- Volume 78, Issue 10, 1997

A set of monoclonal antibodies (MAbs) specific for the attachment (G) glycoprotein of a recently isolated strain of human respiratory syncytial virus (HRSV) is described. Antibody reactivity with a series of HRSV isolates belonging to antigenic groups A and B identified three epitope categories: (i) strain- specific or variable epitopes that were present in a limited set of viruses from the same antigenic group, (ii) group-specific epitopes shared by viruses from the same antigenic group and (iii) conserved epitopes present in all HRSV isolates. Sequence analysis of escape mutants was used to map relevant antigenic sites of the G glycoprotein. Strain-specific epitopes were located preferentially in the variable C-terminal third of the G polypeptide, in agreement with previous studies of the Long strain. However, a new strain-specific epitope was mapped into another variable region, N-terminal to the cluster of cysteines in the G protein ectodomain. In contrast, the group-specific and conserved epitopes were located in the central conserved region of the G protein primary structure. These results, together with previous analysis of the Long strain, provide a detailed antigenic map of the HRSV attachment protein. Some mutants selected with group-specific antibodies contain multiple A-G substitutions (hypermutations) and lack one or two of the four cysteines which are conserved in all HRSV isolates. The genetic mechanism implicated in the generation of the hypermutated viruses and its relevance for the natural history of HRSV are discussed.

Neutralization and haemagglutination-inhibition (HI) of a type A influenza virus by a panel of five monoclonal IgGs, their F(ab′)2s, Fabs and Fabs anti-mouse Fab were compared. The MAbs were specific for antigenic sites A, B and D of the haemagglutinin. Activities of the IgGs varied by up to 6-fold on a molar basis, apart from the HI activity of HC58 which was > 100-fold lower. This was not due to low functional affinity as HC58 had the second highest value (nM) as determined by an equilibrium method with whole virions. Conversion to the F(ab′)2 reduced neutralization and HI by only 2- to 6-fold, indicating that the Fc region had little involvement in these processes. However, all Fabs had low neutralization and HI activity compared with their IgGs, neutralization being reduced by 86 to > 1912-fold, and HI by 13 to > 69-fold. Although decreased, their affinities remained high, in the nM range. Neutralization and HI by three of the Fabs (HC2, HC3W and HC61) were restored by the addition of anti-Fab IgG; however, HC10 Fab antiFab IgG still had no detectable neutralization activity but gave HI, and HC58 FabManti-Fab IgG had no detectable HI activity but neutralized to the same extent as its IgG. The different properties of the antibodies are discussed in the light of their known mechanisms of action: HI by steric blocking of attachment of virus to the red cell receptor, and neutralization by the inhibition of post-attachment events (HC2, HC10 and HC61). The data demonstrate just how variable are the antiviral properties of individual IgGs.

The relationship between the efficiency of the neutralization process and the affinity of five monoclonal IgG antibodies specific for the haemagglutinin of type A influenza virus has been investigated by determining their neutralization rate constants (Kneut.) and affinities (Kdissoc). We addressed the hypothesis that if antibody affinity alone determined the efficiency of neutralization, then the Kneut.:Kdissoc. ratio would be the same for all antibodies. However, we found that the Kneut.:Kdissoc. ratio varied by up to 125-fold, suggesting that properties unique to the epitope are of major importance in determining the efficiency of neutralization. These data suggest that vaccines should preferentially stimulate antibodies to epitopes that mediate the most efficient neutralization, and that a high Kdissoc. should be an important but secondary consideration.

The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immuno- precipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled- down in the NSI-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.

Batken virus, isolated from mosquitoes and ticks, was tentatively classified as a member of the family Bunyaviridae. Here we show that Batken virus is inhibited by the interferon-induced Mx1 protein of mice which selectively blocks the growth of orthomyxoviruses, including Thogoto and Dhori viruses. Furthermore, we show that Batken virus multiplication is characterized by accumulation of viral proteins in the nucleus and by budding of viral particles from the cell surface. Serological crossreactions between Batken and Dhori viruses revealed a phylogenetic relationship of these viruses, as previously also proposed by D. K. Lvov. Fragments of the Batken virus glycoprotein and nucleo- protein genes were amplified by RT-PCR. The deduced amino acid sequences were similar to the corresponding Dhori virus sequences. Therefore, Batken virus should be classified into the newly established genus Thogotovirus of the family Ortho- myxoviridae. Finally, our results demonstrate that Mx1 susceptibility of orthomyxoviruses is a reliable marker in the hunt for new family members.

Borna disease (BD) is a transmissible, progressive polioencephalomyelitis primarily of horses and sheep. The genomes of two cell-adapted strains of Borna disease virus (BDV), the aetiological agent of BD, have been cloned and sequenced. According to the structural characterization achieved so far, BDV contains a non-segmented negative-sense 8·9 kb single-stranded RNA genome. In this paper we report the expression, purification and intracellular tracing of a novel non-glycosylated BDV-specific protein with a molecular mass of approximately 10 kDa (BDV p10 protein). The successful isolation of the corresponding mRNA from infected cells, amplification of the genetic region by RT-PCR and its efficient expression as a glutathione S-trans- ferase (GST) fusion protein demonstrated that antibodies specific for the BDV p10 protein are induced in infected animals. In addition, we have produced monospecific antisera against the GST-p10 fusion protein in rabbits. This monospecific antiserum recognized the BDV p10 protein in brain cells of naturally and experimentally infected animals as well as in persistently BDV-infected cells. Antibody- mediated affinity-chromatography using the anti- p10 serum could successfully be applied to purify a ca. 10 kDa antigen from infected animal cells to such an extent that glycosylation of this component could be ruled out.

Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNAin infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30–80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.

We have determined the nucleotide sequence of the 5′ -terminal region of the hepatitis G virus (HGV) genome in 11 hepatitis patients from three cities in China. Phylogenetic analyses revealed that the Chinese isolates were genetically distinct from previously described West African isolates (type 1) and American, European and East African isolates (type 2), with a mean sequence divergence of approximately 10 %. The mean divergence between isolates from Lanzhou, in the northwest of China, and those from Shanghai and Nanjing, on the east coast of China, was 5 % (range 3–7 %). The isolates from Shanghai and Nanjing were closely related to a common strain in Japan, while some of those from Lanzhou were closely related to a southeast Asian type 3 isolate. Thus, the Chinese isolates belong to the type 3 variant of HGV.

A low frequency peripheral blood mononuclear cell (PBMC) subpopulation, referred to as natural interferon-producing (NIP) cells, is described as producing interferon-α (IFN-α) following contact with non-infectious viral structures, namely viral glycoproteins. These cells are characterized in vitro as non-T, non-B, MHC class II and CD4 cells. In this study, NIP cells were analysed in vivo after an intravenous injection of UV-inactivated transmissible gastroenteritis virus in newborn piglets, which resulted in strong serum IFN-α production. Spleno-cytes, but not PBMC, were the IFN-α producers in vivo. Using double immunohistochemical labelling for both IFN-α and leukocyte markers, we established that splenic NIP cells were not T or B cells. The majority were MHC class II and only a minority expressed a macrophage marker. NIP cells were localized in contact with MHC class II-expressing cells and T cells, which suggested that NIP cells might modulate the antiviral immune response.

In previous studies, we demonstrated that the substitution of amino acid triplets for alanines in the carboxy-terminal portion (amino acids 341–352: ATL EEM MTA CQC) of the capsid protein domain (p24) of human immunodeficiency virus type 1 (HIV- 1) partly led to an inhibitory effect on the capacity to form virus-like particles (VLPs). In these experiments, the uncleaved Pr55gag precursor protein was expressed by recombinant vaccinia viruses. We have now investigated the effects of these mutations with respect to a replication-competent HI-provirus system. substitution of amino acids 344–346 (EEM) for alanines, which was previously shown to lead to an inhibition of VLP formation, completely blocked assembly and release of HIV. A substantial reduction of HIV synthesis was also observed in the proviral system after exchange of amino acids 347–348 [MT(A)] which, in contrast, was formerly shown to result in an increased formation of VLPs. Western blot analysis of lysates of cells transfected with these mutated proviral constructs revealed an abnormal intracellular processing pattern of the Pr55gag precursor molecules. Further analyses suggest a structural aberration of these altered polyproteins as the basis for the observed block of virus formation.

We have demonstrated that COS7 cells transiently co-expressing myristylation-defective (Myr ) and protease-defective (PR ) human immunodeficiency virus (HIV) mutants can release infectious virions when co-transfected with an amphotropic murine leukaemia virus envelope protein expression plasmid (SV-A-MLV-env). In contrast, no infectious virions were detected when a PR, noninfectious HIV gag mutant was co-expressed with the Myr mutant, although the Myr mutant could still process the immature core particles in trans. This result indicates that generation of functionally normal Gag proteins is required for virus infectivity in our complementation system. A mutant with a 56- amino-acid deletion in the N-terminal region of the capsid (CA) domain could still complement the PR mutant to generate infectious virions, suggesting that the deletion mutant could provide a functional protease for processing in the PR mutant. This result is consistent with the concept that mutations within the N-terminal region of the CA domain have no major effects on Gag-Pol incorporation into particles.

Recently, the presence of human immunodeficiency virus type 1 (HIV-1) RNA transcripts with negative- strand polarity has been shown in tissue culture models of acute and persistently infected cells. One of these transcripts encodes a 189 amino acid open reading frame. This highly conserved antisense sequence is complementary to the structured Rev-responsive element and extends through the cleavage site of the Env protein. We tested the ability of this antisense RNA to modulate HIV-1 replication and the mRNA profile when expressed stably or transiently in several cell types. Different cell lines and PBLs were transduced by retroviral vectors producing antisense RNA and were then challenged by HIV infection. We have shown that the endogenously expressed antisense RNA containing the natural open reading frame inhibits HIV-1 IIIB and HIV-1ndk replication in these cells. The level of inhibition varied according to the cells, but was significant in all cases. The production of HIV-1 (BRU, IIIB, NDK) mRNAs was also significantly decreased. HIV-2 replication was not inhibited by expression of the antisense RNA. Our results also suggest that this inhibitory effect is due to the antisense RNA and not to the protein which is encoded by this sequence.

Different strains of human immunodeficiency virus type 1 (HIV-1) show considerable divergence in genetic content and biological properties. One property that has been closely correlated with clinical prognosis is the ability to induce syncytia formation in susceptible cells. This ability had been correlated with the V3 loop sequence of major envelope glycoprotein, gp120, but recent reports have questioned this connection. We investigated the contributions of different regions of the env gene to syncytia induction using chimeric viruses that contain part of the genome of a strain that lacks this ability (HIV-1Ba-L) within the genome of a virus that can form syncytia (HIV-1HXB-2). When tested in two cell lines susceptible to both parental viruses, as well as in primary cells, these chimeric viruses demonstrated that the ability to induce syncytia formation was determined by regions of env outside the V3 loop, which encompass residues that contribute to the binding of CD4 by gp120. Further investigation failed to show any difference in the expression of gp120 on the cell surface or cell adhesion molecules by cells infected with SI or NSI variants that would explain the observed differences in the ability to form syncytia. Assays of relative affinity for CD4 indicated that gp120 from SI variants showed a significantly higher affinity for CD4 than gp120 from NSI variants. These observations suggest that areas of the HIV-1 env gene contributing to the CD4 binding site may also contribute to the determination of syncytium- inducing (SI) and non-syncytium-inducing (NSI) phenotypes.

Live-attenuated simian immunodeficiency virus (SIV) protects macaques against challenge with pathogenic SIV. To evaluate the safety of such vaccines, an investigation of whether or not nef- deleted SIV could be reactivated in vivo by immune activation of the host was conducted. In addition, monkeys infected with apathogenic SIV/HIV-1 chimeric viruses, and two control monkeys that had suppressed replication of pathogenic SIV were examined. During the infection virus became undetectable or persisted at a low level of replication in all monkeys. At this time-point 11 monkeys were immune-activated by a vaccinia virus (VV) superinfection. After VV infection up to 80% of their lymphocytes showed expression of the activation markers CD25 and CD69 over 2 weeks. However, only the two non-progressing monkeys infected with pathogenic SIV showed a noticeable but transient enhancement of SIV replication and increased SIV antibody titres. By contrast, in monkeys infected with apathogenic immunodeficiency viruses no change in virus load was observed. Therefore, attenuated immunodeficiency viruses cannot be reactivated in vivo by a VV-induced immune activation.

The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4 and CD8 cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccin- ated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4M and CD8M cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-γ and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.

Thrombocytopenia is a common finding in infection with equine infectious anaemia virus (EIAV), a lentivirus with some homology to human immunodeficiency virus (HIV). The thrombocytopenia of EIA, like that in some HIV patients, appears to have a multifactorial pathogenesis. To investigate the decreased platelet production seen in experimental EIA, the levels of three potential negative regulators of platelet production - tumour necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interferon-α (IFN-α) - were measured in serum and bone marrow of six severe combined immunodefi- cient (SCID) foals and ten immunocompetent EIAV- infected foals. Levels of cytokines in pre-infection foal sera and bone marrow were compared with levels observed during clinical EIA. Mean serum levels of TNF-α and IFN-α were significantly higher (P < 0·05) on days 4 to 0 of thrombocytopenia than before infection. Serum TGF-β was significantly elevated on all days except day 1 of thrombocytopenia. Bone marrow TNF-α levels were significantly increased in infected foals just before clinical thrombocytopenia. TGF-β activity was not different in pre-infection and pre-thrombocytopenia bone marrows, but levels of TGF-β protein as determined by immunohistochemical staining were significantly higher in pre-thrombocytopenia bone marrow. IFN-α activity in bone marrow increased just before thrombocytopenia, but the difference was not significant at P < 0·05. Serum TNF-α levels were 2–2·5 times higher in SCID foals on three of the days prior to thrombocytopenia than in immunocompetent foals. No significant differences were found between the levels in SCID and immunocompetent foals of serum and bone marrow TGF-β or IFN-α at any of the times examined.

The complete nucleotide sequence of the provirus of feline foamy virus (FeFV), strain F-17, was determined, and compared to the available data for human and simian spumaviruses. In addition to the usual retroviral gag, pol and env genes, two open reading frames are present between the env gene and the 3′-LTR, as in the simian spumaviruses, the first being the putative transactivator. The gag gene is predicted to encode a precursor protein of only 53 kDa compared to 70 kDa for simian spumaviruses and a doublet of 70/74 kDa for human spumavirus. The gag gene contains conserved splice acceptor and donor sites suggesting that, like human foamy virus, FeFV expresses its pol gene using a spliced mRNA.The pol and env genes showed greater sequence similarity to their counterparts in the primate spumaviruses than the gag gene and additional open reading frames.

It has been shown that interleukin 4 (IL-4) stimulates the proliferation of cells from patients affected by adult T-cell leukaemia, the haematological malignancy aetiologically associated with human T-lymphotropic virus type I (HTLV-I). In the present study, human neonatal lymphocytes were exposed to HTLV-I in vitro in the presence of IL-4. The results showed that: (i) cultures exposed to HTLV-I in the presence of either IL-4 or IL-2 bound IL-4; (ii) IL-4 did not substitute for IL-2 as a growth factor in cell lines previously infected and maintained in IL-2; (iii) cultures exposed to HTLV-I and maintained in IL-4 or IL-2 became infected; and (iv) IL-4 sustained the growth of HTLV-I-infected cultures for a maximum of 14 weeks. Moreover, HTLV-I-infected cultures grown in IL-4 showed upregulation of the IL-4 message and lower expression of HLA-DR and CD25 when compared with counterpart cultures maintained in IL-2. These results suggest that continuous growth of T-lymphocytes induced in vitro by HTLV-I infection, at least temporarily, requires signals specifically provided by IL-2 and not by IL-4.