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Lyme disease, caused by spirochetes in the Borrelia burgdorferi sensu lato clade within the Borrelia genus, is transmitted by Ixodes ticks and is currently the most prevalent and rapidly expanding tick-borne disease in Europe and North America. We report complete genome sequences of 47 isolates that encompass all established species in this clade while highlighting the diversity of the widespread human pathogenic species B. burgdorferi . A similar set of plasmids has been maintained throughout Borrelia divergence, indicating that they are a key adaptive feature of this genus. Phylogenetic reconstruction of all sequenced Borrelia genomes revealed the original divergence of Eurasian and North American lineages and subsequent dispersals that introduced B. garinii, B. bavariensis, B. lusitaniae, B. valaisiana, and B. afzelii from East Asia to Europe and B. burgdorferi and B. finlandensis from North America to Europe. Molecular phylogenies of the universally present core replicons (chromosome and cp26 and lp54 plasmids) are highly consistent, revealing a strong clonal structure. Nonetheless, numerous inconsistencies between the genome and gene phylogenies indicate species dispersal, genetic exchanges, and rapid sequence evolution at plasmid-borne loci, including key host-interacting lipoprotein genes. While localized recombination occurs uniformly on the main chromosome at a rate comparable to mutation, lipoprotein-encoding loci are recombination hotspots on the plasmids, suggesting adaptive maintenance of recombinant alleles at loci directly interacting with the host. We conclude that within- and between-species recombination facilitates adaptive sequence evolution of host-interacting lipoprotein loci and contributes to human virulence despite a genome-wide clonal structure of its natural populations., Importance: Lyme disease (also called Lyme borreliosis in Europe), a condition caused by spirochete bacteria of the genus Borrelia , transmitted by hard-bodied Ixodes ticks, is currently the most prevalent and rapidly expanding tick-borne disease in the United States and Europe. Borrelia interspecies and intraspecies genome comparisons of Lyme disease-related bacteria are essential to reconstruct their evolutionary origins, track epidemiological spread, identify molecular mechanisms of human pathogenicity, and design molecular and ecological approaches to disease prevention, diagnosis, and treatment. These Lyme disease-associated bacteria harbor complex genomes that encode many genes that do not have homologs in other organisms and are distributed across multiple linear and circular plasmids. The functional significance of most of the plasmid-borne genes and the multipartite genome organization itself remains unknown. Here we sequenced, assembled, and analyzed whole genomes of 47 Borrelia isolates from around the world, including multiple isolates of the human pathogenic species. Our analysis elucidates the evolutionary origins, historical migration, and sources of genomic variability of these clinically important pathogens. We have developed web-based software tools (BorreliaBase.org) to facilitate dissemination and continued comparative analysis of Borrelia genomes to identify determinants of human pathogenicity., Competing Interests: The authors declare no conflict of interest.

In 2023, a second wave of the global mpox epidemic, which is mainly affecting men who have sex with men (MSM), was observed in some countries. Herein, we benefited from a large viral sequence sampling (76/121; 63%) and vast epidemiological data to characterise the re-emergence and circulation of the Monkeypox virus (MPXV) in Portugal during 2023. We also modelled transmission and forecasted public health scenarios through a compartmental susceptible-exposed-infectious-recovered (SEIR) model. Our results suggest that the 2023 mpox wave in Portugal resulted from limited introduction(s) of MPXV belonging to C.1.1 sublineage, hypothetically from Asia, followed by sustained viral transmission and potential exportation to other countries. We estimated that the contribution of the MSM high sexual activity group to mpox transmission was 120 (95% CrI: 30-3553) times higher than that of the low sexual activity group. However, among the high sexual activity group, vaccinated individuals likely contributed approximately eight times less [0.123 (95% CrI: 0.068-0.208)] than the unvaccinated ones. Vaccination was also linked to potential reduced disease severity, with a Mpox Severity Score of 6.0 in the vaccinated group compared to 7.0 in unvaccinated individuals. Scenario analysis indicated that transmission is highly sensitive to sexual behaviour, projecting that a slight increase in the MSM sub-population with high sexual activity can trigger new mpox waves. This study strongly supports that continued vaccination, targeted awareness among risk groups and routine genomic epidemiology is needed to anticipate and respond to novel MPXV threats (e.g. global dissemination of clade I viruses).

Bovine alpha herpesvirus-5 (BoAHV-5) is related to the development of meningoencephalitis in cattle. Very little is known about the molecular pathways involved in the central nervous system (CNS) damage associated with inflammation during BoHV-5 infection in mice. To better identify the specific immunological pathways triggered by BoAHV-5 infection in mice, we evaluated the mRNA expression of 84 genes involved in innate and adaptive immune responses. We compared gene expression changes in the cerebrum from noninfected and infected mice with BoHV-5 at a 1 × 107 TCID50. Then, we analyzed the association of these genes with neurological signs, neuropathology, and activation of glial cells in response to BoHV-5 infection. Three days after BoAHV-5 infection, increased expression of TNF, IL-2, CXCL10, CXCR3, CCR4, CCL5, IFN-γ, IL-10, IRF7, STAT1, MX1, GATA 3 C3, LIZ2, caspase-1 and IL-1b was found. We also observed the upregulated expression of the CD8a, TBX21 and CD40LG genes and the downregulated expression of the CD4 gene after BoAHV-5 infection. In addition, BoHV-5-infected animals showed higher levels of all the evaluated inflammatory mediators (TNF, IFN-γ and IL-10) on day 3 postinfection. BoAHV-5-infected animals showed neurological changes along with meningoencephalitis, neuropil vacuolation, hemorrhage and reactive gliosis. Astrogliosis and microgliosis, indicated by increased expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), were found throughout the neuropil in infected brains. Moreover, cleaved caspase-3 immunopositive glio-inflammatory cells were visualized around some blood vessels in areas of neuroinflammation in the cerebrum. In agreement on that we found higher cleaved caspase-3 and Iba-1 expression evaluated by western blot analysis in the brains of infected mice compared to control mice. In conclusion, our results revealed., Competing Interests: Declaration of Competing Interest All authors wish to confirm that there are no known conflicts of interest associated with this publication., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population., (© 2023. The Author(s).)

The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6 ). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus., (© 2022. The Author(s).)

Up to 27 May 2022, Portugal has detected 96 confirmed cases of monkeypox. We describe 27 confirmed cases (median age: 33 years (range: 22-51); all males), with an earliest symptom onset date of 29 April. Almost all cases (n = 25) live in the Lisbon and Tagus Valley health region. Most cases were neither part of identified transmission chains, nor linked to travel or had contact with symptomatic persons or with animals, suggesting the possible previously undetected spread of monkeypox.

Background: Increased levels of inflammatory cytokines are associated with severe dengue evolution, but the source of such hypercytokinemia is elusive. We investigated the contribution of innate lymphocytes, innate lymphoid cells (ILCs), and natural killer (NK) cells in cytokine production in early dengue infection., Methods: Peripheral blood mononuclear cells of individuals with dengue without warning signs (DWS-) and dengue with warning signs and severe dengue (SD) presentation combined (DWS+) were obtained between 2 and 7 days since fever onset and submitted to flow cytometry without specific antigen stimulation to evaluate cytokines in ILC and NK cell subpopulations., Results: ILCs and NK cells were found to be important sources of cytokines during dengue. ILCs of the DWS+/SD group displayed higher production of interferon gamma (IFN-γ) and interleukin (IL) 4/IL-13 when compared to DWS- individuals. On the other hand, NK Eomes+ cells of DWS- patients displayed higher IFN-γ production levels compared with the DWS+/SD group. Interestingly, when NK cells were identified by CD56 expression, DWS+/SD displayed higher frequency of IL-17 production compared with the DWS- group., Conclusions: These results indicate that ILCs and NK cells are important sources of inflammatory cytokines during acute dengue infection and display distinct profiles associated with different clinical forms., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL) is a rare headache syndrome included in the Classification of Headache of the International Headache Society as a "headache attributed to non-infectious inflammatory intracranial disease." We report one 15-year-old patient with clinical history and cerebrospinal fluid findings compatible with the diagnosis of HaNDL in whom Borrelia lusitaniae was identified in cerebrospinal fluid by polymerase chain reaction.

Borrelia turdi is a spirochete from the Borrelia burgdorferi complex, first reported in Japan, that has been increasingly detected in Europe. This genospecies is mostly associated with avian hosts and their ornithophilic ticks such as Ixodes frontalis. In this study, we isolated B. turdi from five I. frontalis feeding on Turdus merula, Turdus philomelos, Parus major and Troglodytes troglodytes, and one Ixodes ricinus feeding on a T. merula in Portugal. These isolates were genetically characterised according to their 5S-23S rRNA intergenic spacer, 16S rRNA and through typing of seven housekeeping genes (multilocus sequence typing). Multilocus sequence analyses revealed that the strains isolated in our study, although belonging to B. turdi genospecies, are not identical to the B. turdi reference strain Ya501. Instead, our strains are separated into a clear defined group, suggesting that the European samples diverged genetically from the strain originally detected in Japan. Population analysis of 5S-23S rRNA sequences can further resolve subpopulations within B. turdi, but more samples from a large geographical scale and host range would be needed to assess potential phylogeographical patterns within this genospecies.

We examined the presence of borreliae and rickettsiae bacteria in ticks from wild passerine birds on three islands of the Archipelago of the Azores, the westernmost region of Palearctic. A total of 266 birds belonging to eight species from seven families were examined on São Miguel, Santa Maria and Graciosa islands in 2013. Ticks collected from these birds consisted of 55 Ixodes frontalis (22 larvae, 32 nymphs, 1 adult female) and 16 Haemaphysalis punctata nymphs. Turdus merula and Erithacus rubecula were the birds most infested with both tick species. Three T. merula in Santa Maria were infested with 4 I. frontalis infected with Borrelia turdi. No rickettsiae were found in the ticks. We report for the first time the presence of I. frontalis and B. turdi on the Azores islands and we showed that the spatial distribution reaches further west than previously thought., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Argasid ticks of the Ornithodoros erraticus complex are associated with traditional pig-farming practices on the Iberian Peninsula and are also found elsewhere in North Africa, West Africa, and western Asia. The ticks associated with pig farming on the Iberian Peninsula are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe, and their ecology makes them an extremely effective reservoir of both ASFV and the Borrelia species which cause tick-borne relapsing fever (TBRF) in humans. The recent reappearance of ASFV in the European Union, coupled with evidence that Portuguese tick populations continue to harbor Borrelia despite a lack of confirmed human infections, suggest that these populations merit closer attention. In Portugal, a series of surveys over the last twenty-five years indicates that the number of farm sites with tick infestations has declined and suggest that populations are sensitive to changes in farm management, particularly the use of modern pig housing. Various technologies have been suggested for the control of farm-associated Ornithodoros ticks and related species but, in our opinion, farm management changes are still the most effective strategy for population control. Furthermore, we suggest that this species could probably be eradicated from Iberian pig farms., (© 2014 The Society for Vector Ecology.)

In this study a novel Rickettsia from the spotted fever group, isolated from Ornithodoros erraticus soft ticks collected from pigpens in the south of Portugal, is described. After initial screening revealed Rickettsia-positive ticks, isolation attempts were then performed. Successful isolates were achieved by shell-vial technique using Vero E6 cells at 28°C. Molecular characterization of the isolate was performed based on analysis of five rickettsial genes gltA, ompA, ompB, sca1 and htr with their subsequent concatenation along with other rickettsial species resulting in a clustering of the new isolate with Rickettsia felis and Rickettsia hoogstraalii. The degree of nucleotide sequence similarity with other rickettsiae fulfills the criteria for classification of our isolate as a novel species. The name Rickettsia lusitaniae sp. nov. (=CEVDI PoTiRo) is proposed for this new species found in O. erraticus., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1 year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.

A serological survey was conducted in Macao, China, in 753 individuals, with the objective of looking for antibodies to the mosquito, Aedes albopictus (Skuse 1894) (Diptera: Culicidae), and to dengue, before the occurrence of any autochthonous dengue cases. Blood samples were collected at several public health services, a questionnaire was answered, and enzyme-linked immunosorbant assay (ELISA) and Western blot techniques were performed with extracts of mosquito head and thorax (HT). Anti-Aedes albopictus IgG antibodies were present in titres 1:10(2)-1:10(3) in 9%, and in titres 1:10(4)-1:10(5) in 42% of the sera tested. This reactivity was more frequent (59%) in the population which had resided only in Macao in the 2 years previous to the survey, as opposed to those that had also resided in other areas (50%). From the 230 reactive sera selected for immunoblot, 48 (21%) reacted with a wide range of proteins from above 224 kDa to 21 kDa, with different patterns according to individual sera. Proteins in the intervals 35.3-28.7 kDa and 28.7-21.1 kDa were labelled by the greatest number of sera, 15 and 19 respectively. The presence of anti-Aedes albopictus antibodies presented a statistical relation to skin reaction to mosquito bites, but immunoblot patterns did not. Anti-dengue IgG antibodies were found in 48% of the subjects, with a higher proportion in people who had resided out of Macao, or who were nationals from dengue-endemic neighboring countries. Anti-dengue reactivity was in agreement with anti-mosquito reactivity in half of the population. It would be interesting to see if this proportion has changed since dengue became endemic in Macao in 2001.

In Portugal, recent studies have confirmed the presence of Francisella tularensis in Dermacentor reticulatus. Bacterial endosymbionts with significant homology to F. tularensis have been described in several species of ticks. In this work we identified Francisella-like endosymbionts in D. reticulatus ticks (39%), confirming the presence of these bacteria in Portugal. This finding should be considered in future studies using molecular approaches to detect Francisella prevalence in ticks and environmental samples.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

We report the isolation of Borrelia lusitaniae from a 13-year-old female child presenting with a vasculitis syndrome. The patient was treated with doxycycline, 100 mg bid for 20 days, and is in remission after a follow-up of 2 years. These results should alert clinicians to the fact that B. lusitaniae may be pathogenic in humans, highlighting that patients may be seronegative or present with minimal positive antibody titres and clinical signs that are not specific for Lyme borreliosis. In order to prevent the occurrence of more serious disease manifestations via timely treatment, the analysis by molecular methods may be a useful approach when antibody titres are uninformative.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.