- Volume 77, Issue 10, 1996

We have previously shown that in AIDS patients a predominant species of infectious virus can be found which is not neutralized by homologous serum. The presence of the infectious virus was associated with the lack of type-specific antibody directed against the V3 domains of these virions. In contrast to this lack of V3-specific antibody, the other V3 domains of non-infectious virions were well recognized by antibody. To determine whether the lack of a V3-specific antibody response is due to a progressive loss of antibody during human immunodeficiency virus type 1 (HIV-1) infection, we monitored the anti-V3 antibody response in 90 patients over time. Anti-V3 antibodies were monitored by a V3-specific ELISA using 21 different V3 domains as a fusion with glutathione S-transferase (GST-V3) based upon sequences from 11 HIV-1 patient isolates and 10 sequences from an HIV-1 B subtype consensus-like GST-V3 expression library. This strictly heterologous screening showed a loss of V3-specific antibodies in 20 out of the 90 patients tested. To study the in vivo relevance of these findings we analysed V3 antibody loss in two patients. This strictly autologous antibody screening was performed based upon V3 sequences of the patients’ cell-free virions. In both patients the loss of a V3-specific antibody could be detected in parallel to a decline of CD4+ T cells. Moreover, the escape of a distinct V3 variant was shown to correlate closely with the loss of the V3-specific antibody.

To determine whether human immunodeficiency virus type 1 (HIV-1) in faeces is representative of the HIV-1 population in intestinal tissue, we studied HIV-1 V3 variation in faeces, intestinal biopsies and serum from two individuals. Phylogenic analysis of HIV-1 V3-coding RNA in faeces from one individual showed three distinct genotypes. Viruses belonging to all three genotypes were also present in sigmoidal tissue and in serum. Jejunal tissue contained two of these three genotypes. Analysis of the V3-coding RNA in faeces of the other individual showed five distinct genotypes. One of these genotypes was present in all specimens from this individual. Besides this shared genotype, jejunal tissue and serum contained sequences belonging to one other genotype. In addition, one of the other three V3 variants was detected in sigmoidal tissue. For both persons the shared HIV-1 RNA genotypes in faeces and serum displayed a distinctly different frequency distribution. In one individual, the genotype which was detected in a majority of the clones in faeces (59%) and as a minority in serum (11%), was the most abundant genotype in jejunal and sigmoidal tissue (61% and 80%, respectively). For the other individual the genotype that was present in faeces in a significant number of clones (43%) was detected in serum as a minority (8%), whereas this genotype composed 47% of the clones isolated from jejunal tissue. Taken together these data suggest that faeces contain HIV-1 sequences that are derived from local HIV-1 replication in intestinal tissue.

Two chimeric viruses were constructed between human immunodeficiency virus type 1 (HIV-1) and an apathogenic simian immunodeficiency virus (SIVagm3mc) from African green monkeys. One of the chimeras, HE-A391, expressed the HIV-1-derived env, vpu, tat and rev genes and the SIVagm3mc-derived LTR and the gag, pol and vif genes. The other chimera, SE-H13, contained the SIVagm3mc-derived env, tat and rev genes and the HIV-1-derived LTR and the gag, pol, vif and nef genes. Both constructs yielded infectious viruses and their phenotypes (growth-competence and cell-killing capacity) were examined in various CD4+ cells including human and monkey PBMCs. The results indicated that the replicative properties of the chimeras were mainly dependent on the 5′-genomic half of the parental viruses, and the determinant for viral cytopathogenicity was located within the 5′ half of the HIV-1 genome.

Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV infection. In addition, lymphocyte apoptosis has been described in SIV-infected macaques that developed simian AIDS. To investigate further the role of apoptosis in AIDS pathogenesis, we studied lymphocytes of HIV-2-infected cynomolgus macaques that did not develop simian AIDS. We compared apoptosis of lymphocytes from animals infected with non-pathogenic HIV-2 to that in macaques infected with pathogenic SIV. Unfractionated peripheral blood mononuclear cells of SIV- and HIV-2-infected macaques showed evidence of apoptosis by electron microscopy, flow cytometry (terminal dUTP nick end labelling) and visualization of DNA fragmentation. Between 30–50% apoptotic cells could be detected in SIV-infected animals, compared to approximately 30% in HIV-2-infected and 5–12% in uninfected monkeys. However, separation of PBMC into T cell subpopulations revealed striking differences in apoptosis between SIV- and HIV-2-infected macaques. In SIV-infected monkeys both CD4 and CD8 cells underwent apoptosis to a large extent. In contrast, in the HIV-2-infected macaques apoptosis was restricted to the CD8 cell compartment. The lack of apoptosis in CD4 cells of healthy HIV-2-infected macaques implies an important role for CD4 cell apoptosis in AIDS pathogenesis.

A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled α-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled α-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa α subunit and was competed by α-bungarotoxin. Based on binding of rabies virus to the α subunit and the ability of α-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR α subunit.

We have tested whether immunity can be transferred from a mother fish to its fry. Rainbow trout mother fish were inoculated against infectious haematopoietic necrosis virus (IHNV) by intraperitoneal injection of a fragment of the IHNV glycoprotein spanning amino acids 31 to 310. This protein fragment was obtained by isolating the specific cDNA from Japanese IHNV strain HV7601 and expressing it in Escherichia coli. Fry from immunized and control fish were exposed to IHNV at various intervals after hatching, and their mortality monitored. Survival of the fry of immunized fish was significantly greater when exposure to virus occurred 7 days after hatching, and some immunity appeared to persist until at least 25 days after hatching.

Helper T (Th) cells can be classified functionally into two main types. Broadly, Th1 cells play a major role in eliminating viral pathogens, while Th2 cells mediate anti-parasite immunity and allergic responses. These functions are thought to depend on characteristic and distinct patterns of cytokine production. Infection with human respiratory syncytial virus, an important common cold virus, causes transient lymphocytic bronchiolitis in mice. Activated T cells are partly responsible for this disease, but also eliminate the virus. To show whether polarized cytokine production occurs in individual cells during viral bronchiolitis, we sampled murine bronchoalveolar lavage and mediastinal lymph node cells before and after infection. RT-PCR of cellular mRNA and flow cytometric analysis of intracellular cytokine production showed a rapid IFN-γ response at both sites, which persisted for more than 3 weeks in the lung. Most IFN-γ-producing cells were CD8+. Some early CD4+ IFN-γ-producing cells also made IL-10. Only low levels of IL-2, IL-4 and IL-5 mRNA or protein expression were detected at any time at either site. No cytokines were detected in B cell populations at either site. These novel techniques show the true complexity of cytokine production patterns on a cell-by-cell basis, allowing T cells to be reclassified according to function.

The paramyxovirus phospho-(P) and nucleocapsid (NP) proteins are involved in transcription and replication of the viral genome. To study the interaction between NP and P proteins, we established HeLa cell lines that constitutively expressed the NP and/or P proteins of human parainfluenza virus type 2 (hPIV-2). Co-immunoprecipitation assays revealed that the NP and P proteins can form complexes in HeLa cells expressing both proteins (HeLa-NP + P cells) and in mixed cell lysates of HeLa-NP and HeLa-P cells. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with NP protein. The results indicate that two independent NP-binding sites exist on P protein: one is located in the N-terminal part of the protein, aa 1–47, and the other in the C-terminal part, aa 357–395. In addition, cells co-expressing NP and P proteins with N-terminal deletions showed immunofluorescence staining patterns (granular pattern) similar to those found in hPIV-2-infected cells. However, cells co-expressing NP and P proteins with C-terminal deletions showed a different immunofluorescence staining pattern (diffuse pattern), indicating that the C-terminal region is required for granule formation.

Several years ago, we reported that a Sendai virus (SeV) defective genome (DIH4UV) could be rescued in vivo with human parainfluenza virus type 1 (hPIV1) and bovine PIV3 but not by measles virus or vesicular stomatitis virus. It was concluded that the cis-acting RNA sequences were conserved within the SeV/PIV1/PIV3 group but that interactions between the polymerase complex (P-L) and the template protein N were unique for each virus. We have re-examined these conclusions using proteins expressed from cloned N, P and L genes for SeV and PIV3. The results demonstrate the specificity of the protein-protein interactions between polymerase and template, and confirm the prediction of the earlier work that PIV3 N, P and L proteins are capable of assembling and replicating SeV minigenomes also expressed from a cDNA clone.

The immune response to a vaccinia virus recombinant expressing the measles virus haemagglutinin (VV-HA) was compared after parenteral or mucosal immunizations in mice. Parenteral immunizations with 106 p.f.u. VV-HA induced HA-specific antibody-producing cells (IgG > IgA) and HA-specific class I-restricted cytotoxic T lymphocytes (CTL) in the spleen. In contrast, intranasal administrations of 106 p.f.u. of VV-HA induced HA-specific spot-forming cells in the spleen (IgG > IgA) and the lungs (IgA > IgG), and HA-specific CTL in the spleen. Co-immunization by the nasal route with VV-HA and cholera toxin enhanced HA-specific immune responses. Oral immunizations with 108 p.f.u. of VV-HA generated low numbers of HA-specific IgA-producing cells in the lamina propria of the gut, and a weak HA-specific CTL activity in the spleen and mesenteric lymph nodes. Oral co-immunization with VV-HA and cholera toxin greatly enhanced the level of HA-specific spot-forming cells in the lamina propria (IgA ≫ IgG). Interestingly, intrajejunal immunizations with 108 p.f.u. VV-HA alone induced high levels of anti-HA IgG-producing cells in the spleen and anti-HA IgA-secreting cells in the lamina propria of the gut. These data show that (i) VV-HA by the nasal route is immunogenic and generates a measles-specific mucosal immune response in the lung, which represents the primary site of replication of measles virus and that (ii) VV-HA can also induce measles-specific immunity in the intestine provided that it is protected from degradation in the gastrointestinal tract, or that cholera toxin is used as an adjuvant.

After immunization with measles virus (MV) several monoclonal antibodies (MAbs) were obtained, which reacted with peptides corresponding to the amino acids 361–410 of the haemagglutinin protein (MV-H). Three of these MAbs (BH6, BH21 and BH216) inhibited haemagglutination, neutralized MV in vitro and protected animals from a lethal challenge of rodent-adapted neurotropic MV. These MAbs reacted with the 15-mer peptides H381 and H386 defining their overlapping region 386–395 as a sequential neutralizing and protective epitope, which can be imitated by a short peptide. H381 and H386 share two Cys residues (C386KGKIQALC394ENPEWA) and for optimal MAb binding of peptide (or MV) disulphide bonds were required in addition to a linear C-terminal extension. Other MAbs bound to peptides C- (BH147, BH195) and N-terminally (BH168, BH171) adjacent to the loop but did not neutralize or protect. When sera from measles patients or from women of child-bearing age were tested with the peptides corresponding to this haemagglutinating and neutralizing epitope (HNE), none of the sera recognized the 15-mer peptides of this region, while some reactivity was found to 30-mers homologous to different wild-type mutants. Its lack of recognition by maternal antibodies and its high degree of conservation would make the HNE loop an attractive candidate to include into a subunit vaccine, which could be administered during early childhood, independent of immune status.

The relationships between different strains of mumps virus were established by determination of the sequence of the HN gene. They closely resemble those established from other portions of the genome, suggesting that the viruses are not recombinants over the areas examined. The relationships were consistent with those established by reaction with monoclonal antibodies raised against the Urabe strain, which has a similar antigenic structure to previously studied laboratory strains, and largely consistent with the specificity of the serological response of naturally infected or vaccinated human subjects.

Recent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.

We have analysed the uptake of influenza C virus and bovine coronavirus (BCV) by a polarized epithelial cell line, Madin-Darby canine kidney (MDCK) cells. Both viruses use N-acetyl-9-O-acetyl-neuraminic acid as a receptor determinant for attachment to cells. Virus binding assays with immobilized proteins indicated that a glycoprotein of 40 kDa is the major surface protein containing the receptor determinant for the two viruses. MDCK cells grown on filters for permeable support were found to have differential sensitivity to infection by these viruses. Both viruses were able to initiate infection via the apical domain of the plasma membrane, but only influenza C virus also accomplished infection via the basolateral plasma membrane. The resistance of MDCK cells to BCV infection from the basal filter chamber was overcome when the cell polarity was abolished by maintaining the cells in calcium-free medium. This finding indicates that the resistance to basolateral infection by BCV is a property of the cell line and not due to a technical problem related to the use of filters. Our results indicate that two viruses which use the same receptor for attachment to cells may differ in their ability to enter polarized cells. The possible involvement of an accessory molecule in the entry of BCV is discussed.

Human aminopeptidase N (hAPN or CD13) and porcine aminopeptidase N (pAPN) are functional receptors for human coronavirus (HCV) 229E and porcine transmissible gastroenteritis virus (TGEV), respectively. However, hAPN cannot function as a receptor for TGEV and pAPN cannot function as a receptor for HCV 229E. In this study, we constructed a series of chimeric hAPN/pAPN genes and expressed the corresponding proteins in transfected cells. Subsequently, we identified the chimeric proteins that can function as a receptor for HCV 229E. The results show that replacement of a small region of pAPN sequence (pAPN amino acids 255–348) with the corresponding hAPN sequence (hAPN amino acids 260–353) converts pAPN into a functional receptor for HCV 229E. The region of hAPN that we have defined in this way does not correspond to the region of pAPN that has been identified as essential for the TGEV-receptor interaction. We conclude that although both viruses use a homologous receptor protein, different regions of the protein are required to mediate susceptibility to infection with HCV 229E and TGEV.

We have determined the nucleotide sequence of the region of the rubella virus genome which encodes amino acids 195–296 of the E1 glycoprotein (E1-195–296) from a panel of 22 rubella viruses obtained from Europe, USA and Asia between 1963–1995. E1-195–296 contains neutralizing and haemagglutinating determinants, and may represent a major antigenic domain. The nucleotide sequence divergence of the 22 rubella viruses compared to the Therien strain sequence ranged from 0.65–7.14%. The greatest sequence divergence occurred in two rubella viruses of Indian origin, and was more than twofold greater than that previously reported for rubella virus. The majority of nucleotide changes occurring in the 22 viruses did not effect the deduced amino acid sequence of E1-195–296. Two rubella viruses isolated from cases of reinfection in pregnancy did not exhibit nucleotide sequence variation resulting in changes in the deduced amino acid sequence of E1-195–296, suggesting that antigenic change within this region of E1 is not associated with rubella reinfection. A rubella virus isolated from a synovial fluid sample exhibited a nucleotide substitution in a putative neutralization domain contained within E1-195–296. Phylogenetic analysis was performed to study the relationship between E1-195–296 coding sequences of the 22 viruses in this report and corresponding sequences of other rubella viruses in the databank.

Hepatitis C virus (HCV) is the aetiological agent responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term. The variability of the HCV genome has posed difficulties in serological detection and vaccine design. The recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of a phage display library using the VH genes of a HCV-infected patient and the VL genes of two non-immune individuals. From this library we were able to obtain specific IgG single-chain Fvs (scFvs) that recognize viral core and envelope proteins by selection on synthetic peptides derived from the core sequence PKARRPEGRTWAQPG and the envelope E2 sequence RPIDDFDQGWGPITY. The specificity of the scFvs was demonstrated by their specific reactions with homologous peptides in ELISA and the specific blocking of scFv binding by homologous peptides, in a dose-dependent manner, in inhibition ELISA. The binding of the anticore 4c2 to homologous peptide was blocked by HCV-positive human sera in an antibody-concentration-dependent manner, suggesting that the scFv recognizes a similar if not identical epitope to those of one or more of the polyclonal antibodies present in the sera.