Avian flu: multiple introductions of H5N1 in Nigeria.

As the avian influenza virus H5N1 swept from Asia across Russia to Europe, Nigeria was the first country in Africa to report the emergence of this highly pathogenic virus. Here we analyse H5N1 sequences in poultry from two different farms in Lagos state and find that three H5N1 lineages were independently introduced through routes that coincide with the flight paths of migratory birds, although independent trade imports cannot be excluded.

Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes.

To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5-45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830-844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(4)] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L(2)T(2))(4), were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(4)] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates.

A comprehensive phylogenetic study based on the hemagglutinin (H) protein of all known African measles virus (MV) isolates is presented. The study includes 64 new H gene sequences from Ghana. Nigeria and South Africa as well as viruses from Zambia and The Gambia for which only incomplete sequencing data were available and that have previously not been genotyped. The results provide further support to the tentative assignment of the Nigerian and Ghanaian viruses to a new genotype B3 within clade B. A distinct geographic distribution pattern emerged with clade B viruses circulating exclusively in African countries north of the equator. All MV strains from southern Africa grouped in clades A and D with the majority of viruses belonging to genotype D4. The viruses considerably differed by their sensitivity to neutralization by monoclonal antibodies (mAb), but three selected antibodies were sufficient to distinguish between African MVs representing four different genotypes.

A naturally occurring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function.

We have investigated the effect of a new Leu196Pro mutation, identified in the MIDAS-like domain of the beta3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on beta3 integrin receptor function. Expression of the mutant beta3Pro196 subunit in CHO cells, either associated with recombinant human alphaIIb or alphav, resulted in normal biosynthesis of beta3 and heterodimerization with alphav or alphaIIb, but selectively interfered with alphaIIbbeta3 maturation and transport to the cell surface. Functional analysis of the beta3 mutant receptors revealed strong inhibition of alphavbeta3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125FAK phosphorylation and fibrin clot retraction, as opposed to normal alphaIIbbeta3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant aIIbbeta3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL196T motif in the beta3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in beta3 is not involved in alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen, while it is essential for alphavbeta3-mediated interaction with this ligand.

B cells specific for bromelain-treated erythrocytes are not derived from adult rat bone marrow.

As part of an evolutionary layered hematopoietic system, the B lymphocyte compartment consists of different lineages of B lymphocytes, which evolve sequentially during ontogeny. In mice, there is ample evidence for the existence of at least two lineages, a layer of B-1 cells (Ly-1 B cells) and the evolutionary more advanced layer consisting of conventional B cells. In a previous study we were unable to detect B-1 cells in the rat as determined by phenotypic markers. Here we studied the possible existence of putative B-1 cells in the rat based on some functional and developmental characteristics as have been described for mouse B-1 cells. We show that B cells secreting antibodies that recognize bromelain-treated mouse red blood cells (BrMRBC) can be identified in rat spleen, whereas these cells (in contrast to DNP-specific B cells) are virtually absent in lethally X-irradiated and bone marrow (BM) reconstituted animals. The number of anti-rMRBC-secreting B cells could not be restored to control levels by reconstitution with fetal liver cells or by cotransfer of 10(7) cells from peritoneal cavity, lymph node or Peyer's patches or up to 2 x 10(8) splenocytes. Although our findings thus suggest that B-1 cells (or B-1 like cells) may be present in rats, formal proof for the existence of such a lineage in rats awaits definition of these cells at the progenitor level.

A simplified immunoassay based on measles virus recombinant hemagglutinin protein for testing the immune status of vaccinees.

Simplified tests based on recombinant antigens are considered to be important for monitoring immunity against measles virus (MV). The hemagglutinin protein (H) is the main target for neutralising and protective antibodies. We produced a recombinant MV-H protein, in a high-yield mammalian expression system based on the Semliki Forest virus replicon. The antigenicity of this recombinant protein was investigated with monoclonal antibodies and its suitability for measuring the immune status of vaccinees was tested in a large cohort by ELISA (H-ELISA). The results were evaluated against neutralisation (NT) and hemagglutination inhibition (HI) titers and MV-specific IgG measured in a commercial whole-virus based ELISA (MV-ELISA, Enzygnost). The H-ELISA correlated better with HI (r=0.78) and NT titers (r=0.80), than the MV-ELISA (HI, r=0.58; NT, r=0.59). In contrast to the MV-ELISA, the H-ELISA detected no false-positive sera (P < 0.02) and the number of false-negative sera was significantly lower in the H-ELISA than in the MV-ELISA (4/378 vs. 15/378; P < 0.025). The performance of the H-ELISA did not deteriorate significantly when, instead of background corrected net values, uncorrected raw O.D. values of the H-antigen were considered, or when early time points (30 min) were evaluated. These results demonstrate that the recombinant H-ELISA detects efficiently non-immune individuals among vaccinees, despite their relatively low MV-antibody levels. A simplified format with single value measurements did not result in loss of sensitivity or specificity and its performance compared favorably with commercial ELISAs based on whole virus.

Serologic evidence of chicken infectious anemia in commercial chicken flocks in southwest Nigeria.

Sera samples from seven poultry farms in southwest Nigeria consisting of 7 broiler, 10 pullet, 1 layer, 1 cockerel, and 1 broiler breeder flocks were tested for the presence of chicken infectious anemia virus (CIAV) antibodies using a commercial enzyme-linked immunosorbent assay kit. Eleven of the 20 flocks (55%) and six out of seven (86%) farms were positive for CIAV antibodies. The seroprevalence largely depended on the age of the flocks. Seroprevalence was higher within the older pullet and layer flocks (83%-100%) than in the younger broiler flocks (0%-83%). In essence, all flocks older than 6 to 8 wk became infected. This is the first report of serologic evidence of CIAV in Subsaharan Africa. Since Southwest Nigeria is the main port of entry of imported chicken and the hub of major poultry breeders, the disease can probably be found throughout the country and beyond. Further studies are necessary to assess economic losses due to CIAV and the cost benefit of countermeasures.

Characterization of rat HSA/CD24 protein bearing the B lineage-restricted epitope recognized by MAb HIS50.

HIS50 MAb recognizes a GPI-linked molecule on the surface of rat cells committed to the B lineage but is absent from cells of other hematopoietic lineages. Eukaryotic expression cloning of the cDNA has revealed that HIS50 recognizes a rat homologue of human CD24 and murine HSA. CD24/HSA exists in multiple glycoforms and plays an important role in hematopoietic and neural cell development. HIS50 is significant in that it is the only, presently available, anti-rat CD24/HSA MAb. Furthermore it is the only reported MAb with specificity restricted to B cell forms of CD24. Here we report Western blot analysis of HIS50 antigen (ag). In rat bone marrow HIS50 MAb recognized MW species in the range of 35-70 kDa. These species were shown by immunomagnetic cell sorting to be all derived from the HIS50+ cell subset. In other tissues with varying HIS50 levels as judged by immunostaining, apparent levels of HIS50 ag in the 35-70 kDa range varied accordingly. Thus, Western blot data corresponded to the immunostaining data of HIS50 ag, and the size distribution of B-restricted forms of rat CD24 appeared as wide as that reported for the more extensively expressed forms of human and murine CD24. N-Deglycosylation of cell lysates from lymphoid organs reduced the signal in the 35-70 kDa range, without appearance of lower MW HIS50-reactive species. Thus, unlike certain epitopes on human and murine CD24, HIS50 epitopes appeared (partially) N-linked carbohydrate dependent. The data reported here provide a basis for the further use of HIS50 MAb in studying the role of the highly heterogeneous CD24 molecules in cell development.

Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa.

In Africa, highly pathogenic avian influenza H5N1 virus was first detected in northern Nigeria and later also in other regions of the country. Since then, seven other African countries have reported H5N1 infections. This study reports a comparison of full-length genomic sequences of H5N1 isolates from seven chicken farms in Nigeria and chicken and hooded vultures in Burkina Faso with earlier H5N1 outbreaks worldwide. In addition, the antigenicity of Nigerian H5N1 isolates was compared with earlier strains. All African strains clustered within three sublineages denominated A (south-west Nigeria, Niger), B (south-west Nigeria, Egypt, Djibouti) and C (northern Nigeria, Burkina Faso, Sudan, Côte d'Ivoire), with distinct nucleotide and amino acid signatures and distinct geographical distributions within Africa. Probable non-African ancestors within the west Asian/Russian/European lineage distinct from the south-east Asian lineages were identified for each sublineage. All reported human cases in Africa were caused by sublineage B. Substitution rates were calculated on the basis of sequences from 11 strains from a single farm in south-west Nigeria. As H5N1 emerged essentially at the same time in the north and south-west of Nigeria, the substitution rates confirmed that the virus probably did not spread from the north to the south, given the observed sequence diversity, but that it entered the country via three independent introductions. The strains from Burkina Faso seemed to originate from northern Nigeria. At least two of the sublineages also circulated in Europe in 2006 as seen in Germany, further suggesting that the sublineages had already emerged outside of Africa and seemed to have followed the east African/west Asian and Black Sea/Mediterranean flyways of migratory birds.

Passive immunity to measles in the breastmilk and cord blood of some nigerian subjects.

Maternal and cord blood collected from 33 Nigerian mother-child pairs were tested for measles-sepcific IgG. All 33 had protective measles antibodies at the time of delivery with a positive correlation of r = 0.87. Determination of the rate of waning of these antibodies revealed that 58 per cent of these children had lost the protective maternal antibody by the age of 4 months and only 3 per cent of the children had enough antibody to protect them between the ages of 6-9 months. Fifty-five colostrum samples from the same mothers and 347 breastmilk samples collected at various periods of breastfeeding also showed that anti-measles IgA had dropped below the protective cut-off within the first 2 weeks of birth. It is evident that the Nigerian child is born with solid anti-measles antibody but the rate of waning has left a large number unprotected before the first dose of the vaccine. There is an urgent need to review the measles vaccination programme in Nigeria to protect these susceptible infants.

Evidence that intestinal IgA plasma cells in mu, kappa transgenic mice are derived from B-1 (Ly-1 B) cells.

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived, Igh-Ca allotype) mu heavy chain and kappa light chain transgenes, specific for trinitrophenyl, on a C57BL background (Igh-Cb allotype). FACS analyses show that the majority of B cells in peripheral lymphoid organs and bone marrow (BM) express transgenic IgM exclusively. A small proportion of the B cells, however, express endogenous IgM, usually concomitant with transgenic IgM. Three criteria establish that the endogenous IgM expressing B cells belong to the B-1 cell lineage. (i) Endogenous IgM expressing B cells in B6-Sp6 mice have the same localization pattern as B-1 cells from normal animals: they are enriched in the peritoneal cavity. (ii) The endogenous IgM+ B cells have the phenotype of B-1 cells: the endogenous IgM+ peritoneal B cells express Mac-1 (CD11b) and low levels of IgD, and most also express CD5 (Ly-1). (iii) B6-Sp6 BM poorly reconstitutes endogenous IgM+ B cells, just as adult BM from normal mice poorly reconstitutes B-1 cells. In contrast, B cells which only express the transgene are readily reconstituted by B6-Sp6 BM. The few endogenous IgM+ cells in the B6-Sp6 BM recipients are located in the peritoneal cavity and have the phenotype of B-1b cells (previously the Ly-1 B sister population), which are known to be reconstituted by adult BM. Two-color immunofluorescence staining of tissue sections from the gut and from isolated gut lamina propria cells shows the presence of many IgA containing cells, about one-third of which simultaneously express cytoplasmic (transgenic) IgM. The C-region of this IgA is produced by endogenous C alpha genes, because the transgene encodes only for C mu. Furthermore, the majority of gut IgA containing cells do not express the idiotype of the transgene, indicating that most of the gut IgA cells are encoded by endogenous VH genes and thus the result of an isotype switch from endogenous IgM expressing B cells. Since the endogenous IgM+ cells are B-1 cells (both B-1a and B-1b), the data strongly indicate that the intestinal IgA plasma cells also belong to the B-1 cell lineage.

Resistance of recent measles virus wild-type isolates to antibody-mediated neutralization by vaccinees with antibody.

The neutralization capacity of sera from Luxembourgian adolescent vaccinees and from Nigerian women with measles-induced immunity to a number of measles virus strains was compared. Although both cohorts were matched for their hemagglutination inhibition and standard neutralization titers, 12 of the 22 late convalescent sera, and only 6 of 24 vaccinees neutralized all viruses. Similarly, only 2 of 20 viruses were not neutralized by at least 75% of late convalescent sera, in comparison to 10 of 20 viruses that resisted neutralization by at least 75% of the vaccinees. The more resistant viruses were not limited to a certain clade. One Nigerian virus was resistant to neutralization by 30% of the late convalescent women and by 75% of vaccinees. These results suggest that qualitative differences in neutralizing antibodies may reduce further protection of infants by passively acquired immunity against wild-type viruses when vaccinated girls become mothers.

Cell type- and differentiation stage-dependent expression of PML domains in rat, detected by monoclonal antibody HIS55.

Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In isolated rat liver nuclei, HeLa cells, and normal human and rat lymph nodes, staining of HIS55 colocalized with that of 5E10. 5E10 recognizes PML nuclear domains, multimolecular complexes of unknown function containing the product of PML gene and at least two other components. HIS55 foci were expressed widely in many tissues but the expression level varied in a cell type-specific manner, with the number of HIS55 nuclear foci ranging from 0 (as in neurons) to over 100 (as in megakaryocytes) and the size ranging from fine (as in cortical thymocytes) to very large (as in urethra epithelium). HIS55 ag expression level also varied among cells of the same lineage, as observed in embryonic development of rat and in the hemopoietic system of adult rat. The expression level of HIS55 foci roughly correlated with the overall rate of protein synthesis of cells, supporting a role of PML domains as transcription regulatory units. The expression of HIS55 foci, however, did not correlate with the growth index of cell populations. Our observations on normal tissues agreed with the hypothesis that PML domain expression is regulated by external, possibly site-dependent factors. We further supported this by demonstrating that PML domains in rat ventral prostate epithelia were upregulated upon castration.

Processing of the DRB1*1103-restricted measles virus nucleoprotein determinant 185-199 in the endosomal compartment.

MHC class II molecules present to CD4+ T cells protein fragments which mostly derive from the extracellular and from the endosomal compartments. Determinants of cytosolic proteins are, however, also displayed by MHC class II molecules following pathways which are still not yet fully characterized. Here we describe the isolation of DRB1*1103-restricted T cell clones specific for the measles virus (MV) nucleoprotein peptide 185-199 (N185). Experiments were then conducted to delineate how this determinant is assembled with DR molecules. In vitro binding analyses indicated that complexes between the N185 peptide and DRB1*1103 protein are optimally constituted at pH 4-4.5. In cellular experiments it was observed that chloroquine, leupeptin and emetine, which are classical inhibitors of presentation of MHC class II-restricted antigens, when added during infection of B cells with MV, prevent presentation of the N185 determinant. In addition, it was found that the N185 determinant is efficiently presented when the nucleoprotein is exogenously provided to B cells, either by blocking MV fusion with the peptide FFG or by the use of purified nucleoprotein. In contrast, it was observed that nucleoprotein recombinant vaccinia virus (vv-N)-infected B cells weakly stimulated N185-specific T cells, indicating that the restricted localization of the nucleoprotein in the cytosol resulted in a poor presentation of the N185 determinant. Taken together, these findings suggest that it is prior to delivery of the nucleoprotein into the cytosol that the N185 determinant is efficiently assembled with newly synthesized DR molecules in the acidic environment of the endosomal compartment.

Immunosorbent assay based on recombinant hemagglutinin protein produced in a high-efficiency mammalian expression system for surveillance of measles immunity.

Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as "gold standards." In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.

High sequence diversity in infectious bursal disease virus serotype 1 in poultry and turkey suggests West-African origin of very virulent strains.

Fifty-eight outbreaks of Infectious bursal disease virus (IBDV) were observed in vaccinated chicken flocks in four Southwestern states of Nigeria between 1995 and 2000. Bursa samples from 40 flocks were found virus-positive in VP2-specific nested RT-PCR. Sequences of the hypervariable region of VP2 were compared to reference strains of the different IBDV variants including also 1988 isolates from Nigeria. Sequence analysis revealed that all 40 Nigerian isolates belonged to the very virulent (vv) variant. The maximum sequence diversity of 5.7% was higher than in all other vvIBDV sequences listed in Genbank (3.6%). Two clusters within Nigerian isolates are unique to this region. Serotype 1 IBDV was also detected in four symptomatic turkey flocks. The turkey isolates were found within 2 of the 3 VV-clusters of chicken isolates. Full length sequence of a turkey isolate (NIE009t) confirmed its close relation to vvIBDV strain D6948NET for both segment A (1.4% sequence diversity) and segment B (2.1%). Thus, turkeys should be considered susceptible to vvIBDV infection. The unusually high sequence diversity of vvIBDV may be an indication of a West-African origin of this virus, from where it spread to other continents.