Antigenic and genetic analyses of influenza B viruses isolated in Lusaka, Zambia in 1999.

Previous studies of the hemagglutinin (HA) genes of various influenza B virus isolates demonstrated the existence of two antigenically distinct virus lineages represented by B/Victoria/2/87 and B/Yamagata/16/88, respectively. Here, we investigated the antigenic and genetic characteristics of influenza B viruses isolated from children living in Lusaka, Zambia between January and May 1999. Antigenic analysis with chicken antiviral sera showed that all the Zambian isolates had the HA protein belonging to B/Yamagata/16/88-related lineage. Furthermore, phylogenetic analyses of the eight RNA segments performed by using the total or partial nucleotide sequences of the two representative Zambian strains (B/Lusaka/270/99 and B/Lusaka/432/99) as well as the previously reported sequences suggested that the Zambian viruses are closely related to the recently circulating reassortants represented by B/Shiga/T30/98 and B/Yamanashi/166/98 which acquired the genes coding for three polymerase proteins (PB2, PB1, and PA), HA, nucleoprotein, and matrix protein from a B/Yamagata/16/88-like parent and the gene encoding nonstructural proteins (NS1 and NS2) from a B/Guandong/8/93-like parent.

Bafilomycin A(1) inhibits rhinovirus infection in human airway epithelium: effects on endosome and ICAM-1.

To examine the effects of bafilomycin A(1), a blocker of vacuolar H(+)-ATPase, on rhinovirus (RV) infection in the airway epithelium, primary cultures of human tracheal epithelial cells were infected with RV14. Viral infection was confirmed by showing that viral RNA in the infected cells and the viral titers in the supernatants of infected cells increased with time. RV14 infection upregulated the production of cytokines and mRNA of intercellular adhesion molecule (ICAM)-1 in epithelial cells. Bafilomycin A(1) reduced the viral titers of RV14 and inhibited the production of cytokines and ICAM-1 before and after RV14 infection. Bafilomycin A(1) reduced susceptibility of epithelial cells to RV14 infection. RV14 increased activated nuclear factor-kappaB in the cells, and bafilomycin A(1) reduced the activated nuclear factor-kappaB. Bafilomycin A(1) decreased the number of acidic endosomes in the epithelial cells. These results suggest that bafilomycin A(1) may inhibit infection by RV14 by not only blocking RV RNA entry into the endosomes but also reducing ICAM-1 expression in the epithelial cells. Bafilomycin A(1) may therefore modulate airway inflammation after RV infection.

Antibody masking renders HIV-1 resistant to cationic membrane filtration through alteration of its electrostatic characteristics.

Previously, it was demonstrated that any human immunodeficiency virus type 1 (HIV-1) strain proliferating in peripheral blood mononuclear cells (PBMCs) in vitro, and resuspended in seronegative plasma, could be captured efficiently (mean > 95%) by a porous polypropylene (PP) membrane modified cationically. We investigated if this cationic membrane could capture HIV-1 obtained from seropositive plasma, and confirmed whether this membrane was effective for the preparation of safe plasma products against HIV-1 transmission. Thirty-six seropositive plasma samples derived from HIV-1 positive cohorts in New York and Lusaka (Republic of Zambia), including 18 cases of acquired immunodeficiency syndrome (AIDS) related complex, AIDS and five terminal cases of AIDS, were filtered through the cationic membrane to determine the reduction of RNA concentration, the gag p24 concentration, and infectious titer. Only a small reduction in RNA concentration (mean < 20%) and almost no decrease in gag concentration (mean < 2%) were obtained, despite the fact that the infectivity was eliminated entirely by the filtration. Due to the possibility that anti-HIV-1 antibodies in patients' plasma combine with HIV-1, laboratory-adapted HIV-1(HTLV-IIIB) was mixed with seropositive plasma to test the effect of antibodies on HIV-1 adsorption, and also to investigate the interfacial electrokinetic potential (zeta-potential) of both intact and plasma-treated HIV-1. The zeta-potential of HIV-1(HTLV-IIIB) in the presence of seropositive plasma was neutral as opposed to negative when stored in seronegative plasma or culture medium. Also the rate of HIV-1 capture by the membrane, as determined by the reduction in RNA concentration, sank from 95% to 20%, the same capture percentage observed when filtering plasma of patients. These findings suggested that in patients' plasma, the antibody-masked HIV-1 comprise most of the viral population, and was not trapped on the cationic membrane because of its electrostatic character. Conversely, the cationic membrane was thought to adsorb antibody-free HIV-1 exclusively. It was suggested that each viral swarm had its own zeta-potential, and this difference in electrostatic character determined the extent of the viral adsorption by the cationic membrane.

Effects of dexamethasone on rhinovirus infection in cultured human tracheal epithelial cells.

To examine the effects of glucocorticoid on rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with either RV2 or RV14. Viral infection was confirmed by demonstrating that viral RNA in infected cells and viral titers of supernatants and lysates from infected cells increased with time. RV14 infection upregulated the expression of mRNA and protein of intercellular adhesion molecule-1 (ICAM-1), the major RV receptor, on epithelial cells, and it increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha in supernatants. Dexamethasone reduced the viral titers of supernatants and cell lysates, viral RNA of infected cells, and susceptibility of RV14 infection in association with inhibition of cytokine production and ICAM-1 induction. In contrast to RV14 infection, dexamethasone did not alter RV2 infection, a minor group of RVs. These results suggest that dexamethasone may inhibit RV14 infection by reducing the surface expression of ICAM-1 in cultured human tracheal epithelial cells. Glucocorticoid may modulate airway inflammation via reducing the production of proinflammatory cytokines and ICAM-1 induced by rhinovirus infection.

Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production.

To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1alpha and IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1alpha and IL-1beta significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-alpha was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.

Safety and efficacy of the neuraminidase inhibitor zanamivir in treating influenza virus infection in adults: results from Japan. GG167 Group.

The study was carried out to evaluate the therapeutic effects of zanamivir, a highly selective, potent and specific inhibitor of influenza A and B virus neuraminidases, in adult patients with acute influenza-like illness. Patients who presented within 36 h of the onset of influenza-like symptoms were randomly assigned to receive one of three treatments, twice daily, for 5 days: 10 mg zanamivir powder for inhalation (zanamivir inhalation group), 10 mg zanamivir powder for inhalation plus 6.4 mg zanamivir nasal spray (zanamivir inhalation plus intranasal group) or placebo (placebo group). The primary end point was the time to alleviation of the three major symptoms (fever, headache and myalgia). The secondary end point was the time to alleviation of five influenza symptoms (fever, headache, myalgia, cough and sore throat). One hundred and sixteen patients with influenza-like illness were recruited to the study. No differences were observed between the two groups of patients who received zanamivir (inhalation group or inhalation plus intranasal group). Patients who received zanamivir recovered significantly faster (median 3 days to recovery) than the patients in the placebo group (median 4 days to recovery; P < 0.01). Topically administered zanamivir was well tolerated. This study confirms that in adults, topically administered zanamivir is well tolerated and is effective in reducing the time to alleviation of influenza symptoms.

Respiratory syncytial virus infection in children with acute respiratory infections in Zambia.

Epidemiological research on respiratory syncytial virus (RSV) infections in children was carried out at the Virology Laboratory, University Teaching Hospital (UTH), in Lusaka, Zambia, from January-December 1996. Specimens including 736 nasal washings and 2424 throat swabs were collected from children with acute respiratory infections (ARI) and tested for RSV by enzyme immunoassay and by virus isolation. RSV was isolated in 62 (4.1%) of 1496 throat swabs collected from March to September and was detected in 99 (16.3%) of 609 nasal washings from March to November. The average RSV isolation rate was 2.6% and the average RSV detection rate was 13.5%. The highest RSV isolation (8.1%) and detection (30.5%) rates were in June 1996. RSV antibody in the 278 serum specimens collected from Zambian children, who were hospitalized in the paediatric ward, UTH, was detected using a standard neutralization test. The antibody positive rate was 60-80% in children > 4 years. It is evident that RSV is one of the main causal agents of ARI in children in Zambia.

Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1beta.

Exacerbations of asthma are often associated with respiratory infection caused by rhinoviruses. To study the effects of rhinovirus infection on respiratory epithelium, a primary target for respiratory viruses, human rhinovirus (HRV)-2 and HRV-14 were infected to primary cultures of human tracheal epithelial cells. Viral infection was confirmed by showing that viral titers of supernatants and lysates from infected cells increased with time and by polymerase chain reaction. HRV-2 and HRV-14 infections upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major rhinovirus receptor, on epithelial cells, and they increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha in supernatants. Antibodies to ICAM-1 inhibited HRV-14 infection of epithelial cells and decreased the production of cytokines after HRV-14 infection, but they did not alter HRV-2 infection-induced production ofcytokines. IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to HRV-14 infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, a neutralizing antibody to IL-1beta significantly decreased viral titers of supernatants and ICAM-1 mRNA expression after HRV-14 infection, but a neutralizing antibody to TNF-alpha was without effect. Immunohistochemical studies revealed that both HRV-14 infection and IL-1beta increased ICAM-1 expression on cultured epithelial cells. These findings imply that HRV-14 infection upregulated ICAM-1 expression on epithelial cells through increased production of IL-1beta, thereby increasing susceptibility to infection. These events may be important for amplification of airway inflammation after viral infection in asthma.

Laboratory diagnosis of acute measles infections in hospitalized children in Zambia.

Laboratory diagnosis of measles infection is rarely performed in developing countries and tends to depend on clinical symptoms alone. We evaluated detection of immunoglobulin M (IgM) antibodies for confirmation of acute measles infection in Zambia. In 149 hospitalized children with clinical diagnosis of measles, IgM antibodies were detected in 88.6% (132/149). The IgM-positive rate increased with time after onset of skin rash and all samples were positive after 4 days. In addition to IgM antibody test, virus isolations from throat swabs using B95a cells were also performed. These were positive in only 20.9% (14/67), and both IgM and virus isolation in combination increased the positive rate to 92.5% (62/67). Vaccinated children had higher neutralizing (Nt) antibody responses and, among IgM-negative patients, all 4 vaccinated children had high Nt antibodies while all 10 unvaccinated children had negative or low Nt results. The IgM antibody test was proved to be a sensitive method for laboratory confirmation of measles virus infection in developing countries.

Epidemiology of influenza virus infections in children with acute respiratory infections in Zambia.

A viral aetiological and epidemiological study of acute respiratory infections (ARI) in children was carried out in Lusaka, Zambia between June 1993 and September 1995. A total of 3,760 throat swab specimens were collected for virus isolation from children under 5 years of age who had ARI and were attending three health centres in Lusaka. Between June and November 1993, 52 cases of the influenza A/H3N2 viruses were isolated. Between May and July 1994, 34 influenza B cases were isolated. In 1995, one A/H3N2 influenza virus was isolated in January and then the same type of influenza virus was isolated from 55 samples between June and August. The isolation rate of influenza virus was highest at 14.3% (20/139) in August 1993, at 15.1% (18/119) in June 1994 and at 25.4% (43/169) in July 1995. This is the first report of a consecutive study of influenza virus infections in Zambia and the results reveal that influenza virus infections are one of the most important pathogens of ARI in children in the cool, dry season (June-August) in this country.

Interspecies transmission of influenza C virus between humans and pigs.

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

Frequent occurrence of genetic reassortment between influenza C virus strains in nature.

Previous studies of the haemagglutinin-esterase (HE) genes of various influenza C isolates suggested the existence of three distinct virus lineages (C/Yamagata/26/81-, C/Aichi/1/81- and C/Mississippi/80-related lineages) in Japan in the 1980s. Here we analysed the genetic properties of three strains (C/Yamagata/5/92, C/Miyagi/3/93 and C/Miyagi/4/93) isolated in Yamagata and Sendai Cities, Japan, in 1992/1993. Comparison of total or partial nucleotide sequences of the seven RNA segments of C/Yamagata/5/92 with those of 11 previous isolates suggested that the 1992 strain is a reassortant which inherited HE, P3, NP and M genes from a C/Mississippi/80-like virus and PB2, PB1 and NS genes from a C/pig/Beijing/115/81-like virus. Furthermore, it became evident that at least two (C/England/83 and C/Yamagata/9/88) of the 11 reference strains are also reassortants.

[The etiology of acute lower respiratory tract infections in infants].

The etiology of acute lower respiratory tract infections (ALRI) was studied in pediatric inpatients under 2 years of age admitted to Chiba Municipal Hospital between June 1994 and March 1995. Eighty-seven patients, 99 episodes were investigated for bacterial infection with the use of blood culture and washed sputum culture, for viral infection with the use of virus isolation, antigen detection and antibody assays, for Mycoplasma pneumoniae infection with the use of antibody assay and for Chlamydia infection with the use of antigen detection. Pathogens were identified in 71 (71%) of the 99 episodes. Evidence of bacterial infection was detected in 43 episodes (43%), viral infection in 37 episodes (37%), Mycoplasma pneumoniae infection in 4 episodes (4%) and Chlamydia infection 3 episodes (3%). The major bacterial pathogens were H. influenzae, M. (B) catarrhalis and S. pneumoniae. RS virus and influenza virus epidemics occurred during the winter. A mixed bacterial and viral infection was documented in 13 episodes (13%). RS virus infection was common in infants up to 6 months old. Mixed bacterial and influenza virus infections were common in 1 or more year old children. Virus isolation was useful for the grasp of the viral epidemic. Bacterial associated infections were common in children under 2 years of age with ALRI. Washed sputum culture and sputum gram stains' were useful for the treatment of infant ALRI.

Prevalence of hepatitis B antigens in human immunodeficiency virus type 1 seropositive and seronegative pregnant women in Zambia.

PIP: During April-October 1992 in Zambia, 1861 pregnant women attending prenatal clinics in 3 urban health centers in Lusaka and 5 district hospitals in various provinces were recruited to examine the association between HIV infection and hepatitis B virus (HBV) infection. 340 (18.3%) tested positive for HIV infection. HIV-positive pregnant women were more likely to test positive for the hepatitis B surface antigen (HBsAg) than were HIV-negative pregnant women, but the difference was not significantly different (7.1% vs. 5.4%; p = 0.23). On the other hand, among the HBsAg-positive pregnant women, HIV- infected women were more likely to test positive for hepatitis B e antigen (HBeAg) than were HIV-negative women (25% vs. 12.3%; p 0.05), suggesting more HBV replication in HIV-infected people. Only women younger than 30 tested positive for HBeAg. If HIV does indeed facilitate HBV replication and increases its rate of vertical transmission, the HBV epidemiological pattern in sub-Saharan African could change. Further studies focusing on the epidemiological impact of HIV on HBV infection in sub-Saharan Africa are needed.^ieng

Hepatitis B virus infection among pregnant women in Zambia.

The prevalence of hepatitis B virus (HBV) markers was studied in pregnant women attending antenatal clinics in Zambia. A total of 2,098 pregnant women were recruited into the study at three urban health centres in Lusaka, the capital city and four district hospitals in rural areas of different provinces of Zambia. The overall prevalence of HBsAg was 6.5% (137/2,098), and HBeAg was present in 16.1% (22/137) of those positive for HBsAg. Antibody positive rate (HBsAb and/or HBcAb) was 51.3% in randomly selected HBsAg negative samples. HBsAg positive rate varied between 3.3% and 13.6% in each study sites. Prevalence for both HBsAg and antibodies to HBV were significantly higher in rural areas (district hospitals) than in urban areas (urban health centres in Lusaka). These data show that although HBV is endemic in Zambia, the prevalence varies from region to region.

Inhibitory effect of pyridobenzazoles on virus replication in vitro.

Twelve species of pyridobenzazoles and pyrimidobenzimidazole were examined as inhibitors of the replication of respiratory syncytial virus (RSV) in HeLa cells. From the pyridobenzazoles studied, 2-benzamido-4-cyano-1-oxo-1H,5H-pyrido[1,2-alpha]benzimidazol emerged as a potent inhibitor of the RSV. Based on its inhibitory effect on the cytopathogenicity of the RSV in HeLa cells, the 50% effective dose was found to be 0.95 micrograms/ml. The cytotoxicity of this compound for HeLa cells was examined by monitoring the incorporation of radiolabeled uridine into cellular RNA. The selectivity index was ca. 30, the same as Virazole.

Measles infection in hospitalized children in Lusaka, Zambia.

A 2-year hospital-based survey of measles infections were carried out at the University Teaching Hospital (UTH) in Lusaka, Zambia from January 1992 to December 1993. During this period, a total of 1066 children with a clinical diagnosis of measles were admitted to the paediatric isolation ward at UTH. Measles cases were seen throughout both 1992 and 1993. However, there was a peak from September to December, 1992. The number of cases decreased with age, and 370 (34.7%) were under 1 year old. It is noteworthy that 203 (19.0%) were less than the 9 months of age which is the recommended time for measles vaccination in Zambia. The overall case fatality rate was 12.6%, and was higher in children aged 0-3 years (14.3%) than in those aged 4 years and above (6.7%). Measles vaccination status could be checked from the child's immunization card for 343 measles cases over 9 months of age, 118 (34.4%) of these having previously received measles vaccine. Vaccinated children had a significantly lower case fatality rate (6.4%) than the unvaccinated group (17.0%). This suggests that while measles vaccine cannot prevent infection, it can reduce the severity of infection.

PIP: During January 1992-December 1993, in Zambia, 1066 children aged 0-15 were admitted to the pediatric isolation ward with measles at the University Teaching Hospital in Lusaka. Measles cases peaked during September-December 1992, which comprised all of the 1992 hot dry season and the beginning of the hot rainy season. There were more measles cases in 1992 than in 1993 (831 vs. 235). 86.9% of all measles cases were 0-5 years old. The overall case fatality rate was 12.6%. Measles cases aged less than 3 were more likely to die than older children (14.3% vs. 6.4%; p = 0.025). The researchers had access to immunization cards in 330 measles cases aged 10 months or older. 34.4% had received measles vaccine earlier. The vaccine efficacy in this group was 44.3%. Vaccinated cases were less likely to die than unvaccinated cases (6.8% vs. 17%; p = 0.009). These findings indicate that the measles vaccine used had a low efficacy, but it minimized the severity of measles and protected against death.

Longitudinal investigation of epidemiologic feature of adenovirus infections in acute respiratory illnesses among children in Yamagata, Japan (1986-1991).

Using human embryonic fibroblast (HEF) and HEp-2 cell cultures, adenoviruses were isolated from 989 (3.7%) out of 26,793 pediatric patients with ARI in Yamagata, Japan from January, 1986 to December, 1991. All isolates were identified as types 1 (Ad1)-6 and no other serotypes were identified. Epidemiologic feature was different depending on the subgenus group. Ad1, 2, 5 and 6 (group C) were endemic and the infections occurred frequently in the summer season. Ad3 (group B) was epidemic in the autumn to winter season, although the virus was isolated every month in non-epidemic season. No seasonal distribution of Ad4 (group E) could be determined because the number of patients was limited. Neutralizing antibody positive ratio for group C were more than 40% at 1-2 years of age and almost 100% by 10 years of age but those for Ad3 (group B) were 40% by 10 years of age. The neutralizing antibodies for Ad4 (group E) or Ad7 (group B) became negative by 10 years of age. With group C infections, most cases were infants and young children less than 2 years of age, but Ad3 infections were older children with the peak at 4 and 5 years of age.