Anaesthesia, surgery, obstetrics, and emergency care in Guyana.

The surgical and anaesthesia needs of low-income countries are mostly unknown due to the lack of data on surgical infrastructure and human resources. The goal of this study is to assess the surgical and anaesthesia capacity in Guyana. A survey tool adapted from the WHO Tool for Situational Analysis to Assess Emergency and Essential Surgical Care was used to survey nine regional and district hospitals within the Ministry of Health system in Guyana. In nine hospitals across Guyana, there were an average of 0.7 obstetricians/gynaecologists, 3.5 non-OB surgeons, and 1 anaesthesiologist per hospital. District and regional hospitals performed an annual total of 1520 and 10,340 surgical cases, respectively. All but 2 district hospitals reported the ability to perform surgery. An average hospital has two operating rooms; 6 out of 9 hospitals reported routine medication shortages, and 4 out of 9 hospitals reported routine water or electricity shortages. Amongst the three regional hospitals, 16.1% of pregnancies resulted in Caesarean section. Surgical capacity varies by hospital type, with district hospitals having the least surgical capacity and surgical volume. District level hospitals routinely do not perform surgery due to lack of basic infrastructure and human resources.

Difference between the abilities of human Fcgamma receptor-expressing CV-1 cells to neutralize American and Asian genotypes of dengue virus 2.

Sera from patients involved in a Peruvian outbreak of dengue virus serotype 1 infection cross-neutralized the American genotype of dengue virus serotype 2 up to 100-fold more efficiently than they did the virulent Asian genotype of dengue virus serotype 2, as determined by a plaque reduction neutralization test (PRNT) with CV-1 fibroblasts modified to express human Fcgamma receptor CD32. The concordant preferential immune enhancement of the Asian genotype of dengue virus serotype 2 in human monocytes suggests that such a modification might strengthen the correlation between the PRNT titer and protection.

Influence of the human high-affinity IgG receptor FcgammaRI (CD64) on residual infectivity of neutralized dengue virus.

We examined dengue virus immune complex-phagocyte interaction with respect to a single Fc receptor class using a transient expression system involving the high-affinity human macrophage receptor, FcgammaRI. We found that New Guinea C strain dengue 2 virus formed well-defined plaques in normal and transfected COS cells and we analyzed the structural determinants of FcgammaRI-mediated binding and internalization of dengue 2 virus immune complexes by expressing native or truncated forms of the receptor in COS cells, alone or with its accessory gamma chain signaling unit, which bears an immunoreceptor tyrosine-based activation motif (ITAM). The residual infectivity of dengue 2 virus treated with neutralizing human antiserum was strikingly higher in FcgammaRI-bearing COS cells than in controls. Compatible with the IgG subclass specificity of FcgammaRI, this difference was abrogated quantitatively by treatment of FcgammaRI-transfected cells with human IgG1 but not IgG2 myeloma protein. The magnitude of receptor-mediated plaque formation after cotransfection with gamma chain was also significantly higher than in controls but was less than that observed with FcgammaRI transfection only, a difference probably explained by reduced levels of FcgammaRI expression in gamma chain cotransfectants. Deletion of the FcgammaRI cytoplasmic domain had no effect on receptor-mediated immune complex infectivity. We conclude that the FcgammaRI extracellular domain is sufficient for internalization of infectious dengue virus immune complexes through a mechanism that does not involve classical ITAM-dependent signaling.

Yellow fever virus envelope protein has two discrete type-specific neutralizing epitopes.

Two monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type-specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161, 474-478, 1987) using a second YF-type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.

Replication of yellow fever virus in the mouse central nervous system: comparison of neuroadapted and non-neuroadapted virus and partial sequence analysis of the neuroadapted strain.

Serial passage of yellow fever virus (YF17D) in mouse brain enhances neurovirulence, causing a reduction in survival time after intracerebral inoculation of adult mice. To study the biological and genetic basis for this phenomenon, we compared neurovirulence properties of the neuroadapted Porterfield strain (PYF) to a YF17D strain generated from a full-length YF cDNA template (YF5.2iv). Adult mice were infected by olfactory bulb inoculation, which results in widespread distribution of virus throughout the central nervous system. Although PYF and YF5.2iv spread rapidly throughout the neuraxis, maximal titres of PYF in the brain and spinal cord were 1000- to 10,000-fold higher than those of YF5.2iv. Paralysis and death occurred earlier with the PYF strain. Several cDNA clones of the E/NS1 region of the PYF strain were sequenced. Three predicted amino acid changes were consistently observed in the envelope protein of the PYF strain compared to YF5.2iv. Common substitutions were also identified in NS1 and NS2A. The potential contribution of these genetic differences to neurovirulence was evaluated by generating recombinant, intertypic PYF/YF5.2iv viruses. Physical signs of disease and mean spinal cord titres after inoculation of one recombinant were not different from the YF5.2iv parent. Our data indicate that PYF and YF5.2iv differ significantly in their virulence properties, however, common amino acid substitutions in the E/NS1 region of the PYF strain do not determine its enhanced neurovirulence. Other regions of the viral genome may contribute dominant effects on the virulence properties of the PYF strain.

Neutralizing F(ab')2 fragments of protective monoclonal antibodies to yellow fever virus (YF) envelope protein fail to protect mice against lethal YF encephalitis.

Monoclonal antibodies (MAbs) prepared against the yellow fever virus (YF) vaccine strain 17D (17D YF) envelope E protein were used to investigate Fc piece involvement in antibody-mediated protection against YF encephalitis in mice 17D YF passaged either in Vero cells or in mouse brain (P-YF) to increase neurovirulence was used. To avoid uncertainty concerning antibody clearance and blood-brain barrier penetration, and to directly compare protective activity with neutralization in vitro, pre-formed antibody-virus complexes were injected intracerebrally or assayed for plaque formation in parallel. F(ab')2 fragments of an IgG2a MAb that strongly neutralized both YF strains retained molar equivalent neutralizing activity in vitro, but did not protect. However, further incubation of such F(ab')2-virus antibody complexes with rabbit IgG, but not F(ab')2 anti-mouse IgG resulted in protection. To unambiguously test for Fc piece involvement in this model we derived an IgG2a isotype switch variant from a protective IgG1 MAb-secreting hybridoma and prepared F(ab')2 fragments of the derivative. Intact and fragmented antibodies exhibited weak neutralizing activity. The variant antibody failed to protect against P-YF, but against considerably less neurovirulent 17D YF its protective capacity was 10-fold higher than that of its IgG1 parent. F(ab')2 fragments of the variant did not protect. Together, these results provide strong evidence of an in vivo protective function for the anti-virion antibody Fc piece and indicate that in vitro neutralizing activity as a predictor of antibody protective capacity is dependent on Fc piece integrity and isotype.

Immunization of monkeys with baculovirus-dengue type-4 recombinants containing envelope and nonstructural proteins: evidence of priming and partial protection.

Groups of rhesus monkeys were immunized with baculovirus-dengue type-4 (DEN-4) recombinant-infected cell extracts. One recombinant contained all of the DEN-4 structural proteins and two nonstructural (NS) proteins (C-M-E-NS1-NS2a), while the other was a fusion protein containing a portion of the respiratory syncytial virus G glycoprotein and DEN-4 envelope glycoprotein (RSVG-E). Both preparations were immunogenic; all monkeys receiving either immunogen responded with the production of antivirion antibodies in enzyme immunoassays. All except one monkey receiving the recombinant b(C-M-E-NS1-NS2a) made antibodies to NS1. One monkey that received b(RSVG-E) showed the production of low levels of neutralizing antibodies. Following challenge with unmodified DEN-4 virus, seven of nine monkeys in the immunized group became infected and were viremic for a mean of 4.1 days. The control, sham-inoculated monkeys were also viremic; the mean number of days of viremia in this group was 4.7 days. The remaining monkeys in the immunized group (n = 7), although not protected, had evidence of priming. Hemagglutination inhibition antibody responses following challenge indicated an anamnestic response in this group of animals. Based on these results, it was concluded that future immunization schedules should be altered to optimize immune responses and that immunization with more potent and purified immunogens would probably result in higher seroconversion rates and antibody levels in monkeys.

The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against YF encephalitis in mice.

The mechanism by which antibodies to the flavivirus nonvirion protein NS1 protect mice against encephalitis is unknown but their binding to cell surface NS1 raises the possibility of involvement of an Fc piece-directed immune function. To investigate this, we prepared the F(ab')2 moiety of a protective IgG2a monoclonal antibody (Mab) against yellow fever virus (YF) NS1 and isolated IgG2a- and IgG2b-secreting isotype switch variants from a hybridoma that secretes a nonprotective IgG1 anti-YF NS1 Mab. Each Mab, complexed to NS1, bound to macrophage Fc receptor (FcR) but only the IgG2a and IgG2b Mabs sensitized YF-infected cells to complement-mediated cytolysis. Passively transferred IgG2a Mabs, but not F(ab')2, IgG1, or IgG2b Mabs, interfered with replication of YF in mouse brain. IgG2a Mabs protected mice against YF encephalitis but passive transfer of a mixture of variant IgG2a and parent IgG1 Mabs, or of unrelated IgG2a and IgG1 Mabs directed at the same NS1 epitope, resulted in markedly reduced protection, findings that may bear on flavivirus vaccine design. IgG2a Mab interfered with CNS YF replication in, and protected, C3-depleted mice, but not mice treated with high-dose cyclophosphamide to eliminate antibody-dependent killer cell activity. Taken together, these results indicate that epitope specificity alone may be inadequate to account for protection by anti-NS1 antibody and are consistent with involvement of an FcR-dependent protective mechanism that is governed by antibody isotype.

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses.

Two recombinant baculoviruses were constructed by inserting via the transfer vector pAcYM1 the genes coding for the structural proteins of dengue (DEN)-2 virus downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of DEN-specific envelope (E) glycoprotein. Recombinant DEN-2 E glycoproteins were processed and transported to the surface of Spodoptera frugiperda Sf9 cells infected with both viruses. We show that about one-third of the E glycoprotein minus its whole C-terminal hydrophobic anchor domain was secreted into an endoglycosidase H-resistant form. The type-specific neutralizing epitopes were conserved in the recombinant proteins as shown with a panel of monoclonal antibodies.

Attenuation of wild-type yellow fever virus by passage in HeLa cells.

During the 1960s three different research groups reported that passage of wild-type yellow fever (YF) virus [strain Asibi (YF-Asibi)] in HeLa cells resulted in attenuation of the virus for monkeys so that the virus no longer caused viscerotropic disease. We have repeated and extended this observation to analyse the process of attenuation of YF virus during cell culture passage. A large plaque (LP) variant of YF-Asibi virus became attenuated for both monkeys and mice following six serial subcultures in HeLa cells (YF-Asibi-LP HeLa p6). Thus, attenuation was probably due to a genetic change in the virus population rather than to selective enrichment of a pre-existing variant of YF-Asibi-LP virus. No evidence was obtained to implicate defective interfering particles in the attenuation process. Comparison of the YF-Asibi-LP viruses before and after passage in HeLa cells, using a panel of envelope protein-reactive monoclonal antibodies (MAbs), showed that MAbs which specifically neutralize YF-Asibi-LP virus, and not YF 17D-204 vaccine virus, also neutralized YF-Asibi-LP HeLa p6. This indicated that the epitopes involved in the biological process of neutralization were not altered during attenuation. However, two MAbs that recognize envelope protein epitopes did distinguish between HeLa- and non-HeLa-passaged YF-Asibi-LP virus. One of these (MAb 117) which is YF wild-type-specific, recognized YF-Asibi-LP virus but not YF-Asibi-LP HeLa p6 virus, whereas the other (MAb411), which is YF vaccine-specific, recognized YF-Asibi-LP HeLa p6 virus but not YF-Asibi-LP virus. These results suggest that antigenic changes in the viral envelope protein may determine the relative virulence or attenuation of YF virus.

Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis.

The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge.

Protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus non-structural proteins, NS1, NS2a and NS2b.

Recent evidence of a protective immune response to the flavivirus non-structural protein, NS1, has suggested its incorporation into possible recombinant vaccines. The region of the 17D yellow fever virus (YFV) genome encoding the C terminus of envelope glycoprotein and extending to the N terminus of non-structural protein NS3 (NS1-NS2a-NS2b; nucleotides 2030 to 4940) was expressed in vaccinia virus and physical and immunogenic properties of the NS1 moiety were studied. Recombinant NS1 protein, and native YFV NS1, was detected at the surface of infected cells by immunofluorescence and by immune cytolysis after treatment with anti-NS1 antibody and complement. NS1 was also detected in the extracellular medium as a secreted form. Recombinant NS1 was immunoprecipitated as a single protein of approximately the same size as native 17D YFV NS1. Unboiled, both recombinant and native NS1 formed polymers of high Mr. Immunization of mice with this recombinant vaccinia virus stimulated production of non-neutralizing, complement-fixing cytolytic antibody and conferred partial protection against lethal intracerebral inoculation of mice with live 17D YFV.

Immunogenicity of a purified fragment of 17D yellow fever envelope protein.

Information on the immunogenic properties of purified flavivirus proteins may be useful in the development of recombinant or synthetic peptide vaccines. Using a monoclonal antibody, an attempt was made to purify the envelope (E) protein of 17D yellow fever virus (17D YF) by affinity chromatography. The purified material could not be identified as intact E protein but it did bear antigenic determinants of E as determined by selective reactivity with anti-E monoclonal antibodies. Rabbits immunized with this material produced antibodies that neutralized 17D YF and dengue-2 viruses in comparable titers, indicating that cross-reactive antigenic determinants were preserved. Immunization of mice resulted in protection against intracerebral challenge with 17D YF.

Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody.

Among antibodies to flaviviral proteins only those directed at the virion envelope protein (E) or the non-structural glycoprotein NS1 are known to confer protection. To investigate the possible role of complement-mediated cytolysis (CMC) in protection we measured the capacity of anti-NS1, or E monospecific serum or monoclonal antibodies to bind to yellow fever virus (YFV)-infected cells and of anti-NS1 or E serum to sensitize them to CMC. Although both anti-NS1 and anti-E antibody bound to YFV-infected cells, CMC was observed only with anti-NS1 antibody. Greater binding by anti-NS1 antibody suggested the presence of larger amounts of NS1 than E associated with the cell membrane. Using the cell membrane-impermeable, cross-linking reagent BS3, cell surface NS1, but not E, was detected as a homopolymer, a form in which bound antibody might be expected to activate complement more efficiently. Peak titres of progeny virus were reduced 10- to 100-fold when infected cells were treated with complement-fixing, anti-NS1 monoclonal antibody or monospecific, anti-NS1 rabbit serum and complement. Taken together these results are consistent with the hypothesis that CMC subserved by anti-NS1 antibody provides an alternative to direct neutralization of virus in the protective immune response to flaviviral infection.

Comparison of antigenic sites of subtype-specific respiratory syncytial virus attachment proteins.

A panel of 19 monoclonal antibodies (MAbs) were used to probe the antigenic relationships between the G (attachment) proteins of A and B respiratory syncytial virus (RSV) subtypes (GA and GB). At least three and two antigenic sites were present on GA and GB, respectively, including a shared neutralizing site. Most of the antibodies had some degree of complement-independent neutralizing capacity, but in common was a large neutralization-resistant fraction of virus (range 13 to 78%). Passive administration of MAbs to the cross-reactive antigenic site reduced pulmonary virus titres of both A and B subtype virus in the cotton rat model. Protection with subtype-specific MAbs, however, did not always correlate with in vitro neutralizing capacity. The cross-reactive antigenic site appears to be stable to denaturation by polyacrylamide gel electrophoresis and is present on the unglycosylated and partially glycosylated forms of GA and GB by Western blot analysis of infected cell lysates.

Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus.

Non-neutralizing, serotype-specific anti-NS1 monoclonal antibodies partially protected passively immunized mice from lethal dengue 2 virus intracerebral challenge. There was no apparent correlation between complement-fixing activity and protective capacity among individual anti-NS1 monoclonal antibodies. Immunization with specific combinations of non-protective or partially protective antibodies resulted in prolonged survival or reduced mortality. Solid protection, equal to that achieved after immunization with neutralizing polyclonal antibody, was achieved only with an antibody pair which individually fixed complement to high titre with homologous virus. Some groups of mice had increased morbidity after immunization with combinations of protective monoclonal antibodies that bind to overlapping epitopes. These results may affect the design of recombinant dengue vaccines which may require the inclusion of serotype-specific antigenic domains.

Prospects for a virus non-structural protein as a subunit vaccine.

The design of contemporary vaccines to date has been dependent upon our understanding of the process of stimulation of protective immunity by virion proteins. However, studies with the flaviviruses have demonstrated that protective immunity can be elicited by a non-structural protein, NS1. Surprisingly, immunity to infection is stimulated in the absence of neutralizing antibodies. The information concerning NS1-induced immunity is described and is discussed with relevance to the role of the protein in immunity and vaccine development, against flavivirus diseases. The possibility exists that the phenomenon of non-structural protein induced immunity is applicable to the prophylaxis of other virus diseases.

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain).

The location of a major antigenic determinant involved in the neutralization of a flavivirus, yellow fever virus (YF), has been defined in terms of its position in the amino acid sequence of the E protein. Neutralization escape variants of the 17D vaccine strain of YF were selected with two neutralizing monoclonal antibodies. Nucleotide sequencing of the envelope protein genes (E and M) of the variants showed that in each variant there was a single nucleotide change in the E gene leading to a nonconservative amino acid substitution in the E protein at position 71 or 72. The changes are in a region of the E protein which is hydrophilic, rich in cysteine residues, and not conserved between flavivirus subgroups. Since the selecting monoclonal antibodies neutralize attenuated 17D and virulent Asibi strains of YF with equal efficiency (J. J. Schlesinger, M. W. Brandriss, and T. P. Monath, 1983, Virology 125, 8-17), it can be concluded that the neutralization determinant defined for 17D YF is also present in Asibi YF.