified in hedgehogs may pose a potential risk to humans, especially as there is increasing contact between hedgehogs and humans. This study aimed to assess the presence of coronaviruses in hedgehogs in an urban area in Poland.

Results
Twenty-six juvenile (65%) and 14 (35%) adult animals were included in the study. 67.5% were females (27/40), while 32.5% were males (13/40). The mean body weight of juvenile animals was 264.42 ± 104.81 (range 83-467 g), while in the adult group, it reaches 740.07 ± 200.12 (490-1150 g). The presence of coronavirus (CoV) RNA was detected in 10 out of 40 hedgehogs' rectal swabs (25%). Additionally, when examining the organs of animals, coronaviral RNA was detected in 1 out of 18 samples of the lung (5.55%), and it was from a hedgehog previously identified as swab-positive. No viral RNA was identified in the duodenum and kidney collected from hedgehogs. In investigated group, the CoV presence was confirmed in 25% of samples (95% CI: 14.2-40.2). Four adult and two young hedgehogs were found to be underweight; the weight of the other animals was normal. One of ten CoV RNA positive hedgehogs was diagnosed with underweight, but the other animals were in normal condition. An equal number (n = 20) of clinically healthy and sick animals were used in this study (detailed characteristics are presented in Table 2). Most animals (70%) in which the CoV was detected were clinically healthy. The remaining positive hedgehogs were diagnosed with fractures (2/3) and severe ectoparasite infestation (1/3). Eighty % of positive hedgehogs were juveniles, while only 20% were adults, noting that the number of juveniles in our study was almost double that of adults. Females represented 90% (9/10) of positive animals. There was no significant relationship between clinical status, gender, age, and detection of hedgehog coronaviruses ( Table 1). The odds ratio calculated for gender indicates a 6 times higher (95% CI 0.671-53.685) likelihood of having positive results in females compared to the males. After controlling for age, the odds of being bCoV RNA positive was 2.67 times higher in juvenile animals (95% CI 0.481-14.789).
Phylogenetic analysis. Topology of the phylogenetic tree based on the obtained sequence fragments of the RdRp gene revealed that ten of the positive hedgehogs resulted in being infected with merbecoviruses ( Fig. 1A). Phylogenetic analysis showed that CoVs from Polish hedgehogs grouped together with other representatives of Hedgehog coronavirus 1 (HedCoV1), species identified in Western Europe. All 10 Polish hedgehog CoV strains were grouped within the same subgroup (Fig. 1B). The alignment of sequences of detected CoV strains showed identity between 98.1 and 100% and possessed the highest similarity (96.3-97.0%) with the viruses from German hedgehogs of subgroup 1 (GenBank numbers KC545384, KC545386). They also revealed high nt sequence similarity with Italian (95.4-96.8%) and German strains of subgroup 2 (93.5-95.0%) (MW246795-802, MT024741, and NC039207, KC545385, respectively). The nt homology of Polish hedgehog CoVs to strains sampled in Great Britain (MK679660) and China (MT002834-35, MK907286-87) were 92.8-93.3% and 83.5-84.7%, respectively.

Discussion
Hedgehogs have been indicated as a possible wild reservoir of emerging CoVs with potential public health implications 14 . This study confirmed the presence of betacoronavirus (bCoV), subgenus Merbecovirus in hedgehogs in Poland for the first time. Our preliminary results suggest that CoVs are quite common in hedgehogs, while 25% of animals tested in our study were CoVs positive (95% CI: 14.2-40.2). The CoV RNA has also been confirmed in 1 out of 18 samples of the lung. No viral RNA was identified in other samples tested (duodenum and kidney). The presence of bCoVs in hedgehogs has been reported previously in other European countries: France 15 , Germany 14 , Italy 2 , Great Britain 16 , but their prevalence were generally higher than in our study (Germany 58.9%, France 50%, Italy 58%). In one study from Great Britain, the detected prevalence was lower (10.8%). However, it should be remembered that the presence of different viruses, including CoVs, may depend on many factors, such as sampling season, sample handling, and most of all, the detection method used 26 . In hedgehogs, additional variability in their prevalence may be caused by the fact that they hibernate, becoming inactive for  www.nature.com/scientificreports/ a few months. After this period, their metabolism does not immediately reach the normative one. It could be expected that viruses in such hedgehog state replicate slowly and are not shed/shed in lower quantity than in the active state. However, the relatively high CoV RNA occurrence in hedgehogs included in our research may suggest that they play an important role as a wild reservoir of CoVs.
In accordance with previous reports, we did not observe any correlation between CoVs positivity and the health condition of animals 2,14,16 . CoVs positive hedgehogs did not reveal the clinical signs of CoV infection. One of ten CoV RNA positive hedgehogs was diagnosed with underweight, but the other animals were in good condition. Coronaviridae family appear to include viruses, a few of which cause disease. The number of detected microorganisms that are not correlated with any disease has increased significantly with the widespread use of molecular diagnostic tools. This also applies to coronaviruses detected in, i.e. bats or wild birds 27,28 . On the other www.nature.com/scientificreports/ hand, they could be opportunistic pathogens, the virulence of which is revealed under unfavourable conditions or in co-infections with other pathogens. In the present study, there was also no significant relationship between gender and age and detection of hedgehog CoVs, but juveniles and females had a higher likelihood of having positive results compared to the adult and males. It could be related to observing a much stronger amplification of CoV RNA observed in juvenile and lactating female bats of the species Myotis sp. 29 . In addition, juveniles are susceptible to all infections and only develop immunity over the years due to contact with different infectious agents. The CoVs detected in hedgehogs in our study were highly homologous to each other and similar to others from Europe in the compared fragment of the genome (93.5 to 97.0% homology). They differed most from the CoVs detected in Erinaceus amurensis in China (83.5 to 84.7% homology). It is unknown if these differences in the studied genome fragment sequence result from geographical segregation or reflect "species-dependent" grouping. However, it should be noted that in our study, hedgehogs came from a relatively small area. On the other hand, when the same genome fragment was compared, a "species-dependent" grouping of gamma-and deltacoronaviruses identified in wild birds was observed 30 . The progenitors of the three coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS-CoV), which caused the most important epidemics in humans, seem to originate from bats 22,24 . During passaging in different intermediate organisms, these progenitors evolving through mutation and/or recombination gained genetic features that enabled infection and transmission between humans 21,22 . Perfect conditions for the emergence of viruses with new characteristics are during co-infection with two or more viruses when homologous recombination occurs 18,20 . Such emerging viruses could acquire the possibility of host switch, more efficacious replication, and/or more virulent. That is why it is important to monitor the prevalence of CoVs in animals already known as hosts for CoVs (e.g., bats, hedgehogs) and other yet unexplored. Such studies have recently identified novel sarbecovirus in a British horseshoe bat, confirming that these viruses are distributed not only in Asian rhinolophids and revealed that the opportunities for emerging zoonoses are persistent and globally threatening 31 . As bats and hedgehogs are phylogenetically closely related and both are insectivorous animals 17,32 , future studies on the circulation of CoVs in these species may shed light on the diversity and evolution of merbecoviruses.
Considering all the above aspects, the proper management of hedgehogs admitted to wildlife rehabilitation centres, especially multi-species, seems extremely important. At present, it cannot be excluded that Merbecovirus strains detected in hedgehogs may recombine with other CoVs (e.g., from bats or other animals kept in such places), leading to new viruses with potential for interspecies transmission (e.g., from bats to humans). It should also be remembered that these animals usually return to their original areas of distribution after a period of rehabilitation/treatment or rearing, potentially spreading these possibly newly emerged viruses into the environment.
Future studies involving the higher number of hedgehogs from various regions of Poland and other wildlife species are planned to verify the results presented here. It also seems important to obtain whole-genome sequences of the identified merbecoviruses, and such attempts are currently underway. We followed the ARRIVE guidelines during the study.

Materials and methods
Rectal swabs were collected from 40 hedgehogs brought to the Wildlife Rehabilitation Centre (WRC) in Poznan after being found sick, injured, or too young to survive on their own for various diagnostic purposes. All hedgehogs were found on the urban area of the city of Poznan (16° 55′ E; 52° 25′ N), Wielkopolskie Voivodeship, Poland. When admitted to the WRC, hedgehogs were kept in isolation until sample collection (within 12 h of admission) to reduce the possibility of nosocomial infections. Between August 2020 and December 2020, the 40 individual rectal swabs were collected using swabs with transport medium (UTM: Viral Transport, COPAN Diagnostics, USA) and stored at − 80 °C until laboratory examinations. In addition, samples of duodenum, lung, and kidney from 18 hedgehogs that were dead at admission died or were euthanised (with the use of xylazine and ketamine administered intramuscularly followed by intravenous pentobarbital administration) for ethical reasons (spine, bone fractures) during their stay at WRC, were collected and stored at − 80 °C until analyses. The following information for each hedgehog was recorded: delivery date, gender, body weight, age, health status. Detailed characteristic of animals used in the study is presented in Table 2.

Molecular detection of coronavirus RNA.
Hedgehogs' tissues were homogenised in a sterile phosphatebuffered saline (Biomed, Lublin, Poland), obtained suspensions (10% w/v) were clarified by centrifugation (15 min at 3000×g and 4 °C and the supernatant used for RNA isolation. All swab transport media were also centrifuged before the process of RNA isolation. The RNA from 200 ul of the obtained fluids was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The samples were tested for CoV presence using an RT-PCR assay in a nested configuration using primers with a few degenerate nucleotides 33 . The assay amplifies a 555-nucleotide gene fragment of the non-structural protein 12 RNA dependent RNA polymerase (nsp12 RdRp), one of the components of viral replicase machinery present in all coronaviruses of mammalian and avian origin. All reactions were performed in a final volume of 25 µl. For RNA transcription and in the first step of DNA amplification, the One-Step RT-PCR kit (Qiagen, Hilden, Germany) was used. The Platinum Taq DNA Polymerase kit (Invitrogen, Carlsbad, USA) and the PCR product in dilution of 1:5 (v/v) were used in the second step. The vaccine IBV 4/91 strain (Nobilis IB 4/91, MSD Animal Health, USA) served as a positive control. The PCR-positive samples were identified using agarose gel electrophoresis.

Data availability
The viral sequences obtained in this study were deposited at NCBI Genbank, accession numbers: MZ605015-MZ605024. Other relevant data analysed during this study are included in this manuscript.