Influence of climate change on the incidence and impact of arenavirus diseases: a speculative assessment.

The current worldwide incidence of viral haemorrhagic fevers caused by arenaviruses is briefly reviewed. The recently published Assessment Report of the Intergovernmental Panel on Climate Change has described the changes in global climate that are expected to occur over the course of the present century and beyond. Climate modelling and forecasting have not yet reached the stage where confident predictions of regional changes at the level of a virus endemic area can be made. However, in the regions where pathogenic arenaviruses now circulate, significant effects are likely to include increases in surface temperature, changes in the extent and distribution of rainfall, the occurrence of extreme weather events, glacier retreat, and coastal flooding as a result of sea level rise. The possible impact of these changes on the geographical location and the incidence of arenavirus diseases and its human impact are discussed.

Tick-borne virus diseases of human interest in Europe.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.

Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains.

Different types of microcarriers were assessed for the large-scale culture of influenza virus in the Madin-Darby canine kidney (MDCK) cells. Both porous and solid carriers were examined. A higher titre of influenza A/PR8/34 virus was recovered from cultures using solid (1.3x10(9) PFU per ml) rather than porous carriers (4.0x10(8) PFU per ml). High titres of virus (1.0x10(9) PFU per ml) were also obtained from roller bottle cultures of MDCK cells and the traditional culture technique using embryonated hens' eggs (3.9x10(9) PFU per ml). We found that solid carriers composed of dextran with a positive charge are the most suitable carriers for the large-scale growth of influenza A virus in MDCK cells using serum-free media.

Capsid protein diversity among Norwalk-like viruses.

We have determined the complete capsid gene sequence of 20 Norwalk-like viruses (NLVs) collected predominantly from outbreaks in the UK between 1989 and 1996. These comprised nine genogroup I and eleven genogroup II strains. Phylogenetic analysis of these and 15 published sequences suggest seven genomic sub-groups within genogroup I, including three previously described. In genogroup II, eight sub-groups were apparent, of which four were novel. Amino acid identities between strains of distinct genogroups ranged from 37 to 44% while varying between 61 and 100% for strains within a genogroup. Separate phylogenetic analyses of the N-terminus and central variable region of the capsid showed good correlation. Sequence divergence between strains was greatest within the central variable region, with amino acid sequence identities as low as 28% within a genogroup. These 15 genomic sub-groups provide a framework for further investigations of genetic and antigenic relationships within this calicivirus clade.

Induction of immunity using oral DNA vaccines expressing the measles virus nucleocapsid protein.

In this study, we have examined the feasibility of immunisation against measles with plasmid DNA administered by the oral route. After the oral administration, in two 50 microg doses, of poly(DL-lactide-co-glycolide) (PLGA)-encapsulated DNA expressing measles virus nucleoprotein, increasing titres of N-specific serum IgG antibodies were observed in three of ten C3H/He mice over a period of three months. In comparison, oral vaccination of mice with a replication-defective recombinant adenovirus expressing the same transgene induced serum IgG in all animals tested. We also obtained preliminary indication of adjuvant-like activity of PLGA particles when coadministered intraperitoneally (i.p.) with naked plasmid DNA. These experiments demonstrate that oral delivery of either PLGA-encapsulated plasmid DNA or viral vectored DNA is capable of eliciting strong immune responses in mice. We propose that oral administration of biodegradable microparticles offers a novel strategy for future vaccine design for the safe delivery of DNA to mucosal surfaces.

Expression of neurofilament L-promoter green-fluorescent protein constructs in immortalized Schwann cell-neuron coculture.

The neurofilament L/68 protein (NF-L/68) gene is expressed in the immature Schwann cell phenotype but suppressed after myelin-formation. We have investigated conditions which regulate the activity of the NF-L/68 promoter in green fluorescent protein reporter constructs expressed in the immortal rat Schwann cell strain SCL4.1/F7 in coculture with neurons. Constructs expressed in a plasmid vector containing both the full-length promoter and the 3' proximal 107 bp sequence which includes the cyclic AMP response element (CRE), were active in SCL4.1/F7 cells, but were suppressed as the cells underwent spontaneous growth-arrest. Interaction of SCL4.1/F7 with axons accelerated downregulation of expression from both constructs, however expression of the full-length promoter continued in some cells until the onset of myelin-formation. Expression of the NFL/68 construct recommenced when demyelination was induced in culture by exposure to human sera from patients with paraproteinemic gammopathy. We have demonstrated a method to study the regulation of gene expression patterns in single Schwann cells interacting with neurons and shown that different promoter regions may be controlled by axon-related and -unrelated factors.

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles.

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Oral delivery of micro-encapsulated DNA vaccines.

Oral delivery of vaccines is an attractive alternative to injection. It is a non-invasive procedure which allows access to the gut-associated lymphoid tissues (GALT). Immunisation at GALT results in mucosal immune responses, which may be of particular importance in protection against infection at mucosal surfaces, as well as systemic immune responses. Vaccine antigens can be protected in the gut by encapsulation in poly(DL-lactide-co-glycolide) (PLG) microparticles. Their uptake into the immune inductive tissues of the GALT is mediated by M cells, which selectively phagocytose particles less than 10 microns in diameter. We have developed a method for the PLG encapsulation of plasmid DNA. Encapsulated DNA, expressing the insect protein luciferase under the transcriptional control of the human cytomegalovirus immediate early promoter, was administered to mice by intraperitoneal injection or oral gavage. Intraperitoneal injection of encapsulated DNA elicited good serum IgG and IgM responses and a modest IgA response. Oral administration stimulated good serum antibody titres in all three classes, and in addition, significant levels of mucosal IgA. PLG encapsulation thus has the ability to protect plasmid DNA against degradation after administration, and to facilitate its uptake into appropriate cells for the subsequent expression and presentation of antigen, in such a way as to elicit both systemic and mucosal antibody responses. This may have major implications for the design of novel vaccines and delivery strategies.

Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins.

Immune suppression during measles accounts for most of the morbidity and mortality associated with the virus infection. Experimental study of this phenomenon has been hampered by the lack of a suitable animal model. We have used the cotton rat to demonstrate that mitogen-induced proliferation of spleen cells from measles virus-infected animals is impaired. Proliferation inhibition is seen in all lymphocyte subsets and is not dependent on viral replication. Cells which express the viral glycoproteins (hemagglutinin and fusion protein) transiently by transfection induce proliferation inhibition after intraperitoneal inoculation, whereas application of a recombinant measles virus in which measles virus glycoproteins are replaced with the vesicular stomatitis virus G protein does not have an antiproliferative effect. Therefore, in vivo expression of measles virus glycoproteins is sufficient and necessary to induce inhibition of lymphocyte proliferation.

Poly(DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration.

We have developed a method for the encapsulation of plasmid DNA in poly(DL-lactide-co-glycolide) microparticles. Encapsulated DNA, expressing the insect protein luciferase under the transcriptional control of the human cytomegalovirus immediate early promoter, was administered to mice by intraperitoneal injection or oral gavage. Intraperitoneal injection of encapsulated DNA elicited good serum IgG and IgM responses, and a modest IgA response. Oral administration stimulated good serum antibody responses in all three classes, and in addition, significant levels of mucosal IgA. PLG encapsulation thus has the ability to protect plasmid DNA against degradation after administration, and to facilitate its uptake into appropriate cells for the subsequent expression and presentation of antigen, in such a way as to elicit both systemic and mucosal antibody responses. These findings may have major implications for the design of novel vaccines and delivery strategies.

Immunization of mice with plasmid DNA expressing the measles virus nucleoprotein gene.

The measles virus (MV) nucleocapsid (N) protein gene has been inserted into a plasmid vector so as to place the gene under the control of the strong constitutive human cytomegalovirus major immediate early promoter. On intramuscular injection of pMV64 DNA into C3H/He mice, seroconversion with increasing titers of N-specific serum IgG antibodies was observed over a period of 3 months. However, when 3-week-old mice were immunized by intramuscular injection of pMV64 in a two-dose schedule, and challenged intracranially with a rodent-adapted measles virus strain (CAM/RB) at 5 weeks of age, no significant protective response was seen. The lack of effective protection evoked by DNA immunization in this model, where MV challenge must take place before 8 weeks of age, may be due to inefficient induction of cell-mediated immunity resulting from expression in muscle tissue, compounded by a relatively slow rise in immune response compared with that seen with the recombinant adenovirus.

Reactions of Ugandan antisera with peptides encoded by V3 loop epitopes of human immunodeficiency virus type 1.

The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 seropositive Ugandans, including asymptomatic persons and AIDS patients, sampled between 1986 and 1992. Most (71%) of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus), 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. Eighteen percent of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. We conclude that analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

PIP: The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 HIV seropositive Ugandans, including 123 asymptomatic persons and 107 AIDS patients, mostly mothers attending prenatal clinics, sampled between 1986 and 1992. 71% of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus); 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); and 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. Although 70% of the 1986 sera reacted with the Uganda A consensus peptide, only 49% of the 1991/92 sera reacted with this peptide (p 0.005). 20% of the 1991/92 sera, compared with only 7% of the 1986 sera, did not react with any of the peptides (p 0.05). There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. 18% of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. The analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

Lassa virus activity in Guinea: distribution of human antiviral antibody defined using enzyme-linked immunosorbent assay with recombinant antigen.

More than 3,100 households in 27 selected villages distributed in the main geographic regions of Guinea were surveyed for the presence of Lassa virus-specific IgG antibodies (LVA), using an enzyme-linked immunosorbent assay (ELISA) with Lassa virus nucleocapsid protein expressed in insect cells infected with a recombinant baculovirus as antigen. The highest prevalence of LVA (25-55%) was found among inhabitants of tropical secondary forest (areas near Gueckedou, Yomou, and Lola) and guinea savannah (Faranah and Kindia areas), near the southern frontiers with Sierra Leone and Liberia. A much lower prevalence (4-7%) was found among inhabitants of mountainous (Pita, Labe, and Mali) and coastal (Boffa, Boké) areas. We found no discernible differences in LVA prevalence between males and females or among various age groups. Testing of 406 hospital staff members of the eight central hospitals in these areas for LVA revealed a similar distribution of seropositivity among hospitals in various prefectures. The highest prevalence of LVA in hospital staff (29-40%) was in the Gueckedou and Lola hospitals. Sera of LVA-positive persons were tested via Western blot analysis. Antibodies bound predominantly to NP and GP2 proteins.

Current progress towards vaccines for arenavirus-caused diseases.

The arenaviruses are primarily viruses of rodents, but some members of the group cause severe disease (Argentine and Bolivian haemorrhagic fevers and Lassa fever) when transmitted to humans in the specific areas of the world where they are enzootic. Current research of relevance to the provision of vaccines against these diseases, which highlights many of the problems encountered generally in the development of vaccines, is reviewed here. Although one of the classical approaches to vaccine production, the use of inactivated preparations of virus of varying degrees of purity, has produced no results of promise, attenuation of a virulent strain of Junin virus by passage in cultured cells has yielded a vaccine strain currently being tested for efficacy in protecting against Argentine haemorrhagic fever in the human population at risk. The experimental evidence for protection in animal model systems by related, apparently non-pathogenic, viruses and by recombinant vaccinia viruses expressing arenavirus proteins is discussed, together with some of the potential difficulties of these approaches.

Sequence of the nucleocapsid protein gene of Machupo virus: close relationship with another South American pathogenic arenavirus, Junín.

The sequence of the nucleocapsid (N) protein of Machupo virus (causative agent of Bolivian haemorrhagic fever) has been determined, and used to infer a phylogenetic relationship to other arenaviruses. The relationship of the virus to Junín and Tacaribe viruses, together with previous demonstrations of antigenic similarity and cross-protection by heterologous viruses, suggest that vaccines developed against Argentine haemorrhagic fever might also be effective against the Bolivian disease.

Sequence analysis of the V3 loop regions of the env genes of Ugandan human immunodeficiency proviruses.

Ugandan strains of human immunodeficiency virus type 1 (HIV-1) were isolated by cocultivation of peripheral blood lymphocytes from infected individuals with cord blood lymphocytes. Sequences from the V3 region of the env gene were amplified by the polymerase chain reaction (PCR) from chromosomal DNA obtained from low passage virus cultures. The PCR products from 13 Ugandan isolates were cloned into a phagemid vector and sequenced. Many isolates contained divergent V3 loop sequences and adjacent regions: diversity was associated with codon deletions or duplications and with nucleotide substitutions, especially G----A transitions. Proviruses from some of the cultures showed extensive diversity within the V3 loop sequences but others were more homogeneous. The V3 loop apices were conserved in 6 of the Ugandan proviruses and these were very similar to the equivalent regions of several Zairean proviruses. The V3 loop apices of African isolates of HIV-1 are divergent from those of North American isolates. The possible biological consequences of this divergence are discussed.