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.

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.

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.

SARS-CoV-2 Isolates Show Impaired Replication in Human Immune Cells but Differential Ability to Replicate and Induce Innate Immunity in Lung Epithelial Cells.

The primary target organ of coronavirus disease 2019 (COVID-19) infection is the respiratory tract. Currently, there is limited information on the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect and regulate innate immunity in human immune cells and lung epithelial cells. Here, we compared the ability of four Finnish isolates of SARS-CoV-2 from COVID-19 patients to replicate and induce interferons (IFNs) and other cytokines in different human cells. All isolates failed to replicate in dendritic cells, macrophages, monocytes, and lymphocytes, and no induction of cytokine gene expression was seen. However, most of the isolates replicated in Calu-3 cells, and they readily induced type I and type III IFN gene expression. The hCoV-19/Finland/FIN-25/2020 isolate, originating from a traveler from Milan in March 2020, showed better ability to replicate and induce IFN and inflammatory responses in Calu-3 cells than other isolates of SARS-CoV-2. Our data increase the knowledge on the pathogenesis and antiviral mechanisms of SARS-CoV-2 infection in human cell systems. IMPORTANCE With the rapid spread of the coronavirus disease 2019 (COVID-19) pandemic, information on the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and regulation of innate immunity in human immune cells and lung epithelial cells is needed. In the present study, we show that SARS-CoV-2 failed to productively infect human immune cells, but different isolates of SARS-CoV-2 showed differential ability to replicate and regulate innate interferon responses in human lung epithelial Calu-3 cells. These findings will open up the way for further studies on the mechanisms of pathogenesis of SARS-CoV-2 in human cells.

High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783.

Tick-borne encephalitis virus (TBEV) is a medically important arbovirus, widespread in Europe and Asia. The virus is primarily transmitted to humans and animals by bites from ticks and, in rare cases, by consumption of unpasteurized dairy products. The aim of this study was to sequence and characterize two TBEV strains with amplicon sequencing by designing overlapping primers. The amplicon sequencing, via Illumina MiSeq, covering nearly the entire TBEV genome, was successful: We retrieved and characterized the complete polyprotein sequence of two TBEV strains, Hochosterwitz and 1993/783 from Austria and Sweden, respectively. In this study the previous phylogenetic analysis of both strains was confirmed to be of the European subtypes of TBEV (TBEV-Eu) by whole genome sequencing. The Hochosterwitz strain clustered with the two strains KrM 93 and KrM 213 from South Korea, and the 1993/783 strain clustered together with the NL/UH strain from the Netherlands. Our study confirms the suitability and rapidness of the high-throughput sequencing method used to produce complete TBEV genomes from TBEV samples of high viral load giving high-molecular-weight cDNA with large overlapping amplicons.

Serpentoviruses: More than Respiratory Pathogens.

In recent years, nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full postmortem examinations on 30 serpentovirus reverse transcription-PCR (RT-PCR)-positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus infects not only a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts, etc.), but also intravascular monocytes, intralesional macrophages, and endothelial cells. With next-generation sequencing we obtained a full-length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection-associated respiratory or systemic disease did not reveal sequence association to phenotypes; however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that comprises important zoonotic pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Serpentoviruses belong to the same order as the above-mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order Nidovirales.

Orthohantavirus Isolated in Reservoir Host Cells Displays Minimal Genetic Changes and Retains Wild-Type Infection Properties.

: Orthohantaviruses are globally emerging zoonotic pathogens. While the reservoir host role of several rodent species is well-established, detailed research on the mechanisms of host-othohantavirus interactions has been constrained by the lack of an experimental system that is able to effectively replicate natural infections in controlled settings. Here we report the isolation, and genetic and phenotypic characterization of a novel Puumala orthohantavirus (PUUV) in cells derived from its reservoir host, the bank vole. The isolation process resulted in cell culture infection that evaded antiviral responses, persisted cell passaging, and had minor viral genome alterations. Critically, experimental infections of bank voles with the new isolate resembled natural infections in terms of viral load and host cell distribution. When compared to an attenuated Vero E6 cell-adapted PUUV Kazan strain, the novel isolate demonstrated delayed virus-specific humoral responses. A lack of virus-specific antibodies was also observed during experimental infections with wild-type PUUV, suggesting that delayed seroconversion could be a general phenomenon during orthohantavirus infection in reservoir hosts. Our results demonstrate that orthohantavirus isolation on cells derived from a vole reservoir host retains wild-type infection properties and should be considered the method of choice for experimental infection models to replicate natural processes.

Differences in Tissue and Species Tropism of Reptarenavirus Species Studied by Vesicular Stomatitis Virus Pseudotypes.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion's surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential.

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020.

The first case of coronavirus disease (COVID-19) in Finland was confirmed on 29 January 2020. No secondary cases were detected. We describe the clinical picture and laboratory findings 3-23 days since the first symptoms. The SARS-CoV-2/Finland/1/2020 virus strain was isolated, the genome showing a single nucleotide substitution to the reference strain from Wuhan. Neutralising antibody response appeared within 9 days along with specific IgM and IgG response, targeting particularly nucleocapsid and spike proteins.

Introduction and Dispersal of Sindbis Virus from Central Africa to Europe.

Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe, as well as in Africa and the Middle East, collected over 58 years. These were analyzed together with 30 additional published whole SINV-I genomes using Bayesian analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southward on two separate occasions in the 1960s and 1970s. Another introduction from central Africa to southern/central Europe seems to have occurred, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where the most divergent SINV-I strains also originated.IMPORTANCE This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular.

Genome Sequences of RIGVIR Oncolytic Virotherapy Virus and Five Other Echovirus 7 Isolates.

We report here the nearly complete Illumina-sequenced consensus genome sequences of six isolates of echovirus 7 (E7), including oncolytic virotherapy virus RIGVIR and the Wallace prototype. Amino acid identities within the coding region were highly conserved across all isolates, ranging from 95.31% to 99.73%.

Mapping of human B-cell epitopes of Sindbis virus.

Mosquito-transmitted Sindbis virus (SINV) causes fever, skin lesions and musculoskeletal symptoms if transmitted to man. SINV is the prototype virus of genus Alphavirus, which includes other arthritogenic viruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) that cause large epidemics with a considerable public health burden. Until now the human B-cell epitopes have been studied for CHIKV and RRV, but not for SINV. To identify the B-cell epitopes in SINV-infection, we synthetised a library of linear 18-mer peptides covering the structural polyprotein of SINV, and probed it with SINV IgG-positive and IgG-negative serum pools. By comparing the binding profiles of the pools, we identified 15 peptides that were strongly reactive only with the SINV IgG-positive pools. We then utilized alanine scanning and individual (n=22) patient sera to further narrow the number of common B-cell epitopes to six. These epitopes locate to the capsid, E2, E1 and to a region in PE2 (uncleaved E3-E2), which may only be present in immature virions. By sequence comparison, we observed that one of the capsid protein epitopes shares six identical amino acids with macrophage migration inhibitory factor (MIF) receptor, which is linked to inflammatory diseases and to molecular pathology of alphaviral arthritides. Our results add to the current understanding on SINV disease and raise questions of a potential role of uncleaved PE2 and the MIF receptor (CD74) mimotope in human SINV infection.

Enterovirus strain and type-specific differences in growth kinetics and virus-induced cell destruction in human pancreatic duct epithelial HPDE cells.

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1 diabetes. In addition, the capability for rapid adaptation to different cell types suggests that, on occasion, enterovirus strains with different pathogenetic properties may arise from less pathogenic ancestors.

Molecular evolution and epidemiology of echovirus 6 in Finland.

Echovirus 6 (E-6) (family Picornaviridae, genus Enterovirus) is one of the most commonly detected enteroviruses worldwide. The aim of this study was to determine molecular evolutionary and epidemiologic patterns of E-6. A complete genome of one E-6 strain and the partial VP1 coding regions of 169 strains were sequenced and analyzed along with sequences retrieved from the GenBank. The complete genome sequence analysis suggested complex recombination history for the Finnish E-6 strain. In VP1 region, the phylogenetic analysis suggested three major clusters that were further divided to several subclusters. The evolution of VP1 coding region was dominated by negative selection suggesting that the phylogeny of E-6 VP1 gene is predominantly a result of synonymous substitutions (i.e. neutral genetic drift). The partial VP1 sequence analysis suggested wide geographical distribution for some E-6 lineages. In Finland, multiple different E-6 lineages have circulated at the same time.

High seroprevalence of enterovirus infections in apes and old world monkeys.

To estimate population exposure of apes and Old World monkeys in Africa to enteroviruses (EVs), we conducted a seroepidemiologic study of serotype-specific neutralizing antibodies against 3 EV types. Detection of species A, B, and D EVs infecting wild chimpanzees demonstrates their potential widespread circulation in primates.

Amino acids of Coxsackie B5 virus are critical for infection of the murine insulinoma cell line, MIN-6.

It was shown recently that 15 successive passages of a laboratory strain of the Coxsackie B virus 5 in a mouse pancreas (CBV-5-MPP) resulted in apparent changes in the virus phenotype, which led to the capacity to induce a diabetes-like syndrome in mice. For further characterization of islet cell interactions with a passaged virus strain, a murine insulinoma cell line, MIN-6, was selected as an experimental model. The CBV-5-MPP virus strain was not able to replicate in MIN-6 cells in vitro but required adaptation over a few days for progeny production and the generation of cytopathic effects. In order to determine the genetic characteristics required for virus growth in MIN-6 cells, the whole genome of the MIN-6-adapted virus variant was sequenced, and critical amino acids were identified by comparing the sequence with that of a virus strain passaged repeatedly in the mouse pancreas. The results of site-directed mutagenesis demonstrated that only one residue, amino acid 94 of VP1, is a major determinant for virus adaptation to MIN-6 cells.

Enterovirus 94, a proposed new serotype in human enterovirus species D.

The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo. The complete genome of one environmental isolate, the complete coding sequence of one clinical isolate and complete VP1 regions from the other isolates were sequenced. These isolates had 66.6-69.4% nucleotide similarity and 74.7-76.6% amino acid sequence similarity in the VP1 region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses including sequences of the 5' UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a monophyletic group within the species HEV-D. No evidence of recombination was found between EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin alphavbeta3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades.

Mutations N34S and P55S of the SPINK1 gene in patients with chronic pancreatitis or pancreatic cancer and in healthy subjects: a report from Finland.

OBJECTIVE: Mutations in the Kazal type 1 serine protease inhibitor (SPINK1) gene have recently been associated with chronic pancreatitis (CP), an established risk factor for pancreatic cancer. The aim of this study was to investigate the frequency of the SPINK1 gene mutations (N34S and P55S) in patients with CP, or pancreatic cancer, and in healthy subjects in Finland. MATERIAL AND METHODS: The N34S and P55S mutations were determined by PCR amplification followed by solid-phase minisequencing in 116 patients with CP and in 188 with pancreatic cancer. In patients with CP, alcohol was the aetiological factor in 87 (75%), pancreas divisum in 4 (3%), gallstones in 5 (5%) and 20 patients (17%) had an idiopathic disease; 459 healthy individuals were enrolled as controls. RESULTS: The frequency of the N34S mutation was significantly higher in patients with CP (14/116, 12%) than in controls (12/459, 2.6%) (p<0.0001). There was no difference in the frequency of the P55S mutation between patients with CP (1/116, 0.9%) and controls (6/459, 1.3%). The N34S mutation was present in 9 (10%) out of 87 patients with alcoholic CP, and in 5 (25%) patients with idiopathic CP. No SPINK1 mutations were found in patients with CP caused by anatomical variations or gallstones. Among the 188 patients with a pancreatic malignant tumour, the N34S mutation was present in 7 cases (3.7%). The frequency of the N34S mutation in healthy controls in this study was significantly higher than earlier reported in other countries (p=0.03). CONCLUSIONS: The SPINK1 N34S mutation was significantly associated with an increased risk of CP. The association of the N34S mutation with alcoholic CP was marginally stronger than in earlier studies, whereas in the Finnish population in general, this mutation was significantly more frequent than reported elsewhere.