State-of-the-Art Virology Research in Norway.

Norway is situated in a remote and sparsely inhabited part of the world with about 5 [...].

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

Dynamics of SARS-CoV-2 Spike-IgG throughout Three COVID-19 Vaccination Regimens: A 21-Month Longitudinal Study of 82 Norwegian Healthcare Workers.

To facilitate interpretation of clinical SARS-CoV-2 anti-spike IgG analyses post-vaccination, 82 healthcare workers were followed through three vaccination-regimens: two regimens were comprised of two doses of BNT162b2 three or six weeks apart, followed by a dose of mRNA-vaccine, and in the other regimen, the first dose was replaced by ChAdOx1 nCov-19. After each dose, anti-spike IgG was compared between regimens. As many participants became infected, anti-spike IgG persistence was compared between infected and uninfected participants. Thirteen to twenty-one days after the first dose, seroconversion, and the median anti-spike IgG level in the ChAdOx1 group was significantly lower than in the BNT162b2 groups (23 versus 68 and 73 AU/mL). The second dose caused a significant increase in anti-spike IgG, but the median level was lower in the BNT162b2-short-interval group (280 AU/mL), compared to the BNT162b2-long-interval (1075 AU/mL) and ChAdOx1 (1160 AU/mL) group. After the third dose, all groups showed increases to similar anti-spike IgG levels (2075-2390 AU/mL). Over the next half year, anti-spike IgG levels declined significantly in all groups, but appeared to persist longer after post-vaccination infection. This is the first three-dose study with one dose of ChAdOx1. Despite initial differences, all vaccine regimens gave similarly high antibody levels and persistence after the third dose.

Risk factors, immune response and whole-genome sequencing of SARS-CoV-2 in a cruise ship outbreak in Norway.

OBJECTIVE: To improve understanding of SARS-CoV-2-transmission and prevention measures on cruise ships, we investigated a Norwegian cruise ship outbreak from July to August 2020 using a multidisciplinary approach after a rapid outbreak response launched by local and national health authorities. METHODS: We conducted a cross-sectional study among crew members using epidemiologic data and results from SARS-CoV-2 polymerase chain reaction (PCR) of nasopharynx-oropharynx samples, antibody analyses of blood samples, and whole-genome sequencing. RESULTS: We included 114 multinational crew members (71% participation), median age 36 years, and 69% male. The attack rate was 33%; 32 of 37 outbreak cases were seropositive 5-10 days after PCR. One PCR-negative participant was seropositive, suggesting a previous infection. Network-analysis showed clusters based on common exposures, including embarkation date, nationality, sharing a cabin with an infected cabin-mate (adjusted odds ratio [AOR] 3.27; 95% confidence interval [CI] 0.97-11.07, p = 0.057), and specific workplaces (mechanical operations: 9.17 [1.82-45.78], catering: 6.11 [1.83-20.38]). Breaches in testing, quarantine, and isolation practices before/during expeditions were reported. Whole-genome sequencing revealed lineage B.1.36, previously identified in Asia. Despite extensive sequencing, the continued transmission of B.1.36 in Norway was not detected. CONCLUSIONS: Our findings confirm the high risk of SARS-CoV-2-transmission on cruise ships related to workplace and cabin type and show that continued community transmission after the outbreak could be stopped by implementing immediate infection control measures at the final destination.

Should SVGp12 Be Used for JC Polyomavirus Studies? Comment on Prezioso et al. COS-7 and SVGp12 Cellular Models to Study JCPyV Replication and MicroRNA Expression after Infection with Archetypal and Rearranged-NCCR Viral Strains. Viruses 2022, 14, 2070.

A recent paper in Viruses investigates the impact of the JC polyomavirus (JCPyV) microRNA on the replication of different JCPyV strains. Unfortunately, one of the cell lines used, the human fetal glial cell line SVGp12, is productively infected by the closely related BK polyomavirus (BKPyV), which may confound results. Scientists need to take this into account and the potential pitfalls.

Serological Evidence of Hepatitis E Virus Infection in Semi-Domesticated Eurasian Tundra Reindeer (Rangifer tarandus tarandus) in Norway.

Hepatitis E virus (HEV) is a common cause of viral hepatitis in humans. In developing countries, HEV-infections seem to be mainly associated with pigs, but other animal species may be involved in viral transmission. Recently, anti-HEV antibodies were detected in Norwegian wild reindeer. Here, we investigated anti-HEV seroprevalence in Norwegian semi-domesticated reindeer, animals in closer contact with humans than their wild counterparts. Blood samples (n = 516) were obtained from eight reindeer herds during the period 2013-2017 and analysed with a commercial enzyme-linked immunosorbent assay designed for detecting anti-HEV antibodies in livestock. Antibodies were found in all herds and for all sampling seasons. The overall seroprevalence was 15.7% (81/516), with adults showing a slightly higher seroprevalence (18.0%, 46/256) than calves (13.5%, 35/260, p = 0.11). The seroprevalence was not influenced by gender or latitude, and there was no temporal trend (p > 0.15). A positive association between the presence of anti-HEV antibodies and antibodies against alphaherpesvirus and pestivirus, detected in a previous screening, was found (p < 0.05). We conclude that Norwegian semi-domesticated reindeer are exposed to HEV or an antigenically similar virus. Whether the virus is affecting reindeer health or infects humans and poses a threat for human health remains unknown and warrants further investigations.

Antibody response in a family with COVID-19. / Antistoffrespons hos en familie med covid-19.

BACKGROUND: Robust serological assays for SARS-CoV-2 are essential for determining prior infection and the suitability of donated convalescent plasma for plasma therapy. We compared two in-house and three commercial serological assays in a family cohort with SARS-CoV-2-infected members. CASE PRESENTATION: Three individuals in a family of five developed COVID-19 confirmed by PCR, following a trip abroad. Three to four weeks after the onset of symptoms, three commercial ELISAs and an in-house immunofluorescence test demonstrated antibodies to SARS-CoV-2. An in-house neutralisation test also demonstrated neutralising antibodies. INTERPRETATION: The in-house assays and one commercial assay gave congruent results, which were also consistent with the initial PCR and/or clinical evaluation, indicating prior infection in three of the five family members. The other commercial assays indicated possible infection in a fourth family member, but this result was likely due to cross-reactivity. The neutralising antibodies suggest complete or partial immunity against reinfection.

BK Polyomavirus Evades Innate Immune Sensing by Disrupting the Mitochondrial Network and Promotes Mitophagy.

Immune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.

Does the Evidence Support the Existence of the Simian Polyomavirus SV40 Vp4 Viroporin?

The simian polyomavirus SV40 was reported to express Vp4, an N-terminally truncated form of the minor capsid proteins Vp2 and Vp3. Since a missense mutation of the putative Vp4 start codon (Vp2M228I) was found to give reduced progeny release and delayed lysis, Vp4 was claimed to be a viroporin. However, two independent research groups, including our own, were unable to replicate these findings. In contrast, we found no Vp4 expression in SV40-infected cells and no reduction in progeny release for Vp4-deficient virus, and finally, we found that the single amino acid substitution unavoidably introduced into the overlapping Vp2/Vp3 genes during Vp4 mutagenesis reduced early steps but not virus release. Remarkably, the existence of the viroporin Vp4 still seems to be widely accepted, which presumably is preventing important research on polyomavirus release. With this perspective, we will review and comment on the most important experiments that led to the disputed announcement of the viroporin Vp4.

Early fulminant BK polyomavirus-associated nephropathy in two kidney transplant patients with low neutralizing antibody titers receiving allografts from the same donor.

BACKGROUND: BK Polyomavirus (BKPyV) causes premature graft failure in 1 to 15% of kidney transplant (KT) recipients. High-level BKPyV-viruria and BKPyV-DNAemia precede polyomavirus-associated nephropathy (PyVAN), and guide clinical management decisions. In most cases, BKPyV appears to come from the donor kidney, but data from biopsy-proven PyVAN cases are lacking. Here, we report the early fulminant course of biopsy-proven PyVAN in two male KT recipients in their sixties, receiving kidneys from the same deceased male donor. CASE PRESENTATIONS: Both recipients received intravenous basiliximab induction, and maintenance therapy consisting of tacrolimus (trough levels 3-7 ng/mL from time of engraftment), mycophenolate mofetil 750 mg bid, and prednisolone. At 4 weeks post-transplant, renal function was satisfactory with serum creatinine concentrations of 106 and 72 µmol/L in recipient #1 and recipient #2, respectively. Plasma BKPyV-DNAemia was first investigated at 5 and 8 weeks post-transplant being 8.58 × 104 and 1.12 × 106 copies/mL in recipient #1 and recipient #2, respectively. Renal function declined and biopsy-proven PyVAN was diagnosed in both recipients at 12 weeks post-transplant. Mycophenolate mofetil levels were reduced from 750 mg to 250 mg bid while tacrolimus levels were kept below 5 ng/mL. Recipient #2 cleared BKPyV-DNAemia at 5.5 months post-transplant, while recipient #1 had persistent BKPyV-DNAemia of 1.07 × 105 copies/mL at the last follow-up 52 weeks post-transplant. DNA sequencing of viral DNA from early plasma samples revealed apparently identical viruses in both recipients, belonging to genotype Ib-2 with archetype non-coding control region. Retrospective serological work-up, demonstrated that the donor had high BKPyV-IgG-virus-like particle ELISA activity and a high BKPyV-genotype I neutralizing antibody titer, whereas both KT recipients only had low neutralizing antibody titers pre-transplantation. By 20 weeks post-transplant, the neutralizing antibody titer had increased by > 1000-fold in both recipients, but only recipient #2 cleared BKPyV-DNAemia. CONCLUSIONS: Low titers of genotype-specific neutralizing antibodies in recipients pre-transplant, may identify patients at high risk for early fulminant donor-derived BKPyV-DNAemia and PyVAN, but development of high neutralizing antibody titers may not be sufficient for clearance.

Correction to: Seroprevalence of hepatitis E virus (HEV) in a general adult population in Northern Norway: the Tromsø study.

Unfortunately, the supplement tables are missing in the original article. The missing files have been included here.

Seroprevalence of hepatitis E virus (HEV) in a general adult population in Northern Norway: the Tromsø study.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the world but only a few cases have been diagnosed in Norway. To investigate the HEV exposure rate in a presumed low-risk area, we have conducted a population-based study of anti-HEV IgG seroprevalence in Northern Norway. A total of 1800 serum samples from 900 women and 900 men, age 40-79 years, were randomly selected from the 21,083 participants in the 7th Tromsø Study, representing the 32,591 inhabitants of the Tromsø municipality that were ≥ 40 years. All samples were analyzed by ELISA-1 (recomWell HEV IgG). Samples testing positive or borderline, as well as a 1.5-fold excess of negative samples, were retested by ELISA-2 (DiaPro HEV IgG). If still borderline or a result discordant from ELISA-1, the sample was retested by ELISA-3 (Wantai HEV IgG) and strip-immunoassay (recomLine HEV IgG). Anti-HEV IgG was detected in 205 individuals (11.4%), yielding an estimated seroprevalence of 10.4% in the age-matched population of Tromsø. Using logistic regression analysis followed by multivariable backward elimination analysis, increasing age (OR 1.036 per year; p < 0.001) and higher education (OR 2.167; p < 0.001) were found as potential risk factors, whereas travel abroad or eating of red meat were not. Our results indicate that HEV-infection is common in Northern Norway and suggest that HEV testing should be included in the evaluation of elevated liver enzymes.

Progressive multifocal leukoencephalopathy. / Progredierende multifokal leukoencefalopati.

Progressive multifocal leukoencephalopathy is a rare, opportunistic infection of the central nervous system caused by the John Cunningham virus (JCV). There is no effective antiviral treatment available, and restoring immunocompetence is essential for survival. If this occurs too quickly, however, the inflammatory response may prove fatal. This is an up-to-date review of the disorder, intended for clinicians responsible for immunomodulatory therapy.

The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release.

The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form, VP3, are both important for viral entry. The closely related simian virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses, including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells, we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, wild-type SV40 and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently led to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40-infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis. IMPORTANCE: Almost a decade ago, SV40 was reported to produce a late nonstructural protein, VP4, which forms pores in the nuclear membrane, facilitating progeny release. By performing transfection studies with unaltered BKPyV and SV40 and their respective VP4-deficient mutants, we found that VP4 is dispensable for progeny release, contrary to the original findings. However, infection studies demonstrated a counterintuitive reduction of infectivity of certain VP4-deficient mutants. In addition to the isoleucine-substituted SV40 mutant of the original study, we included alanine-substituted VP4-deficient mutants of BKPyV (VP2M229A) and SV40 (VP2M228A). These revealed that the reduction in infectivity was not caused by a lack of VP4 but rather depended on the identity of the single amino acid substituted within VP2/3 for VP4 start codon mutagenesis. Hopefully, our results will correct the longstanding misconception of VP4's role during infection and stimulate continued work on unraveling the mechanism for release of polyomavirus progeny.

Human BK Polyomavirus Plasmid pBKV (34-2) (Dunlop) Contains Mutations Not Found in the Originally Published Sequences.

The plasmid pBKV (34-2) (ATCC 45025) contains the entire BK polyomavirus Dunlop genome. Sequencing revealed 12 point mutations compared to the GenBank sequence, but only 4 point mutations compared to the published sequence. The origin of these differences is unknown, but may impact virological as well as diagnostic research and development.

Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

BK polyomavirus (BKPyV)-associated hemorrhagic cystitis (PyVHC) complicates 5 to 15% of allogeneic hematopoietic stem cell transplantations. Targeted antivirals are still unavailable. Brincidofovir (BCV; previously CMX001) has shown inhibitory activity against diverse viruses, including BKPyV in a primary human renal tubule cell culture model of polyomavirus-associated nephropathy. We investigated the effects of BCV in BKPyV-infected and uninfected primary human urothelial cells (HUCs), the target cells of BKPyV in PyVHC. The BCV concentrations causing 50 and 90% reductions (EC50 and EC90) in the number of intracellular BKPyV genome equivalents per cell (icBKPyV) were 0.27 µM and 0.59 µM, respectively. At 0.63 µM, BCV reduced viral late gene expression by 90% and halted progeny release. Preinfection treatment for only 24 h reduced icBKPyV similarly to treatment from 2 to 72 h postinfection, while combined pre- and postinfection treatment suppressed icBKPyV completely. After investigating BCV's effects on HUC viability, mean selectivity indices at 50 and 90% inhibition (SI50 and SI90) calculated for cellular DNA replication were 2.7 and 2.9, respectively, those for mitochondrial activity were 8.9 and 10.4, those for total ATP were 8.6 and 8.2, and those for membrane integrity were 25.9 and 16.7. The antiviral and cytostatic effects, but less so the cytotoxic effects, were inversely related to cell density. The cytotoxic effects at concentrations of ≥10 µM were rapid and likely related to BCV's lipid moiety. After carefully defining the antiviral, cytostatic, and cytotoxic properties of BCV in HUCs, we conclude that a preemptive or prophylactic approach in PyVHC is likely to give the best results.

Efficient uptake of blood-borne BK and JC polyomavirus-like particles in endothelial cells of liver sinusoids and renal vasa recta.

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells that mediate high-capacity clearance of soluble waste macromolecules and colloid material, including blood-borne adenovirus. To explore if LSECs function as a sink for other viruses in blood, we studied the fate of virus-like particles (VLPs) of two ubiquitous human DNA viruses, BK and JC polyomavirus, in mice. Like complete virions, VLPs specifically bind to receptors and enter cells, but unlike complete virions, they cannot replicate. 125I-labeled VLPs were used to assess blood decay, organ-, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and -cytochemistry. BK- and JC-VLPs rapidly distributed to liver, with lesser uptake in kidney and spleen. Liver uptake was predominantly in LSECs. Blood half-life (∼1 min), and tissue distribution of JC-VLPs and two JC-VLP-mutants (L55F and S269F) that lack sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. Liver uptake was not mediated by scavenger receptors. In spleen, the VLPs localized to the red pulp marginal zone reticuloendothelium, and in kidney to the endothelial lining of vasa recta segments, and the transitional epithelium of renal pelvis. Most VLP-positive vessels in renal medulla did not express PV-1/Meca 32, suggesting location to the non-fenestrated part of vasa recta. The endothelial cells of these vessels also efficiently endocytosed a scavenger receptor ligand, formaldehyde-denatured albumin, suggesting high endocytic activity compared to other renal endothelia. We conclude that LSECs very effectively cleared a large fraction of blood-borne BK- and JC-VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. In addition, we report the novel finding that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism.

Antiviral effects of artesunate on JC polyomavirus replication in COS-7 cells.

The human JC polyomavirus (JCPyV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). A growing number of patients with induced or acquired immunosuppression are at risk for infection, and no effective antiviral therapy is presently available. The widely used antimalarial drug artesunate has shown broad antiviral activity in vitro but limited clinical success. The aim of this study was to investigate the effect of artesunate on JCPyV replication in vitro. The permissivity for JCPyV MAD-4 was first compared in four cell lines, and the monkey kidney cell line COS-7 was selected. Artesunate caused a concentration-dependent decrease in the extracellular JCPyV DNA load 96 h postinfection, with a 50% effective concentration (EC50) of 2.9 µM. This effect correlated with a decreased expression of capsid protein VP1 and a reduced release of infectious viral progeny. For concentrations of <20 µM, transient reductions in cellular DNA replication and proliferation were seen, while for higher concentrations, some cytotoxicity was detected. A selective index of 16.6 was found when cytotoxicity was calculated based on cellular DNA replication in the mock-infected cells, but interestingly, cellular DNA replication in the JCPyV-infected cells was more strongly affected. In conclusion, artesunate is efficacious in inhibiting JCPyV replication at micromolar concentrations, which are achievable in plasma. The inhibition at EC50 probably reflects an effect on cellular proteins and involves transient cytostatic effects. Our results, together with the favorable distribution of the active metabolite dihydroartemisinin to the central nervous system, suggest a potential use for artesunate in patients with PML.

Treatment of progressive multifocal leukoencephalopathy with interleukin 7.

IMPORTANCE: No reliable treatment options are known for progressive multifocal leukoencephalopathy with underlying immunodeficiency. We describe successful compassionate use of recombinant human interleukin 7 in a patient with idiopathic CD4+ T-cell lymphocytopenia. OBSERVATIONS: After the diagnoses of progressive multifocal leukoencephalopathy and idiopathic CD4+ T-cell lymphocytopenia were established, a 61-year-old man was treated with recombinant human interleukin 7 on November 1, 2012. Except for an episode of epilepsia partialis continua on January 16, 2013, a gradual clinical improvement was observed until March. Abnormalities shown on magnetic resonance imaging regressed; JC virus DNA in plasma, likely originating from the brain based on sequencing data, cleared; and increases in peripheral CD4+ T cells and JC virus intrathecal antibodies were observed. One year after treatment, the CD4+ T-cell count returned to baseline and the clinical improvement waned, possibly due to the patient's complex epilepsy. On the latest evaluation on January 14, 2014, the patient's condition was unchanged, with no signs of ongoing central nervous system infection. CONCLUSIONS AND RELEVANCE: The present case argues strongly for proof of the treatment concept. However, deeper insight into the JC virus and its pathogenesis and the immune response during central nervous system infection as well as further clinical studies are needed before recombinant human interleukin 7 can be recommended for the treatment of other cases of immunodeficiency and progressive multifocal leukoencephalopathy.