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O Hungnes

Publications and source records attributed to O Hungnes.

17 recordsLinked to original sources

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines.

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.

Animals↗

High prevalence of TT virus-related DNA (90%) and diverse viral genotypes in Norwegian blood donors.

Early estimates of the prevalence of TTV viremia in healthy adults of developed countries were in the order of 1--10 %, while similar estimates in Third World countries were considerably higher. Using three different PCRs, TTV-related DNA was detected in serum from 180 out of the 201 Norwegian blood donors tested, indicating that these viruses are almost universally present in adults. Sequence analysis revealed heterogeneity similar to what is found world-wide. The data suggest that the previous discrepancy in prevalences might be related to a lower serum concentration of virus in developed countries. The high prevalence adds evidence to the benign nature of the virus.

Adolescent↗

Intersubtype recombinant HIV type 1 involving HIV-MAL-like and subtype H-like sequence in four Norwegian cases.

Suspected epidemiological links between three cases of human immunodeficiency virus type 1 (HIV-1) infection were verified by the finding of a shared unique virus genotype. A probable male index case was not available for testing. Case 1 was a female sexual partner of the index case. Case 2 was an adult son of case 1. Case 3 was a female sexual partner of case 2. The link to the index case was substantiated by the subsequent finding of another female sexual contact of the index case, harboring the same HIV-1 genotype as the three other cases. To characterize the genotype further, the complete provirus nucleotide sequence was obtained directly from blood cell DNA of case 3. HIV cultivated from case 3 demonstrated CCR5 dependence, an extreme slow-low phenotype, and some genotypic features not present in its directly sequenced counterpart. Most of the gag, pol, and vif genes of these viruses clustered with one of the earliest African HIV-1 strains, MAL, previously classified as a recombinant between the subtypes A, D, and I. Most of the rest of the genome was related to subtype H, albeit with less than 90% identity in most regions. These viruses are the only ones shown to display extensive similarity with MAL in the gag-pol region and among the first HIV-1 recombinants described involving subtype H. We postulate that the gag-pol genes of MAL and these viruses are derived from a common ancestor that is not necessarily intersubtype recombinant in the pol region.

Adult↗

Molecular epidemiology of viral infections. How sequence information helps us understand the evolution and dissemination of viruses.

Viruses evolve much faster than cellular organisms. Together with recent advances in nucleic acid sequencing and biocomputing, this allows us to distinguish between related strains of viruses, and to deduce the relationships between viruses from different outbreaks or individual patients. Databases of nucleotide sequences contain a large number of viral sequences with which novel sequences from local outbreaks can be compared. In this way the dissemination of viruses can be followed both locally and globally. We here review the biological and technological background to the use of virus nucleic acid sequences in epidemiological studies, and provide examples of how this information can be used to monitor human viruses. Molecular studies are particularly valuable for understanding the dissemination and evolution of viruses. The knowledge obtained is useful in epidemiological reconstructions, in real-time surveillance, and may even enable us to make predictions about the future developments of viral diseases.

Animals↗

Mutations designed to alter the stability of a putative RNA duplex in the HIV-1 pol gene.

In the HIV-1 integrase coding region there is a polypurine tract (PPT) involved in the initiation of provirus plus-strand synthesis. Upstream of this PPT there is a 15-nucleotide inverted repeat (IR) complementary to most of the PPT. We have constructed one mutant with five amino acid-neutral U to C and A to G changes in the IR and one mutant with corresponding amino acid-neutral changes in the PPT. Each set of changes abolished the complementarity and suppressed the replication of HIV-1 slightly. The combination of these ten changes restored the complementarity, and doubled the calculated free energy of the putative duplex between the IR and the PPT. This double mutant did not replicate under normal conditions, possibly because the reverse transcriptase was unable to penetrate the duplex. However, when high loads of the double mutant were added to permissive cells, replicative HIV did occasionally appear. The resurrected virus harvested from these cells replicated consistently, even though the ten nucleotide changes were left unchanged. There were no compensatory mutations in the vicinity of the IR/PPT or in the reverse transcriptase gene.

Animals↗

Sequence analysis of HIV-1 group O from Norwegian patients infected in the 1960s.

Three Norwegians, a couple and their daughter, died from AIDS in 1976 after up to 10 years of clinical manifestations of HIV infection (Lindboe et al., 1986, Acta Pathol. Microbiol, Immunol. Scand. 94, 117-123; Frøland et al., 1988, Lancet i, 1344-1345). We here demonstrate the presence of HIV DNA in autopsy materials from the father and the daughter. In phylogenetic analysis, the obtained sequences of the HIV pol and vif genes clustered with the HIV-1 group O clade. The genotyping was confirmed by detection of antibodies against HIV-1 group O in blood samples from the father and the mother. That these and other early isolates of HIV-1 are very similar to the presently circulating viruses and not intermediates between the present subtypes, verifies that the latest common ancestor of HIV-1 existed long before the emergence of the present epidemic. The presence of HIV-1 group O 30 years ago suggests that the limited spread of these viruses, compared to HIV-1 group M viruses, is not due to a later emergence of the group O viruses.

Base Sequence↗

Sequence comparison and mutational analysis of elements that may be involved in the regulation of DNA synthesis in HIV-1.

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the viral RNA (the 3' PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3' side of the PPT to a C did not affect the phenotype.

Base Sequence↗

Mutations in the central polypurine tract of HIV-1 result in delayed replication.

The reverse transcription of HIV-1 generates a linear genomic DNA with a single-stranded gap. The gap is believed to be the result of plus-strand priming from a second, centrally located, polypurine tract (PPT). A mutant containing four amino-acid-neutral purine-to-pyrimidine changes within the central PPT did not replicate as fast as wild-type virus. Another mutant with the entire 15-bp PPT deleted was replication-deficient. The results indicate that plus-strand priming at the central PPT is important for viral replication, possibly by ensuring efficient DNA synthesis.

Base Sequence↗

Survival of HIV-1 activity after disinfection, temperature and pH changes, or drying.

A recently developed assay for measuring infectious HIV-1 particles was used to determine the stability of the virus under various storage conditions as well as the effect of commonly used disinfectants. At the optimum pH of 7.1 the half life of the virus ranged from approx. twenty-four hours at 37 degrees C to no significant loss over 6 months at -75 degrees C. Drying the virus on a glass surface or freezing caused a 5-12 fold and 4-5 fold decrease of activity, respectively. The dried preparations, however, were about as stable as when stored in a buffered solution. A solution of iodine and detergent (2% Jodopax) was the only disinfectant examined which removed all detectable HIV-1 activity. Isopropanol and ethanol were more potent than acetone; however, all three solvents left some viable particles after a 30 min treatment with 70% solutions.

1-Propanol↗

The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA.

During reverse transcription the synthesis of plus strand viral DNA is initiated from an RNA polypurine primer immediately upstream of the U 3 region. The polypurine tract (PPT) sequence at this site is in HIV-1 also present in the middle of the genome. Here we demonstrate that a subpopulation of linear unintegrated HIV-1 DNA has a discontinuity in the plus strand within less than 50 bp from this central PPT, consistent with its utilization as a plus strand initiation site.

DNA Replication↗

Modified oligopeptides designed to interact with the HIV-1 proteinase inhibit viral replication.

The human immunodeficiency virus 1 (HIV-1) codes for a proteinase that cuts viral proteins at specific sites. We have tested 13 modified oligopeptides related to these cleavage sites to see if they inhibit viral replication. To indicate whether a decrease in replication could be due to a general inhibition of cell metabolism, we also measured the effect of the peptides on cellular protein synthesis. Three of the peptides tested (Ac-Gln-Asn-Sta-Val-NH2, Ac-Gln-Asn-Sta-Val-Val-NH2, and Ac-Glu-Asn-Sta-Ile-NH2) inhibited HIV-1 replication at concentrations that did not inhibit protein synthesis. Ac-Gln-Asn-Sta-Val-NH2 was the most potent, causing an approximately 40% decrease in viral replication, measured as the synthesis of HIV-1 antigens and the formation of infectious particles.

Amino Acid Sequence↗

An assay for quantifying infectious HIV particles.

A method for assessing the number of infectious particles in preparations of HIV has been developed. Virus was mixed with cells to allow binding of virus. The cells were then cast in an agar gel to block any further transfer of virus between the cells. After 4 days of incubation the cells initially infected with HIV expressed viral antigens. The percentage of infected cells was then determined by indirect immunofluorescence. The method was developed for HIV, but is presumably suitable for any virus that can replicate in cells not attached to a surface.

Agar↗

Virus quantification by immunofluorescence of cells grown in agar.

We have developed a method for assessing the number of infectious viral particles by measuring what we call fluorescence initiating units (FIV). The present work has been done with HIV, but the methods should be applicable to other viruses as well. Briefly described, cells are mixed with virus and then cast in an agar gel to block further transfer of virus. After a period of incubation sufficient to allow infected cells to express virus antigens, the percentage of infected cells is determined by indirect immunofluorescence.

Cell Line↗

The proteinase inhibitor pepstatin A inhibits formation of reverse transcriptase in H9 cells infected with human immunodeficiency virus 1.

Retroviruses depend on a virus-encoded proteinase. As this enzyme is an interesting target for antiviral therapy, we examined the effect of various low-molecular-weight proteinase inhibitors, as well as a few oligopeptides related to the proteolytic cleavage sites, on the replication of HIV-1 in H9 cells. The increase in reverse transcriptase activity during incubation was assumed to reflect viral replication. Cellular DNA synthesis was measured to quantitate the adverse effects of the inhibitors on the cells. Only one of the substances tested, pepstatin A, had an appreciable selective effect on viral replication. Substances that decreased DNA synthesis generally caused an equally large decrease in reverse transcriptase activity.

CD4-Positive T-Lymphocytes↗

Perforated hepatocytes as a system for studying intracellular proteolysis.

Isolated rat hepatocytes were treated either by electroshock or enterotoxin in order to perforate the plasma membrane. Under appropriate conditions perforated cells could be incubated for a couple of hours without further disruption. The rate of proteolysis was higher in the perforated cells than in homogenized cells. ATP, a thiol reagent and Ca2+/Mg2+ stimulated proteolysis. By using inhibitors, it was shown that the proteolysis was partly lysosomal, and that all four classes of endoproteinases were involved.

Acid Phosphatase↗