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Ake Lundkvist

Publications and source records attributed to Ake Lundkvist.

At least 19 recordsLinked to original sources
PubMed2006

Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered from patients and rodents.

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.

Animals↗
PubMed2006

Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand.

Sixty one tissue samples from several rodent species trapped in five provinces of Thailand were examined for the presence of hantaviral markers by enzyme-immunoassay and immunoblotting. Four samples, all from the great bandicoot rat Bandicota indica, were confirmed positive for the hantaviral N-antigen. Two of them were trapped in Nakhon Pathom province, the other two in Nakhon Ratchasima province, approximately 250 km from the other trapping site. When analysed by RT-nested PCR, all four rodents were found positive for the hantaviral S- and M-segment nucleotide sequences. Genetic analysis revealed that the four newly described wild-type strains belong to Thailand hantavirus. On the phylogenetic trees they formed a well-supported cluster within the group of Murinae-associated hantaviruses and shared a recent common ancestor with Seoul virus.

Animals↗
PubMed2006

Study of the serological response after vaccination against tick-borne encephalitis in Sweden.

The antibody response to vaccination against tick-borne encephalitis (TBE) with FSME-Immun Inject (Immuno AG/Baxter) was studied in 535 persons, mainly adults, attending a vaccination centre in Stockholm, Sweden. Emphasis was laid on long-term follow-up. Antibody activity was measured by three different serological test systems: a commercial ELISA kit, a hemagglutination inhibition (HI) test and a neutralization test (RFFIT). The neutralization test proved to be the most sensitive assay for the detection of the vaccine response, which was demonstrable in the majority of vaccinees (>90% after three and >98% after four and five vaccinations, respectively). ELISA and HI were less sensitive for antibody measurement during primary immunization. Neutralizing antibody activity persisted prior to the third dose in 77% of the vaccinees and prior to the fourth to sixth doses in 89-95% of the vaccinees. ELISA activity, but no neutralizing activity, was found in some individuals. Based on our data and previous experience of vaccine failures after two doses, a more condensed three-dose vaccination schedule may be advantageous and ought to be tested. The persistence of neutralizing antibodies justifies further studies of the antibody responses after the fourth dose for periods beyond the recommended 3-year booster intervals.

Adolescent↗
PubMed2006

High prevalence of influenza A virus in ducks caught during spring migration through Sweden.

As part of our ongoing screening of wild birds in Northern Europe, 358 mallards (Anas platyrhynchos) and 203 shelducks (Tadorna tadorna) were caught in southern Sweden during the spring 2003. Faecal samples were analyzed by real time RT-PCR for the presence of influenza A virus. In contrast to what has been found in North American studies; Eurasian spring migrating ducks passing through Sweden had a relatively high prevalence of influenza A virus.

Animals↗
PubMed2006

Clearance of circulating Epstein-Barr virus DNA in children with acute malaria after antimalaria treatment.

Children living in malaria-endemic regions have a high incidence of Burkitt lymphoma (BL), the etiology of which involves Plasmodium falciparum malaria and Epstein-Barr virus (EBV) infections. In the present study, we compared EBV DNA loads in plasma and saliva samples from Ugandan children with acute malaria (M+) at the time of diagnosis and 14 days after antimalaria treatment, children without malaria (M-), and children with BL. EBV DNA was detected, by real-time polymerase chain reaction, in 31% of the plasma and in 79% of the saliva samples from children in the M+ group. Antimalaria treatment led to clearance of plasma viral load in 85% of the cases but did not affect the levels in saliva. There was a significant difference in plasma EBV loads across the groups. The lowest levels were detected in samples from the M- group, increased levels were detected in samples from the M+ group, and levels reached the highest values in samples from children with BL. The same trend was evident in the frequency and levels of anti-BZLF1 antibodies, which is indicative of viral reactivation. In the M+ group, the positive plasma samples clustered around 7-9 years of age, the peak incidence of BL. The clearance of circulating EBV after antimalaria treatment suggests a direct relationship between active malaria infection and viral reactivation.

Adolescent↗
PubMed2006

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice.

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.

Animals↗
PubMed2006

Nitric oxide and peroxynitrite have different antiviral effects against hantavirus replication and free mature virions.

Reactive nitrogen intermediates (RNI), like nitric oxide (NO) and peroxynitrite, have antiviral effects against certain viruses. Hantaviruses, like other members of the Bunyaviridae family, have previously not been shown to be sensitive to RNI. In this study, we compared the effects of NO and peroxynitrite on hantavirus replication and free mature virions in vitro, and of inducible nitric oxide synthase (iNOS) in hantavirus-infected suckling mice. The NO-generating compound S-nitroso-N-acetylpenicillamine (SNAP), as well as cytokine-induced NO, strongly inhibited hantavirus replication in Vero E6 cells, while pretreatment of free virions with SNAP only had a limited effect on their viability. In contrast, 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, inhibited virus replication only to a very low extent in vitro, but pretreatment of virus with SIN-1 led to a considerably lowered viability of the virions. Infections of various human cell types per se did not induce NO production. The viral titers in iNOS(-/-) mice were higher compared to the controls, suggesting that NO inhibits hantavirus replication in vivo. In conclusion, we show that NO has strong antiviral effects on hantavirus replication, and peryoxynitrite on mature free virions, suggesting that different RNI can have different effects on various parts of the replication cycle for the same virus.

Animals↗
PubMed2006

Type I interferon inhibits Crimean-Congo hemorrhagic fever virus in human target cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of severe hemorrhagic fever occurring sporadically in parts of Africa, Asia, Southeast Europe, and the Middle East. Its recent recognition as a potential agent of bioterrorism/biowarfare highlights the need for effective antiviral therapy. In this study, it is shown that human endothelial cells are permissive to CCHFV. It is also shown that interferon-alpha inhibits the growth of CCHFV in human endothelial and hepatoma cells, reducing virus yields by a factor of 100-1,000. By using a siRNA approach, it was demonstrated that the interferon-induced MxA GTPase is a major factor mediating the antiviral effect against CCHFV, in agreement with previous findings showing that recombinant MxA inhibits CCHFV replication by interacting with the viral nucleocapsid protein. The identification of intrinsic cellular resistance factors that block CCHFV replication may help in designing novel antiviral agents.

Antiviral Agents↗
PubMed2006

Loss of cell membrane integrity in puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin.

Hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome are two diseases caused by hantaviruses. Capillary leakage is a hallmark of hantavirus infection. Pathogenic hantaviruses are not cytotoxic, but elevated levels of serum lactate dehydrogenase (LDH), indicative of cellular damage, are observed in patients. We report increased levels of serum perforin, granzyme B, and the epithelial cell apoptosis marker caspase-cleaved cytokeratin-18 during Puumala hantavirus infection. Significant correlation was observed between the levels of LDH and perforin and the levels of LDH and caspase-cleaved cytokeratin-18, suggesting that tissue damage is due to an immune reaction and that epithelial apoptosis contributed significantly to the damage.

Apoptosis↗
PubMed2006

Serological assays for the detection of human andes hantavirus infections based on its yeast-expressed nucleocapsid protein.

BACKGROUND: The objective of the study was to develop and evaluate IgM and IgG ELISAs and an IgG Western blot test for the serological detection of human infections with Andes virus (ANDV), the major cause of hantavirus cardiopulmonary syndrome (HCPS) in South America. METHODS: The entire nucleocapsid (N) protein-encoding sequence of ANDV (strain AH-1) was cloned and expressed in the yeast Saccharomyces cerevisiae. The polyhistidine-tagged recombinant N (rN) protein of ANDV was purified by nickel-chelation chromatography and characterized by its reactivity with different N-specific monoclonal antibodies. To detect an antibody response directed against ANDV in humans, indirect IgM and IgG ELISAs and an IgG Western blot test based on ANDV rN antigen were developed. The evaluation of the tests was performed using a negative serum panel and 63 blinded sera from Argentina and Chile, containing acute-phase and convalescent sera from HCPS patients. RESULTS: The specificities and sensitivities for the IgM and IgG ELISAs were demonstrated to be very high. The IgG ELISA data were confirmed by the IgG Western blot assay based on the same rN antigen. Almost all anti-ANDV-positive sera reacted to higher endpoint titers with N protein of ANDV than with those of Sin Nombre, Laguna Negra or Puumala virus. The cross-reactivity of anti-ANDV-N IgG-positive sera to rN proteins of other hantaviruses was found to be increased with time after the onset of HCPS. CONCLUSION: The high sensitivity of the novel assays should facilitate early diagnosis of ANDV infections and might contribute to a successful treatment of HCPS patients.

Antibodies, Monoclonal↗
PubMed2006

Tickborne encephalitis virus, Norway and Denmark.

Serum from 2 Norwegians with tickborne encephalitis (TBE) (1 of whom was infected in Denmark) and 810 Norwegian ticks were tested for TBE virus (TBEV) RNA by reverse transcription-polymerase chain reaction. Sequencing and phylogenetic analysis were performed. This is the first genome detection of TBEV in serum from Norwegian patients.

Animals↗
PubMed2005

HFRS causing hantaviruses do not induce apoptosis in confluent Vero E6 and A-549 cells.

Hantaviruses are known to cause little or no cytopathic effect in vitro, but have been suggested to cause apoptosis. To determine whether different hantaviruses would induce apoptosis to varying degrees, confluent Vero E6 cells were infected with the hemorrhagic fever with renal syndrome (HFRS) causing viruses Hantaan, Dobrava, Saaremaa, and Puumala. However, no difference was found in the percentage of adherent cells, or of cells with condensed nuclei, between non-infected and virus-infected cells at 3, 6, 9, or 12 days after infection. Furthermore, no differences in the percentage of cells with inter-nucleosomal cleavage of DNA between uninfected and Hantaan infected cells could be detected using the TUNEL assay. Possibly, slightly more apoptotic cells, but never more than 5%, were detected after Hantaan infection of non-confluent cells as compared to the negative control. Earlier reported results that Tula hantavirus induces significant apoptosis on Vero E6 cells were also verified, suggesting that non-pathogenic hantaviruses might differ from HFRS-causing strains regarding induction of apoptosis. In conclusion, the results indicated that the HFRS-causing hantaviruses might induce a very low level of apoptosis in dividing cells, but not at all in confluent cells.

Animals↗
PubMed2005

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man.

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.

Antibodies, Viral↗
PubMed2005

Optimized diagnosis of acute dengue fever in Swedish travelers by a combination of reverse transcription-PCR and immunoglobulin M detection.

The dengue viruses (genus Flavivirus, family Flaviviridae) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset.

Acute Disease↗
PubMed2005

Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus.

Nitric oxide (NO) is an important signaling molecule between cells which has been shown to have an inhibitory effect on some virus infections. The purpose of this study was to examine whether NO inhibits the replication cycle of the severe acute respiratory syndrome coronavirus (SARS CoV) in vitro. We found that an organic NO donor, S-nitroso-N-acetylpenicillamine, significantly inhibited the replication cycle of SARS CoV in a concentration-dependent manner. We also show here that NO inhibits viral protein and RNA synthesis. Furthermore, we demonstrate that NO generated by inducible nitric oxide synthase, an enzyme that produces NO, inhibits the SARS CoV replication cycle.

Animals↗
PubMed2004

Vaccination of C57/BL6 mice with Dobrava hantavirus nucleocapsid protein in Freund's adjuvant induced partial protection against challenge.

Dobrava hantavirus (DOBV) causes a severe form of hemorrhagic fever with renal syndrome (HFRS) for which there is no therapy or vaccine available. We compared the immunogenicity and protective efficacy of recombinant DOBV nucleocapsid protein (rDOBV N) given with Alum or Freund's as adjuvant, or PBS, in C57/BL6 mice. All mice given Alum or Freund's seroconverted as did 6/8 mice given rDOBV N with PBS. Reciprocal geometric mean total IgG-titers were 5380, 18,100, and 800, respectively, while the mean IgG1/IgG2a ratios were 17.5, 9.25, and 12, respectively. Furthermore, ELIspot assays showed higher levels of IL-4 producing peripheral blood mononuclear cells (PBMCs) in the group given Alum as compared to the other groups. Interestingly, only mice receiving rDOBV N with Freund's adjuvant were protected from challenge (75% protected), indicating that the strong Th2-type of immune response induced by Alum against rDOBV N did not induce protection in mice.

Adjuvants, Immunologic↗
PubMed2004

High yields of stable and highly pure nucleocapsid proteins of different hantaviruses can be generated in the yeast Saccharomyces cerevisiae.

Recently, the high-level expression of authentic and hexahistidine (His)-tagged Puumala (strain Vranica/Hällnäs) hantavirus nucleocapsid protein derivatives in the yeast Saccharomyces cerevisiae has been reported [Dargeviciute et al., Vaccine, 20 (2002) 3523-3531]. Here we describe the expression of His-tagged nucleocapsid proteins of other Puumala virus strains (Sotkamo, Kazan) as well as Dobrava (strains Slovenia and Slovakia) and Hantaan (strain Fojnica) hantaviruses using the same system. All nucleocapsid proteins were expressed in the yeast S. cerevisiae at high levels. The nucleocapsid proteins can be easily purified by nickel chelate chromatography; the yield for all nucleocapsid proteins ranged from 0.5 to 1.5 mg per g wet weight of yeast cells. In general, long-term storage of all nucleocapsid proteins without degradation can be obtained by storage in PBS at -20 degrees C or lyophilization. The nucleocapsid protein of Puumala virus (strain Vranica/Hällnäs) was demonstrated to contain only traces of less than 10 pg nucleic acid contamination per 100 microg of protein. The yeast-expressed nucleocapsid proteins of Hantaan, Puumala and Dobrava viruses described here represent useful tools for serological hantavirus diagnostics and for vaccine development.

Animals↗