Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses.

Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.

Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera.

The recently established virus family Flaviviridae contains at least 68 recognized members. Sixty-six of these viruses were tested by cross-neutralization in cell cultures. Flaviviruses were separated into eight complexes [tick-borne encephalitis (12 viruses), Rio Bravo (six), Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S (four), dengue (four) and Modoc (five)] containing 49 viruses; 17 other viruses were not sufficiently related to warrant inclusion in any of these complexes.

Passive immunization of mice with monoclonal antibodies raised against tick-borne encephalitis virus. Brief report.

Adult Balb/c mice were passively immunized with monoclonal antibodies (100 micrograms/mouse) raised against tick-borne encephalitis (TBE) virus then challenged 24 hours later s.c. with 10 LD50 of TBE virus (Nëudorfl isolate). None of the mice showed evidence of premature death although all except one of the monoclonal antibodies tested are capable of enhancing the infectivity of TBE virus in the Fc receptor-bearing mouse macrophage-like cell line P 388 D 1. The ability of monoclonal antibodies to neutralize TBE virus in vitro, and to fix complement was examined, and of these properties only a single monoclonal antibody, which was able to neutralize virus, was also able to protect mice against virus challenge.

The effect of pH on the early interaction of West Nile virus with P388D1 cells.

The interaction between the flavivirus West Nile virus (WNV) and cells of the mouse macrophage-like cell line, P388D1, was assayed by transmission electron microscopy, by following the association of [35S]methionine-labelled virus with cells, and by using a radiobinding assay with an 125I-labelled F(ab')2 fragment of a monoclonal antibody directed against the major viral envelope surface glycoprotein. Using electron microscopy, both fusion and endocytosis were observed at pH 6.4, but at pH 8.0 only endocytosis was observed. When 35S-labelled WNV was bound to the P388D1 cell surface at 0 degrees C, less virus eluted on warming to 37 degrees C at mildly acidic than at alkaline or neutral pH values. The monoclonal antibody fragment had an increased affinity for cell surface viral E glycoprotein after prebound WNV was warmed at mildly acidic pH values. It is proposed that the warming of cell-virus mixtures at low pH results in fusion with a consequent reduction in elution of virus and an increase in the recognition of cell surface-expressed viral envelope glycoprotein by labelled antibody.

The uncoating and infectivity of the flavivirus West Nile on interaction with cells: effects of pH and ammonium chloride.

Infectivity of the West Nile virus (WNV; Flaviviridae) was inactivated on exposure for brief periods (90 s) to pH 6.6 and below. This inactivation was not due to decreased interaction between cells and acid-treated virus. The RNA of [3H]uridine-labelled virus particles prebound to the cell surface before acidic pH treatment underwent rapid uncoating within 1 min at 37 degrees C at the same pH values that inactivated virus particles. The uncoating of [3H]uridine-labelled virus particles was also studied over longer time periods after synchronized internalization by P388D1 cells. At pH 7.6 uncoating occurred rapidly after a reproducible time lag of 1 min on warming to 37 degrees C and was essentially complete by 15 to 30 min after the start of internalization, leaving uncoated RNA in an infectious form. In contrast, at pH 6.2 viral uncoating occurred rapidly without any time lag and the uncoated RNA appeared to be far less infectious than that uncoated at pH 7.6. Ammonium chloride could almost totally inhibit both the infectivity and uncoating of virus particles on synchronized internalization into P388D1 cells, with a pH optimum of 8.0. These results suggest that the uncoating of virus particles is dependent on an acidic pH, although the location of uncoating (prelysosomal endosome or plasma membrane) decides whether the uncoated RNA will be infectious or not. Essentially the same results were obtained when infections were carried out in the presence of enhancing antibody.

A new mechanism for the neutralization of enveloped viruses by antiviral antibody.

Despite the considerable research that has been carried out into viral neutralization by antiviral antibody, its mechanisms remain poorly understood. Cases have been reported in which antiviral antibody can inhibit viral replication without inhibiting the binding and uptake of virus by susceptible cells. It has been shown that many enveloped viruses enter their target cells by endocytosis and are subsequently located in cellular compartments of increasing acidity. With several enveloped viruses this acidic pH can catalyse a fusion reaction between the membrane of the virus particle and that of a prelysosomal endosome, thus enabling the viral core to enter the cytosol and replication to commence. We have recently demonstrated that such an endosomal fusion event at mild acidic pH is involved in the entry pathway of the enveloped flavivirus, West Nile virus (WNV), into macrophages. We now show that antiviral antibody can neutralize WNV by inhibiting this intraendosomal acid-catalysed fusion step and we speculate on possible implications for the future design of antiviral vaccines.

Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement.

We have measured growth of West Nile virus in mouse primary peritoneal macrophages (resident, thioglycolate elicited, and Mycobacterium bovis BCG activated) and in macrophagelike (P338D1) and nonmacrophage (L929, PS clone D) cell lines infected in the absence or presence of specific antibodies (immunoglobulin G ([IgG], IgM), and complement. Monoclonal antibodies directed against Fc receptors (IgG1/2b, 2.4G2) and type 3 complement receptors (Mac-1) were used to define the role of each receptor. Virus yield depended on a balance between enhancement and neutralization and was influenced by the physiologic state of the macrophage, the receptor pathway of viral entry, the mouse strain and age of donor. BCG-activated macrophages displayed a greater ability to restrict West Nile virus than nonactivated cells only in the presence of antiviral IgM, with or without complement; the Fc receptors for various classes of IgG mediated striking enhancement. These studies identify some of the complex innate and acquired factors that determine the interaction between West Nile virus and primary macrophages in vitro.

pH-dependent fusion between the flavivirus West Nile and liposomal model membranes.

Fusion between purified [3H]uridine-labelled West Nile virus (WNV) particles and liposomes containing RNase, was assayed by degradation of the viral RNA to trichloroacetic acid-soluble material. Fusion of virus with liposomes containing phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol (at a molar ratio of 1:1:1:1.5) was found to be dependent on pH with maximum fusion occurring at pH 6.7 and below. At pH 6.6 fusion was rapid and was essentially complete within 2 min at 37 degrees C. At this time, approximately 50% of the viral RNA had been degraded and increasing the concentration of liposomes or time allowed for fusion increased this percentage only slightly. Fusion was dependent on temperature, was almost totally non-leaky and was not dependent on the presence of divalent cations. The lipid composition of liposomes was found to influence both the pH optimum for fusion and the maximum degree of fusion observed. Electron microscopy was used to visualize the fusion reaction between liposomes and virus particles.

Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry.

The mode of entry of West Nile virus (WNV) into the macrophage-like cell line P388D1 was investigated at the electron microscopical level using synchronized infections. The presence of the antiviral monoclonal antibody F6/16A at a concentration that enhanced viral attachment to P388D1 cells ninefold made no difference to the entry pathway of WNV. In both the absence and presence of F6/16A the initial uptake of single viral particles was mediated by coated pits, and started within 30 s of warming the cells to 37 degrees C. Viral particles later appeared in fully or partially coated vesicles and later in uncoated prelysosomal endocytic vacuoles before degradation in lysosomes. However, aggregates of viral particles (five or more virus particles in cross-section), appeared to be phagocytosed whole by cells in a process which involved aggregates being engulfed by extensions of the plasma membrane. This process exhibited a slower time course than the uptake of single viral particles, becoming prominent 15 to 30 min after warming the cells to 37 degrees C. The involvement of a prelysosomal vacuolar compartment in the entry process was shown by a failure to stain for acid phosphatase. This compartment could be specifically loaded with viral particles when viral internalization occurred at 20 degrees C in the presence of 50 mM-ammonium chloride.

Antibody-dependent enhancement of tick-borne encephalitis virus infectivity.

Fourteen mouse monoclonal antibodies raised against tick-borne encephalitis virus (TBEV) and polyclonal antisera raised against six other flaviviruses, Edge Hill (EHV), Japanese encephalitis (JEV), Langat (LGTV), louping ill (LIV), West Nile (WNV) and yellow fever (YFV), were tested for their ability to enhance the replication of TBEV in cells of the mouse macrophage-like line P388 D1, and for their reactivity in ELISA and haemagglutination inhibition (HI) tests. Irrespective of their specificity for either the 51K or 58K polypeptide present in TBEV-infected cells, 13 of the 14 monoclonal antibodies enhanced the replication of TBEV but not of WNV. The remaining monoclonal antibody, which immunoprecipitated the 58K polypeptide of TBEV enhanced WNV but not TBEV, although it reacted strongly with both viruses in ELISA and HI tests. Only polyclonal antisera against viruses within the tick-borne encephalitis virus complex (TBEV, LGTV and LIV) enhanced TBEV replication, although all the polyclonal antisera reacted with TBEV by ELISA; two (against JEV and WNV) also reacted by HI test and all enhanced the replication of WNV. These findings suggest that with TBEV, enhancement may be TBEV complex-specific rather than flavivirus-specific. Data derived from testing both polyclonal and monoclonal antibodies suggest further that not all antibodies that bind to the envelope glycoprotein of TBEV are able to enhance the replication of TBEV, and that enhancement is epitope-specific.

Flaviviridae.

The family Flaviviridae comprises the genus Flavivirus, which contains 65 related species and two possible members. They are small, enveloped RNA viruses (diameter 45 nm) with peplomers comprising a single glycoprotein E. Other structural proteins are designated C (core) and M (membrane-like). The single strand of RNA is infectious and has a molecular weight of about 4 X 10(6) and an m7G 'cap' at the 5' end but no poly(A) tract at the 3' end; it functions as the sole messenger. The gene sequence commences 5'-C-M-E.... The replication strategy and the mode of morphogenesis are distinct from those of the Togaviridae which are slightly larger and morphologically similar in some respects. Flaviviruses infect a wide range of vertebrates, and many are transmitted by arthropods.

Antibody enhanced viral growth in macrophages.

Antiviral antibody can promote viral entry into macrophages by pathways involving cellular receptors for the Fc portion of immunoglobulin or for complement components. Whether virus taken up through these routes is restricted or results in productive infection depends upon a balance between a number of variables. These include the virus strain and dose, the macrophage source and state of activation, the concentration, class and viral specificity of the antibody, and environmental factors such as time and temperature. Under appropriate conditions viral replication can be enhanced by antiviral antibodies.

Togaviridae.

The family Togaviridae comprises four genera: Alphavirus (with 26 species), Rubivirus (one species), Pestivirus (three species), and Arterivirus (one species). The main characteristics of the member viruses are: (i) the virus particles are spherical, 50-70 nm in diameter, including an envelope with surface projections that incorporate two or three polypeptides, usually glycosylated; (ii) the nucleocapsid comprises a core protein and a single strand of positive-sense RNA, molecular weight about 4 X 10(6); where characterized, the RNA has an m7G 'cap' at the 5' end and is polyadenylated at the 3' end; (iii) maturation occurs by budding of spherical nucleocapsids 30-35 nm in diameter, with proven or presumed icosahedral symmetry, through cytoplasmic membranes. Where characterized, translation of structural proteins occurs on subgenomic messenger RNA(s); these appear to represent the 3' end of the genome. Nearly all alphavirus species are transmitted by mosquitoes. Transmission also occurs transovarially (Alphavirus) or transplacentally (Rubivirus and Pestivirus). Members of a genus are serologically related, but are not related to members of other genera.

Flavivirus infection enhancement in macrophages: radioactive and biological studies on the effect of antibody on viral fate.

35S-labelled West Nile virus was used in radioactive binding, internalization and degradation studies in the macrophage cell line P388D1 in the absence or presence of various concentrations of antiviral antibody. Proteases were used to help distinguish between intracellular and extracellular (bound) virus. It was found that the enhancement in viral infectivity that occurs in the presence of subneutralizing concentrations of antiviral antibody was caused by (i) increased binding of virus to the cell surface and (ii) a higher specific infectivity of antibody-opsonized virus particles, apparently due to a more efficient internalization process. In contrast, little difference was found in the rate of internalization and intracellular degradation for virus particles that did enter the cells. Lysosomotropic amines were capable of markedly inhibiting viral replication in P388D1 cells at an early stage of infection both in the absence and presence of subneutralizing concentrations of antibody, although antibody-mediated enhancement of viral replication remained.