Lena Grillner

Recombinant human monoclonal antibodies against different conformational epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibit its interaction with CD81.

The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81-E2 interaction in vitro; all four were positive at 0.3-0.5 microg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2-CD81 interaction, of which one appeared immunodominant in this donor.

Molecular Epidemiology of Norovirus Infections in Stockholm, Sweden, during the Years 2000 to 2003: Association of the GGIIb Genetic Cluster with Infection in Children

ABSTRACT The incidence of norovirus-associated gastroenteritis and the molecular epidemiology of norovirus strains were studied during three seasons (2000-2001, 2001-2002, and 2002-2003) among patients of all ages, mainly from the Stockholm region in Sweden. A total of 3,252 fecal samples were analyzed by reverse transcription-PCR. The incidences of norovirus infection among adults were 23, 26, and 30% during the three seasons studied and 18, 11, and 15% among children 0 to 15 years of age. During the first season, all norovirus strains detected by PCR were typed either by reverse line blot hybridization or nucleotide sequence analysis. During the two successive seasons, a total of 60 norovirus-positive strains from the beginning, peak, and end of the seasons were selected for nucleotide sequence analysis. We identified two dominant norovirus variants over the seasons: a new norovirus variant, recently described as the GGIIb genetic cluster, dominated among children during the first season, and during the following two seasons, a GGII-4 variant dominated. Our data suggest that norovirus infections are common, not only among adults, but also among children, and that some strains may predominantly affect children.

Development and Implementation of a Molecular Diagnostic Platform for Daily Rapid Detection of 15 Respiratory Viruses

Acute respiratory tract infections are caused by a large number of viruses. Diagnostic methods have until recently been available only for a limited number of these viruses. With the objective to achieve sensitive assays for all respiratory viruses, a rational workflow in the laboratory, and a short turn‐around time, a real‐time PCR diagnostic platform for daily rapid detection of 15 respiratory viruses was developed. The system was evaluated on 585 stored nasopharyngeal aspirates from hospitalized children. Previous analysis by immunofluorescence and virus isolation identified viruses in 37% of the samples while the new PCR diagnostic panel detected 57% virus positive samples. The new platform was introduced in the laboratory in October 2007 and has then fully replaced the standard immunofluorescence assay for rapid detection of viruses and virus isolation. J. Med. Virol. 81:167–175, 2009.

Identification of viral agents associated with diarrhea in young children during a winter season in Beijing, China

Abstract Background Viral diarrhea remains a major cause of childhood morbidity and mortality worldwide. Although rotavirus was extensively studied in China, few comprehensive studies of all viral agents related to diarrhea in children have been conducted. Objectives Our study was performed to investigate the role of enteric viruses in acute diarrhea in our country and to evaluate methods that could be used in routine diagnostics. Study design One hundred stool samples were collected from children under 5 years of age seeking medical care for acute diarrhea during the winter season 2000/2001 in Beijing Childrens Hospital. All specimens were initially screened microscopically for leucocytes/red blood cells. Samples with negative results were analyzed for virus presence using commercial EIAs and/or in-house RT-PCRs. Results At least one viral agent was found in 67% of the specimens. The frequency of rotavirus, astrovirus, norovirus and enteric adenovirus was 59%, 8%, 6% and 2%, respectively. Dual infections were found in 9.0% (6/67) of the positive samples. The results from rotavirus and astrovirus EIAs were concordant with those of rotavirus and astrovirus RT-PCRs. Conclusions Enteric viruses play an important role in pediatric diarrhea during the winter season in China. A combination of microscopic examination of stool samples with specific EIA assays to detect virus antigen in stool specimens may be suitable for routine diagnostics.

Evaluation of the Abbott AxSYM cytomegalovirus (CMV) immunoglobulin M (IgM) assay in conjunction with other CMV IgM tests and a CMV IgG avidity assay

ABSTRACT The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.

Seroconversion to Hepatitis C Virus in Dialysis Patients: A Retrospective and Prospective Study

The prevalence and incidence of hepatitis C virus (HCV) infections were studied in 236 dialysis patients and related to clinical data at two hospitals in Stockholm, Sweden. Patients were followed during 12 months and tested by 1st- and 2nd-generation anti-HCV assays. Time of seroconversion to HCV could be determined by retrospective analysis of stored serum samples. A total of 36 (15%) patients were anti-HCV positive. Time of seroconversion could be determined for 23 patients and was in the majority of cases associated with blood transfusions, but late seroconversion (more than 6 months after transfusion) as well as lack of transfusion in some cases implied that HCV might be transmitted through dialysis equipment. Persistence of elevated alanine amino-transferase levels for more than 6 months occurred in 17% of anti-HCV-positive patients. In conclusion, routes of transmission in dialysis units have to be further evaluated since routes other than transfusion may occur and diagnosis may be delayed in this group of patients probably due to a poor immunological response.

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

Genomic comparison of herpes simplex virus type 1 isolates from Japan, Sweden and Kenya.

One-hundred and seventy-two epidemiologically unrelated herpes simplex virus type 1 (HSV-1) strains isolated in Japan (104 strains) and Sweden (68 strains) were compared by analysis of their genome structures using four restriction endonucleases, BamHI, KpnI, SalI and HindIII. In addition, 32 Kenyan HSV-1 isolates previously compared to Japanese isolates were included for further comparison with the Swedish isolates. Remarkable and statistically significant differences were found between the HSV-1 isolates from the three countries. One-hundred and thirty cleavage sites were examined, and it was shown that isolates from two of the three countries were statistically distinguishable at 27 of these loci. Pairwise comparison between isolates from Japan and Sweden, Kenya and Sweden, and Japan and Kenya revealed variation in 18, 16 and 23 sites, respectively. By considering gains and losses of 19 sites, the total of 204 strains could be classified into 92 distinct cleavage patterns. Isolates from the three countries could be distinguished from each other by the pattern, except for one Swedish and two Kenyan isolates which shared a pattern. Twenty-one fragments that were present or absent only in individual isolates from one or other of the three countries could be detected. These results show that HSV-1 strains from geographically separate countries or anthropologically different races have distinct distributions of endonuclease recognition sites.

Antibodies against Hepatitis C in a Population of Swedish Haemophiliacs and Heterosexual Partners

The prevalence of antibodies against hepatitis C virus in 160 Swedish patients with haemophilia or related coagulation disorders and 13 heterosexual partners was assessed by means of an ELISA method. A high prevalence of HCV antibodies was found in haemophiliacs with chronic hepatitis (87%), with HIV-antibodies (87%), with severe form of the coagulopathy (81%), and among those who at any point had received factor concentrates without viral inactivation (79%) or of foreign origin (84%). On the other hand, none of the partners were seropositive, indicating a low risk of sexual transmission.

Hepatitis A immunity in the Swedish population: A Study of the Prevalence of Markers in the Swedish Population

After a 20-year interval, the prevalence of seroimmunity to Hepatitis A (HA) was again investigated in a statistical sample of the adult Swedish population. Sera from 3382 of the 4800 originally selected persons were tested. The prevalence of antibodies to HA had not changed since the 1960s when only the Scandinavian population was considered. In the oldest population born at the beginning of this century, the presence of antibodies amounted to 69%. It gradually declined to 6% in those born in the 1940s. In the population born after 1950, the percentage of seropositive individuals was only 2%. A slightly higher prevalence was seen in the big cities, compared with the rural areas (13% vs 9%). Persons of non-Scandinavian origin showed a different pattern. Those from other European countries showed a prevalence of about 70% in all the age-groups investigated. Among the young adults of Arabic or Asiatic origin, the figure was > 90%. The conclusion is that the native Swedish population has a low natural exposure to HA, which has not changed during the last 20 years. Prophylaxis before going to countries where the disease is endemic is strongly recommended.