Within-patient emergence of the influenza A(H1N1)pdm09 HA1 222G variant and clear association with severe disease, Norway.

The association between a particular mutation in the HA1 subunit of the influenza virus haemagglutinin, D222G, and severe and fatal disease in cases of influenza A(H1N1)pdm09 in Norway during the 2009 pandemic was investigated using pyrosequencing. The prevalence of the variant among fatal cases was 8/26 and among severe non-fatal cases 5/52. No D222G mutations were found among the 381 mild cases. This difference could not be attributed to sampling differences, such as body location of sampling, or duration of illness. In cases with mutant virus where clinical specimens from different days of illness were available, transition from wild-type to mutant virus was commonly observed (4/5), indicating that the mutant virus emerged sporadically in individual patients. In patients with paired samples from both the upper and lower respiratory tract (n=8), the same viral genotypes were detected in both locations. In most of the D222G cases (11/13), the mutant virus was found as a quasispecies.

Age-dependent prevalence of antibodies cross-reactive to the influenza A(H3N2) variant virus in sera collected in Norway in 2011.

Antibody cross-reactivity to the influenza A(H3N2) variant virus recently reported in the United States, was investigated in Norwegian sera. Seroprevalence was 40% overall, and 71% in people born between 1977 and 1993. The most susceptible age groups were children and people aged around 50 years. The high immunity in young adults is likely to be due to strong priming infection with similar viruses in the 1990s. More research is needed to explain the poor immunity in 45­54 year-olds.

Estimating influenza-related excess mortality and reproduction numbers for seasonal influenza in Norway, 1975-2004.

Influenza can be a serious, sometimes deadly, disease, especially for people in high-risk groups such as the elderly and patients with underlying, severe disease. In this paper we estimated the influenza-related excess mortality in Norway for 1975-2004, comparing it with dominant virus types and estimates of the reproduction number. Analysis was done using Poisson regression, explaining the weekly all-cause mortality by rates of reported influenza-like illness, together with markers for seasonal and year-to-year variation. The estimated excess mortality was the difference between the observed and predicted mortality, removing the influenza contribution from the prediction. We estimated the overall influenza-related excess mortality as 910 deaths per season, or 2.08% of the overall deaths. Age-grouped analyses indicated that the major part of the excess mortality occurred in the > or =65 years age group, but that there was also a significant contribution to mortality in the 0-4 years age group. Estimates of the reproduction number R, ranged from about 1 to 1.69.

Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010.

Infection with the recently emerged pandemic influenza A(H1N1) virus causes mild disease in the vast majority of cases, but sporadically also very severe disease. A specific mutation in the viral haemagglutinin (D222G) was found with considerable frequency in fatal and severe cases in Norway, but was virtually absent among clinically mild cases. This difference was statistically significant and our data are consistent with a possible causal relationship between this mutation and the clinical outcome.

High prevalence of antibodies to the 2009 pandemic influenza A(H1N1) virus in the Norwegian population following a major epidemic and a large vaccination campaign in autumn 2009.

The prevalence of antibodies reactive to the 2009 pandemic influenza A(H1N1) was determined in sera collected before the start of the pandemic, during the early phase, and after the main epidemic wave and nationwide vaccination campaign in Norway. A substantial rise in prevalence of antibodies at protective titres, from 3.2% to 44.9%, was observed between August 2009 and January 2010. The highest prevalence, 65.3%, was seen in the age group of 10-19 year-olds.

Start of the influenza season 2008-9 in Europe - increasing influenza activity moving from West to East dominated by A(H3N2).

The influenza season 2008-9 started in week 49 of 2008 and is so far characterised by influenza virus type A subtype H3N2. Isolates of this subtype that were tested proved susceptible to neuraminidase inhibitors, but resistant to M2 inhibitors. The circulating A(H3N2) viruses are antigenically similar to the component in the current northern hemisphere influenza vaccine.

Pandemic influenza A(H1N1)v: human to pig transmission in Norway?

In Norway there is an ongoing outbreak in pigs of infections with pandemic influenza A(H1N1)v virus. The first herd was confirmed positive on 10 October 2009. As of 26 October, a total of 23 herds have been diagnosed as positive. The majority of the herds seem to have been infected by humans. Sequence analysis of pig viruses from the index farm shows that they are identical or virtually identical to human viruses from the same geographical region.

Oseltamivir-resistant influenza A(H1N1) viruses detected in Europe during season 2007-8 had epidemiologic and clinical characteristics similar to co-circulating susceptible A(H1N1) viruses.

During the 2007-08 influenza season, high levels of oseltamivir resistance were detected among influenza A(H1N1) viruses ina number of European countries. We used surveillance data to describe influenza A(H1N1) cases for whom antiviral resistance testing was performed. We pooled data from national studies to identify possible risk factors for infection with a resistant virus and to ascertain whether such infections led to influenza illness of different severity. Information on demographic and clinical variables was obtained from patients or their physicians. Odds ratios for infection with an oseltamivir resistant virus and relative risks for developing certain clinical outcomes were computed and adjusted through multivariable analysis. Overall, 727 (24.3%) of 2,992 tested influenza A(H1N1) viruses from 22 of 30 European countries were oseltamivir-resistant. Levels of resistance ranged from 1% in Italy to 67% in Norway. Five countries provided detailed case-based data on 373 oseltamivir resistant and 796 susceptible cases. By multivariable analysis, none of the analysed factors was significantly associated with an increased risk of infection with anoseltamivir-resistant virus. Similarly, infection with an oseltamivir-resistant virus was not significantly associated with a different risk of pneumonia, hospitalisation or any clinical complication. The large-scale emergence of oseltamivir-resistant viruses in Europe calls for a review of guidelines for influenza treatment.

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.

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.

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.

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.

Experiences after Twenty Months with Pandemic Influenza A (H1N1) 2009 Infection in the Naïve Norwegian Pig Population.

Pandemic (H1N1) 2009 influenza A virus was detected in Norwegian pigs in October 2009. Until then, Norway was regarded free of swine influenza. Intensified screening revealed 91 positive herds within three months. The virus was rapidly transmitted to the susceptible population, including closed breeding herds with high biosecurity. Humans were important for the introduction as well as spread of the virus to pigs. Mild or no clinical signs were observed in infected pigs. Surveillance of SIV in 2010 revealed that 41% of all the Norwegian pig herds had antibodies to pandemic (H1N1) 2009 virus. Furthermore, this surveillance indicated that pigs born in positive herds after the active phase did not seroconvert, suggesting no ongoing infection in the herds. However, results from surveillance in 2011 show a continuing spread of the infection in many herds, either caused by new introduction or by virus circulation since 2009.

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.

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.

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.

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.