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In man influenza viruses induce a cytolytic T lymphocyte (CTL) activity directed against autologous or HLA-A or -B compatible target cells infected with the immunizing virus. While only type specific CTL are characterized in man, we report here experiments showing intertypic activities of human CTL from donors vaccinated with both A and B type influenza viruses. Their peripheral blood leucocytes (PBL) restimulated in vitro with live influenza virus of one type gave rise to both anti-A and -B activities, when non-infected or Sendaï infected target cells were not lysed. These intertypic activities were restricted by HLA-A or -B antigens and were inhibited by 0KT3 antibody. When u.v.-inactivated viruses were used as restimulating antigen, no intertypic CTL were obtained. The results of competition experiments with cold targets show that no common antigens were recognized by anti-A and anti-B CTL. Moreover the restricting HLA-A or -B molecules seen in association with A or B types viruses appeared different in the same experiment, confirming that different antigens were probably involved for the agents of A and B subgroups. This influenza specific intertypic activity was therefore probably due to an intertypic stimulation of type specific CTL activities, possibly arising at the level of T helper cells. [ABSTRACT FROM AUTHOR]

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC 50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections., Competing Interests: This work is a part of an Open Innovation program instituted by AstraZeneca. M. Preston, D. Smith, A. Davies, and T. Lundbäck are employees and shareholders of AstraZeneca UK Ltd. (M.P., D.S., and A.D.) and AstraZeneca AB (T.L.). M. Fitzek was an employee of AstraZeneca UK Ltd. at the time of this work. All other authors declare that they have no known competing interests.

Background: Norovirus outbreaks cause severe medico-socio-economic problems affecting healthcare workers and patients. The aim of the study was to investigate prevalence of norovirus infection and risk factors for infection in healthcare workers during nosocomial outbreaks., Methods: A cross-sectional study of norovirus infections in healthcare workers was performed in seven outbreak wards in a large university hospital. Packs (swab for rectal sampling, and questionnaire) were posted to healthcare workers on notification of a ward outbreak. Rectal samples were examined with norovirus-specific real-time PCR. Replies from questionnaires were analysed using logistic regression models with norovirus genogroup (G)II positive findings as dependent variable. The results are expressed as odds ratios (OR) with 95% confidence intervals (CI). Sequencing and phylogenetic analyses (1040 nucleotides) were used to characterize norovirus strains from healthcare workers. Cluster analyses included norovirus GII.4 strains detected in ward patients during the ongoing outbreaks., Results: Of 308 packs issued to healthcare workers, 129 (42%) were returned. norovirus GII was detected in 26 healthcare workers (20.2%). Work in cohort care (OR 4.8, 95% CI 1.4-16.3), work in wards for patients with dementia (OR 13.2, 95% CI 1.01-170.7), and having diarrhoea, loose stools or other gastrointestinal symptoms the last week (OR 7.7, 95% CI 2.5-27.2) were associated with increased norovirus prevalence in healthcare workers. Sequencing revealed norovirus GII.4 in healthcare workers samples, and strains detected in healthcare workers and ward patients during a given ward outbreak showed ≥ 99% similarity., Conclusion: Norovirus positive findings in healthcare workers were strongly associated with symptomatic infection, close contact with sick patients, and dementia nursing., (© 2021. The Author(s).)

Background: Hepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3-4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication., Methods: Levels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed., Results: Lower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed., Conclusions: The marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.

Norovirus (NoV) is an important cause of nosocomial gastroenteric outbreaks. This 5-month study was designed to characterize NoV contamination and airborne dispersal in patient rooms during hospital outbreaks. Air vents, overbed tables, washbasins, dust, and virus traps designed to collect charged particles from the air were swabbed to investigate the possibility of NoV contamination in patient rooms during outbreaks in seven wards and in an outbreak-free ward. Symptomatic inpatients were also sampled. Nucleic acid extracts of the samples were examined for NoV RNA using genogroup I (GI) and GII real-time reverse transcription-PCR (RT-PCR). The NoV strains were characterized by RT-PCR, sequencing, and phylogenetic analysis of the RNA-dependent RNA-polymerase-N/S capsid-coding region (1,040 nucleotides [nt]). Patient strains from two outbreaks in one ward were sequenced across the RNA-dependent-RNA-polymerase major capsid-coding region (2.5 kb), including the hypervariable P2 domain. In the outbreak wards, NoV GII was detected in 48 of 101 (47%) environmental swabs and 63 of 108 patients (58%); NoV genotype II.4 was sequenced from 18 environmental samples, dust (n = 8), virus traps (n = 4), surfaces (n = 6), and 56 patients. In contrast, NoV GII was detected in 2 (GII.4) of 28 (7%) environmental samples and in 2 (GII.6 and GII.4) of 17 patients in the outbreak-free ward. Sequence analyses revealed a high degree of similarity (>99.5%, 1,040 nt) between NoV GII.4 environmental and patient strains from a given ward at a given time. The strains clustered on 11 subbranches of the phylogenetic tree, with strong correlations to time and place. The high nucleotide similarity between the NoV GII.4 strains from patients and their hospital room environment provided molecular evidence of GII.4 dispersal in the air and dust; therefore, interventional cleaning studies are justified., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Marked norovirus (NoV) diversity was detected in patient samples from a large community outbreak of gastroenteritis with waterborne epidemiology affecting approximately 2,400 people. NoV was detected in 33 of 50 patient samples examined by group-specific real-time reverse transcription-PCR. NoV genotype I (GI) strains predominated in 31 patients, with mixed GI infections occurring in 5 of these patients. Sequence analysis of RNA-dependent polymerase-N/S capsid-coding regions (∼900 nucleotides in length) confirmed the dominance of the GI strains (n = 36). Strains of NoV GI.4 (n = 21) and GI.7 (n = 9) were identified, but six strains required full capsid amino acid analyses (530 to 550 amino acids) based on control sequencing of cloned amplicons before the virus genotype could be determined. Three strains were assigned to a new NoV GI genotype, proposed as GI.9, based on capsid amino acid analyses showing 26% dissimilarity from the established genotypes GI.1 to GI.8. Three other strains grouped in a sub-branch of GI.3 with 13 to 15% amino acid dissimilarity to GI.3 GenBank reference strains. Phylogenetic analysis (2.1 kb) of 10 representative strains confirmed these genotype clusters. Strains of NoV GII.4 (n = 1), NoV GII.6 (n = 2), sapovirus GII.2 (n = 1), rotavirus (n = 3), adenovirus (n = 1), and Campylobacter spp. (n = 2) were detected as single infections or as mixtures with NoV GI. Marked NoV GI diversity detected in patients was consistent with epidemiologic evidence of waterborne NoV infections, suggesting human fecal contamination of the water supply. Recognition of NoV diversity in a cluster of patients provided a useful warning marker of waterborne contamination in the Lilla Edet outbreak.

Background: Quantification of hepatitis B virus (HBV) DNA and surface antigen (HBsAg) serum levels have become increasingly important for the assessment of clinical stage and response to treatment for chronic hepatitis B. Effective immune clearance results in reduction of viremia by 4-5 log units and HBsAg levels by 2 log, but these processes are not well understood. Thus, it is uncertain to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), an RNA intermediate, contribute to suppression of viremia. Likewise, it is unclear if transcriptional regulation is important for the excessive production of surface antigen (HBsAg) that is a hallmark of HBV infection., Methods: HBV RNA and cccDNA were quantified in 19 liver biopsies from patients with chronic HBV infection, as well as in transfected Huh7.5 cells and in PLC/PRF/5 cells carrying integrated HBV genome., Results: Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R(2) = 0.87, p<0.0001) and HBV DNA levels in serum (R(2) = 0.81, p<0.0001), whereas S-RNA correlated strongly with cccDNA (R(2) = 0.65, p<0.0001) and HBsAg levels (R(2) = 0.57, p = 0.0003). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients (ratio 40 vs. 3, p = 0.01) and in PLC/PRF/5 cells, and was in transfected Huh7.5 cells not influenced by mutations in the HBV core promoter., Conclusion: The reduction of viremia that is observed after loss of HBeAg was mainly explained by reduced cccDNA load in the liver, whereas the contribution of down-regulation of pgRNA transcription was relatively small. Enhanced transcription of S-RNA does not explain excessive production of HBsAg.

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.

The COBAS AMPLICOR hepatitis B virus assay targets a conserved region of the genome and is widely used to monitor treatment of hepatitis B in order to identify emerging resistance. However, the assay failed to recognize increasing viremia levels when YMDD mutations were paralleled by mutations in the segment targeted by the COBAS AMPLICOR probe.

The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at > or =1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (r2 = 0.97).

Hepatitis B virus (HBV) is a major cause worldwide of liver disease, including hepatocellular carcinoma. There are eight known genotypes (A-H), of which genotype A has been divided into two subtypes: A2, prevalent in Europe, and A1, which is prevalent in sub-Saharan Africa, but also occurs in southern Asia. In this study, which includes 14 new complete genomes of non-European genotype A HBV, it was found that West African strains seem to constitute a new subgroup, A3. The high degree of genetic diversity within Africa indicates that genotype A originates from Africa. Based on a 2 % genetic distance between Asian and Somali sequences, it seems that the A1 subtype has spread from East Africa to southern Asia during the last 1000-2000 years. Moreover, it is proposed here that the A2 subtype originates from southern Africa and was imported to Europe around 500 years ago or later. The finding of T-1809/1812 close to the precore start codon and T-1862 and A-1888 in the precore region in HBV e antigen-positive children with signs of a mimimal immune response indicates that these substitutions are stable variants, rather than mutations emerging during infection in individual carriers.

Little is known about coinfection among several hepatitis B virus (HBV) genotypes, although previous reports of recombination and genotype shifts indicate that this should occur. In the present study, we designed a method to identify mixtures of genotype A and another genotype, regardless of whether one of them predominates. Using this method, signs of genotypic coinfection were found in 20 (67%) of 30 hepatitis B e antigen-positive patients treated with interferon (IFN). In 8 of these patients, coinfection or genotype shifts were detectable by direct sequencing or standard preS genotyping. In most of these cases, genotype changes were detected after a >2-log decrease or increase of the HBV DNA level. The presence of genotype mixtures did not significantly influence IFN response. Because quasi-species selection may occur at or shortly after transmission, patients might acquire HBV infection from subjects who appear to be infected with a different genotype. This should be considered when tracing the source of HBV infection.

Mutations that prevent the expression of the hepatitis B e antigen frequently emerge in the immunoreactive phase of infection. The predominant mutation, the precore G-->A-1896 mutation, is restricted by the variability at position 1858 and is rare in strains with cytosine at nucleotide 1858. The C-1858 variant is characteristic of genotype A. It also occurs in genotypes C and F, but not in B, D, or E, explaining the geographical variation in the prevalence of precore mutants. C-1858 strains have been frequently observed in southeast Asia, but have not been phylogenetically characterized. By sequencing eight complete hepatitis B virus genomes, C-1858 variants of east Asian origin were found to constitute a phylogenetic entity within genotype C that probably diverged several hundred years ago. Further study of the distribution of this variant is warranted.

Six genotypes of hepatitis B virus (HBV) have been described. However, relatively few complete genomes originating from East Asia, where most of the world's HBV carriers live, have been studied. We analysed five complete HBV genomes of Vietnamese origin, which in our previous studies had produced atypical genotyping patterns. All five strains had HBsAg sequences with markers for serotype adw. In phylogenetic tree analysis, two of the genomes clustered with genotype C, and three clustered on a separate branch between genotypes A, B and C, suggesting a new genotype. However, these three strains showed signs of recombination in similarity plot and bootscanning analysis. Phylogenetic tree analysis of two segments separately supported recombination between genotype C and a putative new genotype (or possibly a subgroup of genotype A). The segment between nt 1801 and 2865 was clearly of genotype C origin, while the major part of the genome (nt 2866-1800) was placed on a branch close to genotype A. The findings encourage further study of genotypes and recombination in HBV from this geographical region.

The survey was undertaken by ESWI in order to investigate the comparability of the laboratory diagnostic methods and the influenza surveillance systems used in 24 European countries. The results indicate considerable consensus in the general approaches to collection and use of clinical specimens, rapid diagnostic techniques, virus isolation techniques in eggs or/and MDCK cell lines, virus identification and use of inhibition of hemagglutination (IHA) and complement fixation (CF) tests for serological diagnostics. However, the details of the techniques used are somewhat heterogeneous: antigen detection methods (immunofluorescence versus immuno adsorbent assay), isolation methods (eggs versus tissue culture), reagents (locally produced, WHO, commercial) are not always equivalent and results are therefore not really comparable. Some of these discrepancies are due to a lack of resources or a lack of priority for influenza in the country. The greatest differences between individual countries exist in the epidemiological part of surveillance programmes. The mode of collection of influenza related mortality and absentism from work varies considerably in different countries. These findings indicate the need to harmonize viral procedures and surveillance systems in European countries in order to improve validity and comparability of results and as a prerequisite for early information on influenza etiology and spread.

Mutations in the hepatitis B virus (HBV) genome have so far been investigated in cross-sectional or short-term longitudinal studies. Information about long-term changes is lacking due to the difficulty of sampling over long observation periods. In this study, a retrospective approach was used that allowed the analysis of changes in the viral genome from transmission to late stages of infection without the requirement for sampling early during this period. The entire viral genome was sequenced from serum samples of three mothers and their 10 adult children, who presumably had been infected vertically. The emergence of mutations between birth and sampling (mean 26.5 years) was assessed by comparing the individual sequences with the sequence of the strain assumed to have been transmitted. The mean differences from this sequence were 0.02 and 0. 28% in seven asymptomatic and one symptomatic hepatitis B e antigen (HBeAg)-positive carriers, respectively, and 0.62 % in five HBeAg-negative carriers. Mutations occurred throughout the genome and 88% of the mutations caused amino acid substitutions spread over all genes. In HBeAg-negative carriers, the number of nucleotide and amino acid changes was independent of the severity of liver disease and, except the (1762)AGG(1764)-->TGA changes, no specific mutation was associated with liver disease. In conclusion, by using a novel method it was found that the entire HBV genome is extremely stable over long periods of time during the HBeAg-positive phase if the immune response (inflammation) is weak, whereas an average of 20 mutations emerged after development of hepatitis and/or loss of HBeAg without association with clinical outcome.

Virus load and liver damage, as measured by quantitative polymerase chain reaction and histology activity index, were related to genotype and core promoter mutations in 43 chronic hepatitis B virus (HBV) carriers of East Asian origin. T-1762 mutants were more frequent in genotype C strains and were associated with more inflammation (P=.0036) and fibrosis (P=.0088) of the liver but not with hepatitis B e antigen (HBeAg) status or virus load. Conversely, precore mutations were associated with less liver inflammation (P=. 08), which was linked to HBeAg negativity and lower viral replication. Carriers with genotype C were more often HBeAg positive (P=.03) with precore wild type strains and more-severe liver inflammation (P=.009) than were those with genotype B. These findings suggest that pathogenic differences between genotypes may exist and that the T-1762 mutation may be useful as a marker for progressive liver damage but seem to contradict that down-regulation of HBeAg production is the major effect of this mutation.

From an overall point of view, the epidemiological situation of influenza C virus infections in western Europe is hardly known. In some countries like Spain, no epidemiological survey has been carried out to determine whether influenza C virus does or does not circulate and cause infection in the considered geographical area. We thus decided to perform such a study. A total of 191 serum samples was collected from people (from 1.5 to 80 years old) living in Spain in October 1990. These sera were tested for the presence of antibodies to influenza C virus by hemagglutination-inhibition (HI) tests. Significant HI activity was found in 59.3 to 64.9% of the 191 tested sera and titres ranged from 20 to 320. The high prevalence of antibody as well as the highly significant titres indicate an intense circulation of influenza C virus in Spain. A significant difference was found between children/teenagers and adults.

Surveillance of influenza has important practical uses for controlling the spread and the severity of epidemic episodes. The participation of sentinel practitioners directly involved in primary patient care is essential and can be promoted by active surveillance networks working in real time with a strong coordinating structure. The Groupes régionaux d'observation de la Grippe (grog) system, elaborated in France since 1984 on these principles, has allowed a short and middle term prevision of outbreaks and the isolation of a large number of influenza strains.

Methods were developed for the purification of the surface, membrane-bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin. Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16-residue peptide with amino-terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.