During 2004, a highly pathogenic avian influenza A (H5N1) virus caused poultry disease in eight Asian countries and infected at least 44 persons, killing 32; most of these persons had had close contact with poultry. No evidence of efficient person-to-person transmission has yet been reported. We investigated possible person-to-person transmission in a family cluster of the disease in Thailand.

The complete genomes of three human H5N1 influenza isolates were characterized, together with the haemagglutinin (HA) and neuraminidase (NA) genes from two additional human isolates and one chicken isolate. These six influenza isolates were obtained from four different provinces of Thailand during the avian influenza outbreak in Asia from late 2003 to May 2004. All six Thailand isolates contained multiple basic amino acids at the cleavage site in the HA gene. Amino acid residues at the receptor-binding site of the five human viruses were similar to those of the chicken virus and other H5N1 viruses from Hong Kong. The presence of amantadine resistance in the Thailand viruses isolated during this outbreak was suggested by a fixed mutation in M2 and confirmed by a phenotypic assay. All genomic segments of the Thailand viruses clustered with the recently described genotype Z. The Thailand viruses contained more avian-specific residues than the 1997 Hong Kong H5N1 viruses, suggesting that the virus may have adapted to allow a more efficient spread in avian species.

Dengue viruses (DV), composed of four distinct serotypes (DV1 to DV4), cause 50 to 100 million infections annually. Durable homotypic immunity follows infection but may predispose to severe subsequent heterotypic infections, a risk conferred in part by the immune response itself. Antibody-dependent enhancement (ADE), a process best described in vitro, is epidemiologically linked to complicated DV infections, especially in Southeast Asia. Here we report for the first time the ADE phenomenon in primary human dendritic cells (DC), early targets of DV infection, and human cell lines bearing Fc receptors. We show that ADE is inversely correlated with surface expression of DC-SIGN (DC-specificintercellular adhesion molecule-3-grabbingnonintegrin) and requires Fc gamma receptor IIa (FcγRIIa). Mature DC exhibited ADE, whereas immature DC, expressing higher levels of DC-SIGN and similar FcγRIIa levels, did not undergo ADE. ADE results in increased intracellular de novo DV protein synthesis, increased viral RNA production and release, and increased infectivity of the supernatants in mature DC. Interestingly, tumor necrosis factor alpha and interleukin-6 (IL-6), but not IL-10 and gamma interferon, were released in the presence of dengue patient sera but generally only at enhancement titers, suggesting a signaling component of ADE. FcγRIIa inhibition with monoclonal antibodies abrogated ADE and associated downstream consequences. DV versatility in entry routes (FcγRIIa or DC-SIGN) in mature DC broadens target options and suggests additional ways for DC to contribute to the pathogenesis of severe DV infection. Studying the cellular targets of DV infection and their susceptibility to ADE will aid our understanding of complex disease and contribute to the field of vaccine development.

Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis.

Ab-dependent enhancement (ADE) of dengue virus (DENV) infection is mediated through the interaction of viral immune complexes with FcγRs, with notable efficiency of FcγRII. Most human dengue target cells coexpress activating (FcγRIIa) and inhibitory (FcγRIIb) isoforms, but their relative roles in ADE are not well understood. We studied the effects of FcγRIIa and FcγRIIb by transfecting cells to express each individual receptor isoform or through coexpression of both isoforms. We showed that although both isoforms similarly bind dengue-immune complexes, FcγRIIa efficiently internalized virus leading to productive cellular infection, unlike FcγRIIb. We next focused on the main discriminating feature of these isoforms: their distinct intracytoplasmic tails (FcγRIIa with an immunoreceptor tyrosine-based activation motif [ITAM] and FcγRIIb with an immunoreceptor tyrosine-based inhibitory motif [ITIM]). We engineered cells to express “swapped” versions of their FcγRII by switching the cytoplasmic tails containing the ITAM/ITIM motifs, leaving the remainder of the receptor intact. Our data show that both FcγRIIa and FcγRIIb comparably bind dengue immune complexes. However, wild type FcγRIIa facilitates DENV entry by virtue of the ITAM motif, whereas the swapped version FcγRIIa-ITIM significantly inhibited ADE. Similarly, replacing the inhibitory motif in FcγRIIb with an ITAM (FcγRIIb-ITAM) reconstituted ADE capacity to levels of the wild type activating counterpart, FcγRIIa. Our data suggest that FcγRIIa and FcγRIIb isoforms, as the most abundantly distributed class II Fcγ receptors, differentially influence Ab-mediated DENV infection under ADE conditions both at the level of cellular infection and viral production.