Laura Kakkola

Biological and Immunological Relations among Human Parvovirus B19 Genotypes 1 to 3

ABSTRACT The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ∼10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.

Emerging cellular targets for influenza antiviral agents

At the global level, influenza A virus (IAV) is considered a major health threat because it causes significant morbidity. Different treatment and prevention options have been developed; however, these are insufficient in the face of recent IAV outbreaks. In particular, available antiviral agents have limited effectiveness owing to IAV resistance to these virus-directed drugs. Recent advances in understanding of IAV replication have revealed a number of cellular drug targets that counteract viral drug resistance. This review summarizes current knowledge on IAV replication with a focus on emerging cellular drug targets. Interestingly, for many of these targets, compounds for which safety testing has been carried out in humans are available. It is possible that some of these compounds, such as inhibitors of heat shock protein 90, proteasome, importin α5 or protein kinase C, will be used for treatment of IAV infections after careful evaluation in human primary cells and severely ill flu patients.

Human Circovirus TT Virus Genotype 6 Expresses Six Proteins following Transfection of a Full-Length Clone

ABSTRACT The expression profile of the circovirus TTV has not yet been fully characterized. In this paper, we show that following transfection of a full-length viral clone of TTV genotype 6, each of the three virally encoded mRNAs is translated from two initiating AUGs, and therefore, the TTV genome generates at least six proteins. Localization studies of hemagglutinin-tagged versions of these proteins in fixed cells, and green fluorescent protein-tagged versions of these proteins in living cells, expressed following transfection, demonstrated that two were primarily nuclear, two were primarily cytoplasmic, and two were found throughout the cell.

Inhibition of influenza A virus infection in vitro by saliphenylhalamide- loaded porous silicon nanoparticles

Influenza A viruses (IAVs) cause recurrent epidemics in humans, with serious threat of lethal worldwide pandemics. The occurrence of antiviral-resistant virus strains and the emergence of highly pathogenic influenza viruses have triggered an urgent need to develop new anti-IAV treatments. One compound found to inhibit IAV, and other virus infections, is saliphenylhalamide (SaliPhe). SaliPhe targets host vacuolar-ATPase and inhibits acidification of endosomes, a process needed for productive virus infection. The major obstacle for the further development of SaliPhe as antiviral drug has been its poor solubility. Here, we investigated the possibility to increase SaliPhe solubility by loading the compound in thermally hydrocarbonized porous silicon (THCPSi) nanoparticles. SaliPhe-loaded nanoparticles were further investigated for the ability to inhibit influenza A infection in human retinal pigment epithelium and Madin-Darby canine kidney cells, and we show that upon release from THCPSi, SaliPhe inhibited IAV infection in vitro and reduced the amount of progeny virus in IAV-infected cells. Overall, the PSi-based nanosystem exhibited increased dissolution of the investigated anti-IAV drug SaliPhe and displayed excellent in vitro stability, low cytotoxicity, and remarkable reduction of viral load in the absence of organic solvents. This proof-of-principle study indicates that PSi nanoparticles could be used for efficient delivery of antivirals to infected cells.

Obatoclax, saliphenylhalamide and gemcitabine inhibit influenza A virus infection

Background: Novel options should be developed for treatment of IAV infections. Results: Obatoclax, saliphenylhalamide, and gemcitabine target host factors and inhibit IAV and several other viruses infections. Conclusion: These compounds represent potent antiviral agents. Significance: These compounds could be exploited in treatment of severe viral infections. Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

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.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Full-Genome Sequences of Influenza A(H1N1)pdm09 Viruses Isolated from Finnish Patients from 2009 to 2013

ABSTRACT Here we report full-length sequencing of the first large set of influenza A(H1N1)pdm09 virus genomes isolated in Finland between the years 2009 and 2013 and discuss the advantages and needs of influenza virus sequencing efforts.

Comparative Analysis of Whole-Genome Sequences of Influenza A(H1N1)pdm09 Viruses Isolated from Hospitalized and Nonhospitalized Patients Identifies Missense Mutations That Might Be Associated with Patient Hospital Admissions in Finland during 2009 to 2014

ABSTRACT Here, we report 40 new whole-genome sequences of influenza A(H1N1)pdm09 viruses isolated from Finnish patients during 2009 to 2014. A preliminary analysis of these and 186 other whole genomes of influenza A(H1N1)pdm09 viruses isolated from hospitalized and nonhospitalized patients during 2009 to 2014 in Finland revealed several viral mutations that might be associated with patient hospitalizations.