Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Molecular evidence of sporadic Coxiella burnetii excretion in sheep milk, central Portugal.

Coxiella burnetii is the etiologic agent of Q fever, a worldwide zoonosis. Cattle, sheep and goats are considered the main reservoirs of the disease. Transmission to humans occurs mainly through the inhalation of infectious aerosols from milk, faeces, urine, and birth products from infected ruminants. In this study, a 2-year longitudinal approach was performed to ascertain the excretion of C. burnetii in bulk tank milk samples of sheep from a mountain plateau in central Portugal, with sampling conducted during the years 2015 and 2016. From a total of 156 bulk tank milk samples tested by qPCR, only one showed to be positive for C. burnetii (1.28% [95%CI: 0.03-6.94]), from 2015, the first year of collection. Bidirectional sequencing and phylogenetic analysis of IS1111 transposase partial region confirmed the presence of C. burnetii DNA. The presence of C. burnetii in raw milk samples highlights the necessity for additional research to determine if raw milk is a potential source for human infection. Animal health surveillance and prevention measures against this zoonotic disease should be considered.

Presence of Alphacoronavirus in Tree- and Crevice-Dwelling Bats from Portugal.

Coronaviruses (CoVs) are RNA viruses capable of infecting a wide range of hosts, including mammals and birds, and have caused significant epidemics such as the ongoing COVID-19 pandemic. Bats, the second most diverse mammalian order, are hosts for various CoVs due to their unique immune responses and ecological traits. This study investigates CoV prevalence in crevice- and tree-dwelling bats in Portugal, a country with limited prior research on bat CoVs. Using nested RT-PCR and sequencing, we screened 87 stool samples from bats, identifying one sample (1.15%) that was positive for Alphacoronavirus, belonging to Pipistrellus pipistrellus. Phylogenetic analysis revealed close genetic relationships with Alphacoronavirus strains from the same bat species in Europe. The low prevalence suggests habitat-specific differences in viral transmission, with cave-dwelling bats exhibiting higher CoV prevalence due to population density and behaviour. These findings underscore the necessity for sustained surveillance efforts aimed at comprehending CoV dynamics within bat populations, especially concerning the risk of spillover events and viral evolution. Vital to this understanding is the monitoring of bat migration patterns, which serves as a crucial tool for elucidating CoV ecology and epidemiology. Such efforts are essential for ongoing research endeavours aimed at mitigating the potential for future zoonotic disease outbreaks.

Hunting for Answers: Assessing Brucella spp. Seroprevalence and Risks in Red Deer and Wild Boar in Central Portugal.

Between 2016 and 2023, a cross-sectional study was conducted in the central region of Portugal in order to better understand the epidemiology and public health risks resulting from the handling and consumption of game animals infected with Brucella spp. The seroprevalence and risk factors for Brucella spp. seropositivity were evaluated. Antibodies against Brucella spp. were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Results showed that in the 650 serum samples collected from red deer (n = 298) and wild boars (n = 352) in Portugal, 21.7% (n = 141; 95% CI: 18.6-25.1%) tested positive. Wild boar had a significantly higher prevalence (35.5%; 95% CI: 30.5-40.8%) than red deer (5.4%, 95% CI: 3.1-8.6%; p ≤ 0.001). Risk factors for seropositivity were investigated using multivariable logistic regression models. The odds of being seropositive was 8.39 (95% CI: 4.75-14.84; p ≤ 0.001) times higher in wild boar than in red deer. Correlations between sex, age, body condition, and seropositivity could not be observed. The higher seroprevalence in wild boar suggests that this species may primarily contribute to the Brucella spp. ecology in central Portugal.

Seroprevalence Assessment and Risk Factor Analysis of Toxoplasma gondii Infection in Goats from Northeastern Algeria.

T. gondii is the causal agent of toxoplasmosis, a worldwide zoonotic disease relevant in human and veterinary medicine. In Algeria, few reports focused on the presence and circulation of this parasite in the local goat population. The aim of the survey was to evaluate toxoplasmosis seroprevalence and associated risk factors. Sera from 460 goats reared on 72 farms in northeastern Algeria were collected and tested for IgG antibodies to T. gondii by an indirect ELISA. To identify risk factors, a linear regression analysis of the variables was performed. Anti-T. gondii antibodies were found in 94.44% (68/72; 95% CI: 73.34-119.73) of goat farms and in 53.26% (245/460; 95% CI: 46.80-60.36) at the individual level. The multivariable analysis showed that seasonal pasture (OR = 3.804; 95% CI: 3.321-4.358; p = 0.003), presence of water source in pasture area (OR = 4.844; 95% CI: 1.942-7.789; p = 0.0004), use of anthelminthics (OR = 2.640; 95% CI: 1.592-3.146; p = 0.036), number of cats, hygiene, proportion of abortions, number of abortions in the last year, year of sampling, region, and season were the variables significantly associated with T. gondii seropositivity. Abortions in goat herds seem to be related to T. gondii exposure, thus it is crucial to undertake measures and strategies to reduce, control, and prevent toxoplasmosis infection in goats, and thereby in humans, from Algeria.

Detection of Rat Hepatitis E Virus in Pigs, Spain, 2023.

We identified rat hepatitis E virus (HEV) RNA in farmed pigs from Spain. Our results indicate that pigs might be susceptible to rat HEV and could serve as viral intermediaries between rodents and humans. Europe should evaluate the prevalence of rat HEV in farmed pigs to assess the risk to public health.

Molecular detection of Enterocytozoon bieneusi in bats from Portugal.

Enterocytozoon bieneusi is a microsporidia commonly found in the gastrointestinal tract of humans and a wide range of other animals, constituting a major cause of microsporidiosis in humans. Although E. bieneusi has been detected in humans, domestic, and wild animals in Portugal, and its presence in bats has been linked to zoonotic characteristics, its occurrence in bats within the country has not been reported. In this study, we investigated the presence of E. bieneusi in 380 bat fecal samples collected in mainland Portugal through a nested PCR assay targeting the internal transcribed spacer region and the flanking small and large subunits of the ribosomal RNA. Enterocytozoon bieneusi was detected in one bat sample (i.e., 0.26%; Pipistrellus pipistrellus). Additionally, another sample tested positive for Enterocytozoon sp. Phylogenetic analysis of the obtained ITS sequence of E. bieneusi revealed clustering within the potentially zoonotic Group 1. This study represents the first report of E. bieneusi in a bat from Europe. Findings presented here contribute to an enhanced understanding of E. bieneusi epidemiology.

Enterocytozoon bieneusi is the most frequent cause of microsporidiosis in humans. In this study, E. bieneusi, belonging to a potentially zoonotic Group, was detected in 0.26% bat samples from Portugal, highlighting bats' potential role in transmitting this microsporidia to humans and other animals.

Hepatitis E Virus in Individuals Undergoing Heparin Therapy: An Observational Serological and Molecular Study

INTRODUCTION: Heparin is derived from swine and has been suggested as a possible source of HEV. To study the potential risk of HEV infection associated with heparin treatment, two groups of individuals were compared. Sera from heparinized (N=93) and non-heparinized individuals (N=111) were tested for markers of acute HEV infection and anti-HEV IgG seroprevalence. METHOD: An acute HEV case was defined by the presence of anti-HEV IgM and/or HEV RNA. From the 93 heparinized individuals, one was positive for IgM and IgG anti-HEV and two were positive for HEV RNA (for both ORF3 and ORF2), and there were a total of two (2.2%) cases of current or recent HEV infection. From the 111 non-heparinized individuals, three were positive for IgM anti-HEV, one was positive for both IgM and IgG anti-HEV, and none was positive for HEV RNA, and there were a total of three (2.7%) cases of current or recent HEV infection. The difference between HEV cases in the heparinized individuals and the non-heparinized individuals was not statistically significant (2.2% vs. 2.7%; p = 0.799). RESULTS: Concerning IgG anti-HEV, it was detected in 32 individuals from the heparinized group and in 18 from the non-heparinized control group. A statistically significant difference was observed in the presence of anti-HEV IgG in heparinized individuals and controls (p = 0.003). CONCLUSION: This study has not found any association between heparin treatment and acute HEV infection, but has shown the use of therapeutic heparin as a risk factor for IgG anti-HEV seropositivity.

A Thorny Tale of Parasites: Screening for Enteric Protozoan Parasites in Hedgehogs from Portugal.

Enteric protozoan parasites, such as Blastocystis sp., Balantioides coli, Cryptosporidium spp., and Giardia duodenalis, may have implications for both animal and human health.Transmitted through the fecal-oral route, these parasites cause symptoms such as diarrhea, abdominal pain, and weight loss. This study investigated the presence of these enteric protozoan parasites and genetically characterized them in hedgehogs from Portugal. A total of 110 hedgehog stool samples were collected. Molecular detection methods showed an overall occurrence of protozoa in 1.82% (2/110 95% CI: 0.22-6.41) of hedgehogs, with Blastocystis being found in one hedgehog and Cryptosporidium being found in another. No evidence for the presence of B. coli or G. duodenalis was found. This study suggests that there is a need to stay aware of hedgehogs as potential hosts of enteric protozoa. Ongoing research and surveillance efforts are recommended to explore practical prevention and control strategies. The results contribute to the limited knowledge of these parasites in Portuguese hedgehog populations and underscore their potential relevance to both veterinary and public health.

Detection of hepatitis E virus genotype 3 in an Algerian mouse (Mus spretus) in Portugal.

Virus monitoring in small mammals is central to the design of epidemiological control strategies for rodent-borne zoonotic viruses. Synanthropic small mammals are versatile and may be potential carriers of several microbial agents. In the present work, a total of 330 fecal samples of small mammals were collected at two sites in the North of Portugal and screened for zoonotic hepatitis E virus (HEV, species Paslahepevirus balayani). Synanthropic small mammal samples (n = 40) were collected in a city park of Porto and belonged to the species Algerian mouse (Mus spretus) (n = 26) and to the greater white-toothed shrew (Crocidura russula) (n = 14). Furthermore, additional samples were collected in the Northeast region of Portugal and included Algerian mouse (n = 48), greater white-toothed shrew (n = 47), wood mouse (Apodemus sylvaticus) (n = 43), southwestern water vole (Arvicola sapidus) (n = 52), Cabrera's vole (Microtus cabrerae) (n = 49) and Lusitanian pine vole (Microtus lusitanicus) (n = 51). A nested RT-PCR targeting a part of open reading frame (ORF) 2 region of the HEV genome was used followed by sequencing and phylogenetic analysis. HEV RNA was detected in one fecal sample (0.3%; 95% confidence interval, CI: 0.01-1.68) from a synanthropic Algerian mouse that was genotyped as HEV-3, subgenotype 3e. This is the first study reporting the detection of HEV-3 in a synanthropic rodent, the Algerian mouse. The identified HEV isolate is probably the outcome of either a spill-over infection from domestic pigs or wild boars, or the result of passive viral transit through the intestinal tract. This finding reinforces the importance in the surveillance of novel potential hosts for HEV with a particular emphasis on synanthropic animals.

Airborne SARS-CoV-2 is more frequently detected in environments related to children and elderly but likely non-infectious, Norway, 2022.

This study investigates the presence of SARS-CoV-2 in indoor and outdoor environments in two cities in Norway between April and May 2022. With the lifting of COVID-19 restrictions in the country and a focus on vaccination, this research aims to shed light on the potential for virus transmission in various settings. Air sampling was conducted in healthcare and non-healthcare facilities, covering locations frequented by individuals across different age groups. The study found that out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no viable virus detected after RNAse pre-treatment. These positive samples were primarily associated with environments involving children and the elderly. Notably, sequencing revealed mutations associated with increased infectivity in one of the samples. The results highlight the importance of considering children as potential sources of virus transmission, especially in settings with prolonged indoor exposure. As vaccination coverage increases globally, and with children still representing a substantial unvaccinated population, the study emphasizes the need to re-implement mask-wearing mandates indoors and in public transport to reduce virus transmission. The findings have implications for public health strategies to control COVID-19, particularly in the face of new variants and the potential for increased transmission during the autumn and winter seasons.

Occurrence and Phylogenetic Analysis of Zoonotic Enteropathogenic Protist Parasites in Asymptomatic Domestic Ruminants from Portugal.

Enteropathogenic parasites are of significant concern for public health due to their zoonotic potential and their impact on human and animal health. In this study, we investigated their occurrence and characterized these enteropathogens in asymptomatic domestic ruminants from Portugal. A total of 302 stool samples were collected from cattle (n = 166), sheep (n = 73), and goats (n = 63) in various regions of Portugal and tested for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp., and Balantioides coli by PCR. The occurrence of Cryptosporidium spp. was found to be 12.7% (8/63, 95% confidence interval [CI]: 5.65-23.5) in goats; however, no sample was found to be positive for Cryptosporidium spp. in cattle and sheep. For E. bieneusi, 6.35% (4/63; 95%CI: 1.76-15.47) of goats were found to be positive; however, no cattle or sheep were found to be positive. Blastocystis sp. was found in sheep (9.59%; 7/73; 95% [CI]: 0.394-18.76) and goats (12.70%; 8/63; 95% [CI]: 5.65-23.50) but none was found in cattle. No positive results for G. duodenalis or B. coli were detected in this study. This study provides essential baseline information for understanding the silent shedding and epidemiology of these enteropathogens in Portugal, contributing to overall livestock health and related occupational safety. Raising awareness among consumers, veterinarians, and farm owners is crucial to minimize the risk of transmission and promote effective disease control strategies.

Exploring the Fatty Acid Binding Pocket in the SARS-CoV-2 Spike Protein - Confirmed and Potential Ligands.

Severe Acute Respiratory syndrome 2 (SARS-CoV-2) is a respiratory virus responsible for coronavirus disease 19 (COVID-19) and the still ongoing and unprecedented global pandemic. The key viral protein for cell infection is the spike glycoprotein, a surface-exposed fusion protein that both recognizes and mediates entry into host cells. Within the spike glycoprotein, a fatty acid binding pocket (FABP) was confirmed, with the crystallization of linoleic acid (LA) occupying a well-defined site. Importantly, when the pocket is occupied by a fatty acid, an inactive conformation is stabilized, and cell recognition is hindered. In this review, we discuss ligands reported so far for this site, correlating their activity predicted through in silico studies with antispike experimental activity, assessed by either binding assays or cell-infection assays. LA was the first confirmed ligand, cocrystallized in a cryo-EM structure of the spike protein, resulting in increased stability of the inactive conformation of the spike protein. The next identified ligand, lifitegrast, was also experimentally confirmed as a ligand with antiviral activity, suggesting the potential for diverse chemical scaffolds to bind this site. Finally, SPC-14 was also confirmed as a ligand, although no inhibition assays were performed. In this review, we identified 20 studies describing small-molecule compounds predicted to bind the pocket in in silico studies and with confirmed binding or in vitro activity, either inhibitory activity against the spike-ACE2 interaction or antiviral activity in cell-based assays. When considering all ligands confirmed with in vitro assays, a good overall occupation of the pocket should be complemented with the ability to make direct interactions, both hydrophilic and hydrophobic, with key amino acid residues defining the pocket surface. Among the active compounds, long flexible carbon chains are recurrent, with retinoids capable of binding the FABP, although bulkier systems are also capable of affecting viral fitness. Compounds able to bind this site with high affinity have the potential to stabilize the inactive conformation of the SARS-CoV-2 spike protein and therefore reduce the virus's ability to infect new cells. Since this pocket is conserved in highly pathogenic human coronaviruses, including MERS-CoV and SARS-CoV, this effect could be exploited for the development of new antiviral agents, with broad-spectrum anticoronavirus activity.

Prevalence and Risk Factors for Hepatitis E Virus in Wild Boar and Red Deer in Portugal.

Hepatitis E virus (HEV) is a zoonotic foodborne virus with an annual infection prevalence of 20 million human cases, which seriously affects public health and economic development in both developed and developing countries. To better understand the epidemiology of HEV in Central Portugal, a cross-sectional study was conducted from 2016 to 2023 with sera samples from wild ungulates. The seroprevalence and risk factors for HEV seropositivity were evaluated in the present study. Specifically, antibodies against HEV were determined by a commercial enzyme-linked immune-sorbent assay (ELISA). Our results show that in the 650 sera samples collected from 298 wild red deer and 352 wild boars in Portugal, 9.1% red deer and 1.7% wild boar were positive for antibodies to HEV. Regarding age, the seropositivity in juvenile wild ungulates was 1.3%, whereas it was 7.2% in adults. Logistic regression models investigated risk factors for seropositivity. The odds of being seropositive was 3.6 times higher in adults than in juveniles, and the risk was 4.2 times higher in red deer than in wild boar. Both wild ungulate species were exposed to HEV. The higher seroprevalence in red deer suggests that this species may make a major contribution to the ecology of HEV in Central Portugal. Further research is needed to understand how wildlife affects the epidemiology of HEV infections in Portugal.

Antibody-Based Assessment of Coxiella burnetii Circulation in Algerian Goat Herds.

Q fever is a zoonotic disease caused by Coxiella burnetii (C. burnetii), a pathogen with a high capability for infection. The disease primarily affects ruminants, leading to reproductive disorders, but can also be transmitted to humans through contact with infected animals or their products. In Algeria, Q fever is endemic, but little is known about the presence and circulation of C. burnetii in domestic goats. This study aimed to perform a multicentric serological analysis of C. burnetii antibodies in domestic goats from four provinces in the North East Region of Algeria. A total of 504 goat serum samples were collected from 77 herds, and serological analysis was performed using an indirect ELISA. The overall seroprevalence at the herd level was 35.06%, and 8.73% at the individual level. Herds with a history of abortions showed a high seropositivity rate of 88.9%. This research indicates the wide distribution of C. burnetii in goats in this region, suggesting the potential for zoonotic transmission to humans. Further studies and monitoring programs are essential to gain a comprehensive understanding of C. burnetii epidemiology in Algeria and to prevent or mitigate potential outbreaks. Awareness among practitioners and farmers is crucial to address this public health concern effectively.

Hepatitis E Virus in the Wild Boar Population: What Is the Real Zoonotic Risk in Portugal?

Hepatitis E virus (HEV) is an important zoonosis in wild boar. Reported zoonotic cases are mainly associated with the consumption of raw/undercooked meat and/or liver. This study aims to determine the occurrence of HEV in the Portuguese wild boar population. During the hunting season 2021/2022, 123-matched samples (liver, faeces, and blood) were collected from hunted wild boars throughout Portugal. An RT-PCR assay tested liver and faeces samples to detect HEV-RNA. From blood samples, an ELISA test was performed. Only one liver sample was positive for HEV (0,8%) and one other from faeces. A total of 34 sera were seropositive (26.7%). At the same time, in a survey of 106 hunters, 21 consumed/ate the liver of wild boars (19.8%). Only three recognised the possibility of consuming it undercooked. Contrary to previous studies in Portugal, the prevalence of HEV in liver and faeces is low, but the seropositivity is higher. But, when analyzing in detail, it could be observed that an HEV hotspot exists in the southeast of central Portugal and that it is a zoonotic risk for hunters of this region. The data of this study reinforce the importance of including HEV in surveillance programs for wildlife diseases to expand the potential zoonotic information.

Atrial fibrillation cryoablation is an effective day case treatment: the UK PolarX vs. Arctic Front Advance experience.

AIMS: Pulmonary vein isolation (PVI) is the cornerstone of catheter ablation for atrial fibrillation (AF). There are limited data on the PolarX Cryoballoon. The study aimed to establish the safety, efficacy, and feasibility of same day discharge for Cryoballoon PVI. METHODS AND RESULTS: Multi-centre study across 12 centres. Procedural metrics, safety profile, and procedural efficacy of the PolarX Cryoballoon with the Arctic Front Advance (AFA) Cryoballoon were compared in a cohort large enough to provide definitive comparative data. A total of 1688 patients underwent PVI with cryoablation (50% PolarX and 50% AFA). Successful PVI was achieved with 1677 (99.3%) patients with 97.2% (n = 1641) performed as day case procedures with a complication rate of <1%. Safety, procedural metrics, and efficacy of the PolarX Cryoballoon were comparable with the AFA cohort. The PolarX Cryoballoon demonstrated a nadir temperature of -54.6 ± 7.6°C, temperature at 30 s of -38.6 ± 7.2°C, time to -40°C of 34.1 ± 13.7 s, and time to isolation of 49.8 ± 33.2 s. Independent predictors for achieving PVI included time to reach -40°C [odds ratio (OR) 1.34; P < 0.001] and nadir temperature (OR 1.24; P < 0.001) with an optimal cut-off of ≤34 s [area under the curve (AUC) 0.73; P < 0.001] and nadir temperature of ≤-54.0°C (AUC 0.71; P < 0.001), respectively. CONCLUSIONS: This large-scale UK multi-centre study has shown that Cryoballoon PVI is a safe, effective day case procedure. PVI using the PolarX Cryoballoon was similarly safe and effective as the AFA Cryoballoon. The cryoablation metrics achieved with the PolarX Cryoballoon were different to that reported with the AFA Cryoballoon. Modified cryoablation targets are required when utilizing the PolarX Cryoballoon.

Diversity of Rickettsia spp. in ticks from wild mammals of Morocco and Mauritania.

Ticks are known as vectors and reservoirs of rickettsiae and, wildlife vertebrate hosts as suitable dispersers of ticks contributing to the life cycle of rickettsial agents in nature. In the herein study, the presence of rickettsiae was investigated in ticks from wild mammals (Gerbillus and Jaculus, Vulpes rueppellii, Canis anthus, Felis lybica and Felis margarita) in Mauritania and Morocco. Morphological and molecular analysis of ticks allowed their identification as Rhipicephalus sanguineus sensu lato and Hyalomma impeltatum. A total of 126 partially engorged adult ticks, collected from 40 animals, were screened for the presence of rickettsial DNA by conventional PCR targeting the ompB gene, followed by ompA and gltA targets and bidirectional sequencing. As a result of the sequence analyses, that at least three different species of pathogenic spotted fever group rickettsiae were detected. Rickettsia parkeri-like was detected in a R. sanguineus s.l. (n=1) collected from an African wildcat from Morocco. Rickettsia aeschlimannii was detected in a H. impeltatum (n=1) collected from a gerbil rodent. Rickettsia massiliae was detected in R. sanguineus s.l. ticks (n=5) collected from two Ruppells' foxes. The herein study demonstrates that pathogenic Rickettsia species are circulating in Morocco and Mauritania wildlife.

From Spike to Symptom: A Contribution to Decoding the Molecular Mechanisms Underlying SARS-CoV-2 Transmission, Infection, and Disease Pathology.

In December 2019, a novel coronavirus, SARS-CoV-2, was identified in Wuhan, China, from patients with severe pneumonia of unknown origin [...].

First Report of Alphacoronavirus Circulating in Cavernicolous Bats from Portugal.

The emergence of novel coronaviruses (CoVs) has emphasized the need to understand their diversity and distribution in animal populations. Bats have been identified as crucial reservoirs for CoVs, and they are found in various bat species worldwide. In this study, we investigated the presence of CoVs of four cavernicolous bats in six locations in the centre and south of Portugal. We collected faeces, anal, and buccal swab samples, as well as air samples from the locations using a Coriolis air sampler. Our results indicate that CoVs were more readily detected in faecal samples compared to anal and buccal swab samples. No CoVs were detected in the air samples. Phylogenetic analysis showed that the detected viruses belong to the Alphacoronavirus genus. This study represents the first report of Alphacoronaviruses circulating in bats in Portugal and highlights the importance of continuous surveillance for novel CoVs in bat populations globally. Ongoing surveillance for CoVs in bat populations is essential as they are a vital source of these viruses. It is crucial to understand the ecological relationships between animals, humans, and the environment to prevent and control the emergence and transmission of infectious diseases. Further ecological studies are needed to investigate the factors contributing to the emergence and transmission of zoonotic viruses.