- Volume 73, Issue 4, 1992

Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M r of non-N-glycosylated gp60 was 50K.

The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 ± 0.9 s.e.m.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 ± 1.1 s.e.m.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.

Glycoprotein gIII of pseudorabies virus (PrV) is multifunctional. It plays a role in the stable adsorption of the virus to its host cells by interacting with a cellular heparin-like substance. It also affects both release of mature virus from infected cell types and virulence. Thus, although non-essential for growth in vitro, gIII plays a central role in the biology of the virus. The primary attachment of a mutant, PrV2, which has an in-frame internal deletion and expresses a shortened version of gIII, and of wild-type (wt) virus, to MDBK cells has been shown to occur similarly. To ascertain whether different domains of gIII control of the expression of the different biological functions of the gIII protein, we have compared several aspects of virus-host cell interactions of PrV2, of a gIII-null virus, and of wt virus. Our results showed that the deletion of the internal segment of the gIII glycoprotein affects adsorption and virus release differently, i.e. that these two functions of gIII appear to be in dependent of each other. Furthermore, we observed that although the primary adsorption of PrV2 and wt virus to MDBK cells is similar, PrV2 behaved like a gIII-null mutant with respect to virulence. The apparent contradiction between these two findings was resolved when it was found that although PrV2 binds as well as does wt to some cell types, it binds poorly to other cell types. The functional importance of different domains of gIII in virus adsorption thus differs, depending on the cell type with which the virus interacts.

The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV Hällnäs strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the Hällnäs strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a β-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.

In the present study we have investigated the role of the hydrophobic domains of the fowl plague virus (FPV) haemagglutinin (HA) on its intracellular transport and maturation in insect cells. To this end processing of full-length HA (A+) has been compared to that of two truncated forms lacking either the cytoplasmic domain and the transmembrane domain (A−) or lacking the entire HA2 subunit, i.e. the transmembrane domain and the fusion peptide (HA- 2). All glycosylation sites present on A− and HA- 2 were glycosylated, indicating that both truncated forms were completely translocated in the endoplasmic reticulum. Unlike A+, A− and HA- 2 did not form trimers as indicated by cross-linking, gradient centrifugation and studies employing conformation-specific antibodies. Whereas HA- 2 was efficiently secreted, A− was retained in the cells in an apparently membrane-bound form. The data show that the carboxy-terminal transmembrane region is essential for the formation and stability of the trimers of the FPV HA. These observations also indicate that, under certain conditions, the fusion peptide of the FPV HA can serve as a membrane anchor.

The post-translational maturation of the attachment G glycoprotein of human respiratory syncytial virus (RSV) was investigated. The G protein formed homooligomers which sedimented in sucrose gradients at the same rate as the fusion F protein tetramer. Oligomerization of the G protein was insensitive to carbonylcyanide m-chlorophenylhydrazine, showing that this step occurs in the endoplasmic reticulum prior to O-glycosylation which initiated in the trans-Golgi compartment. The sedimentation of the G protein oligomer was essentially unchanged by the subsequent addition of O-linked sugars. This indicated that their contribution to the M r of the G protein is less than that estimated by electrophoretic mobility. It also suggested that O-glycosylation is not an important determinant of G protein oligomerization and, by implication, of polypeptide folding. The G protein is palmitylated. In short labelling pulses, the G protein accumulated as two species of 48K and 50K which contained only N-linked sugars, whose difference in M r was due solely to an N-linked sugar, which both assembled into oligomers, but which differed in the rate of subsequent O-glycosylation. The G protein was not detectably O-glycosylated in the presence of monensin, confirming previous work. In the presence of brefeldin A (BFA), it accumulated as a partially O-glycosylated species (BFA-G) of 68K to 78K. But further analysis by chase incubations following BFA-washout, by lectin-binding, and by glycosidase treatment suggested that BFA-G was not a fully authentic processing intermediate. In particular, some of the O-linked side-chains of the BFA-G protein were found to be sialylated. Rather than being a normal step in processing, this sialylation probably was due to altered distribution or activity of sialyltransferases during BFA treatment and may have resulted in the premature termination of elongation of some of the O-linked side-chains. Thus, these studies (i) indicate that O-glycosylation of the G protein begins in the trans-Golgi compartment and (ii) suggest that O-glycosylation is completed in as a subsequent compartment, but this latter suggestion is complicated by the evidence that the BFA-G protein is not a fully authentic intermediate. Finally, an additional species of 48K to 60K was detected under standard conditions of infection and was found to differ in several ways from the 48K and 50K precursors described above: it was detected only following long labelling periods, it was found in monomers rather than oligomers, and it contained a high content of O-linked sugars. Also, antibodies raised against a peptide from the G ectodomain reacted with the 48K, 50K and mature G proteins but did not react with the 48K to 60K species. This indicated that the latter contained differences in conformation or O-glycosylation which masked that part of the ectodomain. The 48K to 60K species was not detected in virions or secreted from infected cells. Thus, it appeared to be a dead-end by-product rather than a true processing intermediate.

The mutation responsible for the temperature-sensitive (ts) phenotype of mutant tsN19 (complementation group E) of respiratory syncytial virus has been located to the P protein gene. Viral protein synthesis was completely restricted at 39 °C, and the tsN19 P protein did not react with an anti-P monoclonal antibody (MAb) (3–5) at 33 °C. Reversion of temperature sensitivity restored reactivity with MAb 3–5. Nucleotide sequence determination and in vitro expression of cDNA clones of P mRNA derived from wild-type, tsN19 and non-ts revertant-infected cells, revealed that temperature sensitivity and loss of reactivity with MAb 3–5 were consequences of a Gly → Ser amino acid change at position 172. A low M r polypeptide, which represented the C-terminal 93 amino acids of the P protein, was produced by internal initiation in the P open reading frame during in vitro translation, and a similar product was detected transiently in vivo.

Fifty-six monoclonal antibodies (MAbs) directed against human parainfluenza virus type 1 (hPIV-1) were prepared in order to identify the structural proteins of hPIV-1, to examine the immunological relationship between hPIV-1 and Sendai virus (SV), and to determine the antigenic diversity of clinical isolates of hPIV-1. In addition, 41 MAbs characterized previously and directed against SV were used for immunological comparison of SV and hPIV-1 isolates. Of the MAbs against hPIV-1, two reacted with phospho (P) protein, 11 with nucleocapsid protein (NP), 24 with haemagglutinin-neuraminidase (HN) protein and 19 with fusion (F) protein. With the aid of MAbs against hPIV-1 and those against SV showing cross-reactivity with hPIV-1, the structural proteins of hPIV-1 were identified; p83, p56, p34, gp74 and gp60 of hPIV-1 were identified as the P, NP, M, HN and F proteins, respectively. The MAbs against the P protein and NP of hPIV-1 showed limited cross-reactivity with SV, whereas they had high reactivity with clinical isolates of hPIV-1. Interestingly, one MAb against the NP of hPIV-1 lacked reactivity with clinical isolates which were isolated in the 1970s and 1980s. The MAbs against the HN of hPIV-1 also exhibited quite limited reactivity with SV and the clinical isolates; two groups of HN-specific MAbs showed almost no reactivity with the clinical isolates from the 1970s and 1980s, similarly to the NP-specific MAb. However, anti-HN MAbs belonging to the two groups showing specific activities (neuraminidase inhibition and haemolysis inhibition) reacted with almost all clinical isolates. On the other hand, although anti-F protein MAbs had limited reactivity with SV, they showed reactivity with almost all hPIV-1 isolates. The MAbs against the P, NP, M, HN and F proteins of SV also showed limited cross-reactivity with the clinical hPIV-1 isolates, and this reactivity was independent of the time and place of isolation, except for that of the F protein. These results confirm that although hPIV-1 is related to SV, it is antigenically distinct from it.

The nucleotide and deduced amino acid sequences of two genes of phocid distemper virus (PDV) were determined by cDNA cloning and sequencing. The long open reading frame of the gene encoding the nucleocapsid (N) protein is presented. As with other morbilliviruses, the phosphoprotein (P) gene of PDV was found to be located after the 5′ end of the N gene and before the 3′ end of the matrix protein gene. The P gene was shown to have the capacity to encode three distinct proteins, P, V and C, in analogy to other morbilliviruses. The results presented provide evidence for editing of the PDV P mRNA transcript by insertion of G residues. When the nucleotide and deduced amino acid sequences of the N, P, V and C genes were aligned with corresponding sequences of other established members of the morbillivirus genus, compelling homology was found between PDV and canine distemper virus (CDV), whereas there was markedly less similarity between PDV and measles virus or rinderpest virus. On the basis of the alignments presented, the estimated amino acid sequence similarity between the N and P genes of PDV and CDV was 84% and 76%, respectively. These differences at the genomic level indicate that the viruses are two separate entities.

Av01 is a variant of the challenge virus standard strain of fixed rabies virus that was selected with a neutralizing anti-glycoprotein monoclonal antibody, and has a single amino acid change in the glycoprotein. It is avirulent after both intracerebral and peripheral routes of inoculation in adult mice. In this study, Av01 was found to be neurovirulent with stereotaxic brain inoculation in either the striatum or cerebellum of adult mice. Mice that had been inoculated simultaneously with Av01 by the intracerebral and intrastriatal routes recovered. More infectious virus was present in the brains of mice inoculated intrastriatally than intracerebrally, and more neurons contained rabies virus antigen. However, the topographical distribution of infected neurons was similar with both routes. Serum neutralizing antibodies against rabies virus were produced later and in smaller quantities after intrastriatal inoculation. Av01 is probably neurovirulent after stereotaxic brain inoculation because this route produces both a direct site of viral entry into the central nervous system and a low level of immune stimulation.

The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.

Chemical cleavage of the VP6 protein of bovine rotavirus showed that VP6-specific monoclonal antibodies (MAbs) reacted with the amino acid sequence between glycine 48 and asparagine 107. Furthermore, three synthetic peptides (amino acids 48 to 64, 60 to 75 and 91 to 108) containing part of this sequence and 22 consecutive overlapping heptapeptides corresponding to the region between amino acids 48 and 75 were analysed for their immunoreactivity using group-specific MAbs. The MAbs recognized peptides 48–64 and/or 60–75, and a set of overlapping heptapeptides located between residues 53 (asparagine) and 67 (glycine), which have two short sequences in common: IRNW (residues 56 to 59), recognized by MAb RV-133, and (NW)NFD (residues 58/60 to 62), recognized by MAbs RV-50, -1026 and -443. These results indicate that the sequence between amino acid residues 48 and 75 is present in one of the immuno-dominant sites of VP6.

The sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M r 38243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M r 113249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.

The major core protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated.

Human immunodeficiency virus type 1 (HIV-1) infection of the CD4+ SupT and CEM cell lines, blocked in cell replication by the polymerase α inhibitor aphidicolin (APC), was studied. The APC-treated cells showed a lack of viral production, but the presence of single cell killing. High levels of unintegrated viral DNA forms were found in the infected APC-treated cells as compared with untreated cells. Moreover, an increased rate of viral replication occurred in the remaining viable cells following removal of APC. The results indicate that HIV-1 entry and reverse transcription can take place in cells blocked in the S phase of the cell cycle. Replication of infectious progeny virions appears to require de novo cell division. Finally, accumulation of viral DNA in cells during APC treatment can result in cytopathological effects and subsequent enhancement of virus production.

Recombinant interleukin 4 (IL-4) stimulated extracellular (EC) and intracellular (IC) production of human immunodeficiency virus (HIV) from infected human blood-derived monocytes and macrophages when incubated with the cells after but not before virus inoculation. Significant stimulation was observed in 20 of 27 experiments with monocytes (inoculated with HIV immediately after adherence) and 10 of 13 experiments with macrophages (inoculated after 5 days adherence) using a total of 30 normal donors of monocytes and macrophages, and 11 recent isolates of monocytotropic HIV strains (after one passage in mononuclear cells). Marked increases in EC and IC HIV antigen were observed in some experiments, which were comparable with the maximal stimulatory effects of other cytokines such as IL-2. IL-4 also had similar effects on infectious HIV concentration as measured by reverse transcriptase and TCID50 assays. Antibody to IL-4 prevented the stimulatory effect of the cytokine. The proportion of monocytes and macrophages infected by HIV, as determined by in situ hybridization, also increased after incubation with IL-4 for 7 days. The most marked effects were observed with HIV-infected macrophages, for which the proportion of unstimulated infected cells was lower (35 to 45% increasing to 66 to 70% with IL-4 treatment). There was also an increased proportion of cells with high granule concentrations, suggesting that IL-4 increases the intracellular concentration of viral nucleic acids. This was supported by semi-quantitative hybridization experiments showing that total HIV RNA increased in IL-4-stimulated monocytes 48 to 96 h after HIV inoculation. A marked increase in aggregates was observed on day 7 in HIV-infected monocytes treated with IL-4, compared to that in HIV-infected cells alone or IL-4-treated uninfected monocytes. These findings suggest that IL-4 stimulates HIV replication in the early phases of infection and may also facilitate virus transmission by aggregate formation.

Monoclonal antibodies (MAbs) were raised against the transmembrane protein (TM) gp41 of human immunodeficiency virus type 1 (HIV-1, strain HTLV-IIIB). The reactivity of three TM-specific MAbs was investigated in several tests, ELISA, immunostaining of Western blots, immunofluorescence and an alkaline phosphatase-anti-alkaline phosphatase assay. Epitope mapping was done by using overlapping gp41 peptides produced as Escherichia coli fusion proteins and synthetic peptides. In an in vitro assay, all three MAbs showed enhancing effects on HIV-1 infection after single or repeated treatment with the purified MAbs at concentrations of 6 to 25 µg/ml. The enhancing domain is located between amino acids 724 and 752 of the env protein sequence. Homologous peptides based on this sequence were used for analysis of sera from 100 individuals at different stages of HIV infection to evaluate the relevance of antibodies against this region to the prognosis of disease. No antibodies reactive with this region were found in ELISA, indicating that this domain is not immunogenic in humans.

The proposal that replication of human immunodeficiency virus type 1 (HIV-1), mediated by cell-to-cell transmission of the virus, might bypass de novo reverse transcription was tested by using one-step cell-to-cell and cell-free virus infection systems. Two well characterized reverse transcriptase (RT) inhibitors, azidothymidine at 20 µm and phosphonoformic acid at 100 µg/ml, blocked HIV replication completely following both cell-free virus and cell-to-cell transmission infection, as determined from the kinetics of unintegrated viral DNA synthesis and supernatant RT production after virus infection. Our results confirm that de novo reverse transcription is a crucial and mandatory event in HIV-1 replication following cell-to-cell transmission of the virus.