Infective SARS-CoV-2 in Skull Sawdust at Autopsy, Finland.

We assessed the distribution of SARS-CoV-2 at autopsy in 22 deceased persons with confirmed COVID-19. SARS-CoV-2 was found by PCR (2/22, 9.1%) and by culture (1/22, 4.5%) in skull sawdust, suggesting that live virus is present in tissues postmortem, including bone. Occupational exposure risk is low with appropriate personal protective equipment.

Complete coding sequence of an Aedes flavivirus strain isolated from Aedes albopictus collected in Northern Italy.

Complete genome data for the globally distributed Aedes flavivirus (AEFV) is scarce. We analyzed a new Italian AEFV strain isolated from Aedes albopictus. The results demonstrated genetic diversity among Italian AEFVs. The high similarity between AEFV genomes across geographically distant regions suggests long distance spreading via invasive host mosquito species.

Evolution and genetic characterization of Seoul virus in wild rats Rattus norvegicus from an urban park in Lyon, France 2020-2022.

BACKGROUND: Seoul virus (SEOV) is an orthohantavirus primarily carried by rats. In humans, it may cause hemorrhagic fever with renal syndrome (HFRS). Its incidence is likely underestimated and given the expansion of urban areas, a better knowledge of SEOV circulation in rat populations is called for. Beyond the need to improve human case detection, we need to deepen our comprehension of the ecological, epidemiological, and evolutionary processes involved in the transmission of SEOV. METHODOLOGY / PRINCIPAL FINDINGS: We performed a comprehensive serological and molecular characterization of SEOV in Rattus norvegicus in a popular urban park within a large city (Lyon, France) to provide essential information to design surveillance strategies regarding SEOV. We sampled rats within the urban park of 'La Tête d'Or' in Lyon city from 2020 to 2022. We combined rat population genetics, immunofluorescence assays, SEOV high-throughput sequencing (S, M, and L segments), and phylogenetic analyses. We found low structuring of wild rat populations within Lyon city. Only one sampling site within the park (building created in 2021) showed high genetic differentiation and deserves further attention. We confirmed the circulation of SEOV in rats from the park with high seroprevalence (17.2%) and high genetic similarity with the strain previously described in 2011 in Lyon city. CONCLUSION/SIGNIFICANCE: This study confirms the continuous circulation of SEOV in a popular urban park where the risk for SEOV transmission to humans is present. Implementing a surveillance of this virus could provide an efficient early warning system and help prepare risk-based interventions. As we reveal high gene flow between rat populations from the park and the rest of the city, we advocate for SEOV surveillance to be conducted at the scale of the entire city.

Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants may have different characteristics, e.g., in transmission, mortality, and the effectiveness of vaccines, indicating the importance of variant detection at the population level. Wastewater-based surveillance of SARS-CoV-2 RNA fragments has been shown to be an effective way to monitor the COVID-19 pandemic at the population level. Wastewater is a complex sample matrix affected by environmental factors and PCR inhibitors, causing insufficient coverage in sequencing, for example. Subsequently, results where part of the genome does not have sufficient coverage are not uncommon. To identify variants and their proportions in wastewater over time, we utilized next-generation sequencing with the ARTIC Network's primer set and bioinformatics pipeline to evaluate the presence of variants in partial genome data. Based on the wastewater data from November 2021 to February 2022, the Delta variant was dominant until mid-December in Helsinki, Finland's capital, and thereafter in late December 2022 Omicron became the most common variant. At the same time, the Omicron variant of SARS-CoV-2 outcompeted the previous Delta variant in Finland in new COVID-19 cases. The SARS-CoV-2 variant findings from wastewater are in agreement with the variant information obtained from the patient samples when visually comparing trends in the sewerage network area. This indicates that the sequencing of wastewater is an effective way to monitor temporal and spatial trends of SARS-CoV-2 variants at the population level.

Epstein-Barr Virus in the Cerebrospinal Fluid and Blood Compartments of Patients With Multiple Sclerosis and Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood. METHODS: CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls. RESULTS: One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls (p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations. DISCUSSION: EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

Phylogenetic Characterization of Orthohantavirus dobravaense (Dobrava Virus).

We report complete coding sequences of Orthohantavirus dobravaense (Dobrava virus) Igneada strains and phylogenetic characterization of all available complete coding sequences. Our analyses suggested separation of host-dependent lineages, followed by geographic clustering. Surveillance of orthohantaviruses using complete genomes would be useful for assessing public health threats from Dobrava virus.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

ICTV Virus Taxonomy Profile: Kolmioviridae 2024.

Kolmioviridae is a family for negative-sense RNA viruses with circular, viroid-like genomes of about 1.5-1.7 kb that are maintained in mammals, amphibians, birds, fish, insects and reptiles. Deltaviruses, for instance, can cause severe hepatitis in humans. Kolmiovirids encode delta antigen (DAg) and replicate using host-cell DNA-directed RNA polymerase II and ribozymes encoded in their genome and antigenome. They require evolutionary unrelated helper viruses to provide envelopes and incorporate helper virus proteins for infectious particle formation. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Kolmioviridae, which is available at ictv.global/report/kolmioviridae.

Equine dermatitis outbreak associated with parapoxvirus.

Parapoxviruses (PPV) cause skin and mucous membrane lesions in several animal species, and of the five recognized PPVs, at least three are zoonotic. Equine PPV (EqPPV) is the sixth one initially described in humans in the United States and later in a severely sick horse in Finland in 2013-2015. In 2021-2022, a large-scale pustulo-vesicular pastern dermatitis outbreak occurred in horses all over Finland. This study aimed at analysing the outbreak, identifying and describing the causative agent, describing clinical signs, and searching for risk factors. EqPPV was identified as a probable causative agent and co-infections with several potentially pathogenic and zoonotic bacteria were observed. Histopathologically, suppurative and ulcerative dermatitis was diagnosed. Due to the lack of specific tests for this virus, we developed a novel diagnostic EqPPV-PCR with sensitivity of 10 copies/reaction. Based on a large proportion of the genome sequenced directly from clinical samples, very little variation was detected between the sequences of the case from 2013 and the cases from 2021 to 2022. Based on an epidemiological survey, the main risk factor for pastern dermatitis was having racehorses. Approximately one third of the horses at each affected stable got clinical dermatitis, manifesting as severe skin lesions. Skin lesions were also occasionally reported in humans, indicating potential zoonotic transmission. Case stables commonly reported attendance at race events before acquiring the disease. Survey also identified differences in practises between case and control stables. Taken together, these results enable a better preparedness, diagnostics, and guidelines for future outbreaks.

Novel Ozark Orthohantavirus in Hispid Cotton Rats (Sigmodon hispidus), Arkansas, USA.

We report a novel orthohantavirus, putatively named Ozark orthohantavirus, in hispid cotton rats captured within the Ozark Plateau in Arkansas, USA. This virus phylogenetically clusters with other orthohantaviruses that cause severe human disease. Continued orthohantavirus surveillance and virus sequencing are needed to address the potential public health threat of this virus.

Boosted production of antibodies that neutralized different SARS-CoV-2 variants in a COVID-19 convalescent following messenger RNA vaccination - a case study.

Vaccinated convalescents do not develop severe COVID-19 after infection with new SARS-CoV-2 variants. We questioned how messenger RNA (mRNA) vaccination of convalescents provides protection from emerging virus variants. From the cohort of 71 convalescent plasma donors, we identified a patient who developed immune response to infection with SARS-CoV-2 variant of 20A clade and who subsequently received mRNA vaccine encoding spike (S) protein of strain of 19A clade. We showed that vaccination increased the production of immune cells and anti-S antibodies in the serum. Serum antibodies neutralized not only 19A and 20A, but also 20B, 20H, 21J, and 21K virus variants. One of the serum antibodies (100F8) completely neutralized 20A, 21J, and partially 21K strains. 100F8 was structurally similar to published Ab188 antibody, which recognized non-conserved epitope on the S protein. We proposed that 100F8 and other serum antibodies of the patient which recognized non- and conserved epitopes of the S protein, could have additive or synergistic effects to neutralize various virus variants. Thus, mRNA vaccination could be beneficial for convalescents because it boosts production of neutralizing antibodies with broad-spectrum activity.

Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland.

BACKGROUND: Ticks carry microbes, some of which are pathogenic for humans and animals. To assess this One Health challenge, 342 ticks were collected from pet dogs and cats at 10 veterinary clinics in Finland as part of the European project "Protect Our Future Too". METHODS: The tick species were identified, and ticks were screened with quantitative PCR (qPCR) for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Ehrlichia canis, Anaplasma spp., Candidatus Neoehrlichia mikurensis, tick-borne encephalitis virus (TBEV), and Babesia spp. For comparison, a subset of tick DNA (20 qPCR-positive samples) was analysed with 16S next-generation sequencing (NGS). RESULTS: Most ticks were Ixodes ricinus (289, 84.5%), followed by Ixodes persulcatus (51, 14.9%). One hybrid tick (I. ricinus/I. persulcatus, 0.3%) and one Rhipicephalus sanguineus tick (0.3%) were identified. We found one or more of the analysed pathogens in 17% (59/342) of the ticks. The most prevalent pathogen was B. burgdorferi s.l. (36, 10.5%), followed by Anaplasma phagocytophilum (12, 3.5%), B. miyamotoi (5, 1.5%), Babesia venatorum (4, 1.2%), and TBEV (1, 0.3%). Candidatus Neoehrlichia mikurensis DNA was amplified from three (0.9%) ticks. Ehrlichia canis was not detected. In the 16S NGS, six samples produced enough reads for the analysis. In these six samples, we confirmed all the positive qPCR findings of Borrelia spp. and Ca. N. mikurensis. CONCLUSIONS: The high prevalence of pathogenic microorganisms in the ticks of this study emphasizes the importance of awareness of ticks and tick-borne diseases and prevention. Furthermore, the results show that veterinary surveillance can facilitate early detection of tick-borne pathogens and new tick species and draw attention to possible co-infections that should be considered both in symptomatic humans and animals after tick bites.

Partial Genome Characterization of Novel Parapoxvirus in Horse, Finland.

We report a sequencing protocol and 121-kb poxvirus sequence from a clinical sample from a horse in Finland with dermatitis. Based on phylogenetic analyses, the virus is a novel parapoxvirus associated with a recent epidemic; previous data suggest zoonotic potential. Increased awareness of this virus and specific diagnostic protocols are needed.

Superspreading of SARS-CoV-2 Omicron BA.2.23 among vaccinated Finnish adults: symptomatic COVID-19 only contracted by those without recent infection.

An outbreak of SARS-CoV-2 was confirmed after an academic party in Helsinki, Finland, in 2022. All 70 guests were requested to fill in follow-up questionnaires; serologic analyses and whole-genome sequencing (WGS) were conducted when possible.Of those participating - all but one with ≥3 vaccine doses - 21/53 (40%) had test-confirmed symptomatic COVID-19: 7% of those with earlier episodes and 76% of those without. Half (11/21) were febrile, but none needed hospitalisation. WGS revealed subvariant BA.2.23.Compared to vaccination alone, our data suggest remarkable protection by hybrid immunity against symptomatic infection, particularly in instances of recent infections with homologous variants.

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome.

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70-80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, "Why this variation?" remains largely unanswered.

Enhanced Viral Metagenomics with Lazypipe 2.

Viruses are the main agents causing emerging and re-emerging infectious diseases. It is therefore important to screen for and detect them and uncover the evolutionary processes that support their ability to jump species boundaries and establish themselves in new hosts. Metagenomic next-generation sequencing (mNGS) is a high-throughput, impartial technology that has enabled virologists to detect either known or novel, divergent viruses from clinical, animal, wildlife and environmental samples, with little a priori assumptions. mNGS is heavily dependent on bioinformatic analysis, with an emerging demand for integrated bioinformatic workflows. Here, we present Lazypipe 2, an updated mNGS pipeline with, as compared to Lazypipe1, significant improvements in code stability and transparency, with added functionality and support for new software components. We also present extensive benchmarking results, including evaluation of a novel canine simulated metagenome, precision and recall of virus detection at varying sequencing depth, and a low to extremely low proportion of viral genetic material. Additionally, we report accuracy of virus detection with two strategies: homology searches using nucleotide or amino acid sequences. We show that Lazypipe 2 with nucleotide-based annotation approaches near perfect detection for eukaryotic viruses and, in terms of accuracy, outperforms the compared pipelines. We also discuss the importance of homology searches with amino acid sequences for the detection of highly divergent novel viruses.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

Genome Sequence of an Early Imported Case of SARS-CoV-2 Delta Variant (B.1.617.2 AY.122) in Iraq in April 2021.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant was first reported in India. Thereafter, the Delta variant became the most prevalent variant globally. Here, we report the complete genome sequence of an early imported case of a SARS-CoV-2 B.1.617.2 AY.122 strain in Iraq. The strain was obtained from a flight passenger from India to Iraq on 20 April 2021.

Boid Inclusion Body Disease Is Also a Disease of Wild Boa Constrictors.

Reptarenaviruses cause boid inclusion body disease (BIBD), a potentially fatal disease, occurring in captive constrictor snakes boas and pythons worldwide. Classical BIBD, characterized by the formation of pathognomonic cytoplasmic inclusion bodies (IBs), occurs mainly in boas, whereas in pythons, for example, reptarenavirus infection most often manifests as central nervous system signs with limited IB formation. The natural hosts of reptarenaviruses are unknown, although free-ranging/wild constrictor snakes are among the suspects. Here, we report BIBD with reptarenavirus infection in indigenous captive and wild boid snakes in Costa Rica using histology, immunohistology, transmission electron microscopy, and next-generation sequencing (NGS). The snakes studied represented diagnostic postmortem cases of captive and wild-caught snakes since 1989. The results from NGS on archival paraffin blocks confirm that reptarenaviruses were already present in wild boa constrictors in Costa Rica in the 1980s. Continuous sequences that were de novo assembled from the low-quality RNA obtained from paraffin-embedded tissue allowed the identification of a distinct pair of reptarenavirus S and L segments in all studied animals; in most cases, reference assembly could recover almost complete segments. Sampling of three prospective cases in 2018 allowed an examination of fresh blood or tissues and resulted in the identification of additional reptarenavirus segments and hartmanivirus coinfection. Our results show that BIBD is not only a disease of captive snakes but also occurs in indigenous wild constrictor snakes in Costa Rica, suggesting boa constrictors to play a role in natural reptarenavirus circulation. IMPORTANCE The literature describes cases of boid inclusion body disease (BIBD) in captive snakes since the 1970s, and in the 2010s, others and ourselves identified reptarenaviruses as the causative agent. BIBD affects captive snakes globally, but the origin and the natural host of reptarenaviruses remain unknown. In this report, we show BIBD and reptarenavirus infections in two native Costa Rican constrictor snake species, and by studying archival samples, we show that both the viruses and the disease have been present in free-ranging/wild snakes in Costa Rica at least since the 1980s. The diagnosis of BIBD in wild boa constrictors suggests that this species plays a role in the circulation of reptarenaviruses. Additional sample collection and analysis would help to clarify this role further and the possibility of, e.g., vector transmission from an arthropod host.

Characterisation of the RNA Virome of Nine Ochlerotatus Species in Finland.

RNA viromes of nine commonly encountered Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16-60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date.