- Volume 84, Issue 8, 2003

The latency-associated transcript (LAT) promoter of pseudorabies virus (PRV) is unique among viral promoters in that it remains active in trigeminal ganglia during the latent state. It is not known which the viral or host proteins regulate expression of the PRV LAT gene in latently infected neurons. To determine whether host transcriptional proteins in neurons can regulate the PRV LAT promoter in vivo, three transgenic mouse lines containing the PRV LAT promoter (LAP; LAP1 and LAP2) linked to the chloramphenicol acetyltransferase (CAT) gene were generated. All of the transgenic mouse lines, in the absence of the viral proteins, displayed strong expression of the transgene in trigeminal ganglia in addition to other neuronal tissues such as cerebral cortex, cerebellum, hippocampus and olfactory bulb. Expression of the transgene in neurons of trigeminal ganglia was demonstrated by in situ hybridization. These data provide direct evidence that neuronal transcription factors are sufficient to activate the PRV LAP in vivo and that the promoter is neuron-specific.

We describe here the development of a ‘eukaryotic display system’ for heterologous proteins on the viral and host cell surfaces using Bombyx mori nucleopolyhedrovirus (BmNPV). The reporter gene gfp (green fluorescent protein) was fused to either the gp64 gene encoding the full-length BmNPV envelope protein GP64 or to its 5′ region encoding only the N-terminal domain harbouring the signal sequence, and recombinant viruses expressing the corresponding fusion proteins under the strong viral polyhedrin promoter were generated. On infection of the host insect B. mori or the host-derived BmN cells with the full-length GP64–GFP virus, abundant expression of the recombinant protein and its display on the cell surface were achieved. The fusion protein was also a component of the budded virions. Thus, the BmNPV-based display system provides an alternative to the previously established Autographa californica multinucleocapsid nucleopolyhedrovirus display system. The recombinant virus expressing GFP has also been used in preliminary pathological investigations on virus infection in B. mori and provides a simple method for screening for antiviral agents.

We have characterized infection and pathogenesis of an Autographa californica M nucleopolyhedrovirus recombinant, AcMNPV-hsp70/lacZ, carrying the lacZ reporter gene, in penultimate (fifth) instar Spodoptera frugiperda. Bioassays revealed that while <0·1 p.f.u. of budded virus was required to generate an LD50 by intrahaemocoelic injection, approximately 6000 occlusions were required orally to achieve the same mortality in newly moulted fifth instar (50) larvae. In pathogenesis experiments, 78 % of the 50 larvae inoculated orally with 6000 occlusions of AcMNPV-hsp70/lacZ were LacZ-positive at 8 h post-inoculation (p.i.) and 50 % had LacZ signals in tracheal cells indicating that in these larvae infection had been transmitted from the midgut to secondary target cells. At 24 h p.i., maximum numbers of midgut and midgut-associated tracheal foci were observed (mean of 35 foci per infected larva), and 88 % of the larvae were LacZ-positive. The extremely low foci-per-occlusion ratio (0·006) indicated that successful infection of midgut cells was the primary barrier to fatal infection. A second barrier involved the loss of infected tracheal cells associated with the midgut. At 24 h p.i., 88 % of the inoculated larvae had a systemic infection, but in bioassays only 51 % succumbed to polyhedrosis disease. The absence of melanized tracheal cells in the insects throughout the time-course suggested that the larvae that cleared their infections (38 %) did so by a mechanism other than a classical immune response.

Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29–35. In this paper we have shown that SeMNPV ORFs 15–35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15–35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviridae.

Glyptapanteles indiensis polydnavirus (GiPDV) is essential for successful parasitization of the larval stage of the lepidopteran Lymantria dispar (gypsy moth) by the endoparasitic wasp Glyptapanteles indiensis. This virus has not been characterized previously. Ultrastructural studies of GiPDV showed that virions had a rod-like or rectangular form and each contained as many as ten nucleocapsids enclosed by a single unit membrane envelope. Field inversion gel electrophoresis (FIGE) analysis of the virus genomic DNA revealed that GiPDV had a segmented genome composed of 13 dsDNA segments, ranging in size from approximately 11 kb to more than 30 kb. Four genomic segments were present in higher molar concentration than the others. Further characterization of the GiPDV genome yielded several cDNA clones which derived from GiPDV-specific mRNAs, and Northern blot analysis confirmed expression of isolated cDNA clones in the parasitized host. Each was present on more than one GiPDV genomic DNA segment, suggesting the existence of related sequences among DNA segments. It has been proposed previously that in polydnavirus systems, genome segmentation, hypermolar ratio segments and segment nesting may function to increase the copy number of essential genes and to increase the levels of gene expression in the absence of virus replication. The present data support this notion and suggest that GiPDV morphology and genomic organization may be intrinsically linked to the function and evolutionary strategies of the virus.

Adenovirus type 11 (Ad11), a member of the human adenovirus species B (HAdV-B), has a tropism for the urinary tract. The genome of Ad11 was found to comprise 34 794 bp and is 1141 bp shorter than the Ad5 genome of species HAdV-C. The G+C content of the Ad11 genome is 48·9 %, whereas that of Ad5 is 55·2 %. Ad11 and Ad5 share 57 % nucleotide identity and possess the same four early regions, but the E3 region of Ad11 could not be divided into E3A and E3B. The late genes of Ad11 and Ad5 are organized into six and five regions, respectively. Thirty-eight putative ORFs were identified in the Ad11 genome. The ORFs in the late regions, the E2B region and IVa2 show high amino acid identity between Ad11 and Ad5, whereas the ORFs in E1, E2A, E3 and E4, protein IX and the fibre protein show low amino acid identity. The highest and lowest identities were noted in the pre-terminal protein and fibre proteins: 85 % and 24·6 %, respectively. The E3 20·3K and 20·6K ORFs and the L6 agnoprotein were present in the Ad11 genome only, whereas the E3 11·6K cell death protein was identified only in Ad5. All ORFs but the E3 10·3K and L4 pVIII protein vary not only in composition but also in size. Ad11 may have a higher vector capacity than Ad5, since it has a shorter genome and a shorter fibre. Furthermore, in the E3 region, two additional ORFs can be deleted to give extra capacity for foreign DNA.

The human MxA protein is an interferon (IFN)-inducible GTPase with proven antiviral activity against diverse viruses. IFN responsiveness is impaired in chronic hepatitis B virus (HBV) infection. Accordingly, initial experiments showed that, in contrast to parental HepG2 cells, when HepG2-derived 2.2.15 liver cells carrying the HBV genome were treated with IFN, they could not synthesize the MxA protein. Furthermore, MxA expression was reduced in HepG2 cells transiently transfected with the HBV genome. To assess whether HBV-encoded precore/core (preC/C) proteins interact with the IFN-signalling pathway, HepG2, Chang and HeLa cells were transfected with preC/C expression plasmids; the levels of signal transducers remained unaffected. Next, full-length and deletion mutants fused to the CAT reporter gene were tested to investigate whether MxA inhibition occurs at the promoter level. In co-transfection experiments, IFN-induced CAT activity was inhibited by preC/C expression in a dose-dependent manner. Analysis of deletion mutants showed that the region affected by the preC/C proteins comprises IFN-stimulated response elements 2 and 3, upstream of the putative start codon of the MxA promoter. In addition, HBV preC/C proteins interacted directly with the MxA promoter, as shown by electrophoretic mobility shift assays. These results demonstrate a mechanism that HBV probably uses to downregulate an element of the IFN-induced host antiviral responses, which accounts for the impairment observed in HBV-infected patients.

The precore mutation G1896→A occurs frequently in anti-HBe-positive carriers of HBsAg with T1858 in the stem of the encapsidation signal. Hepatitis B virus (HBV) genotype F, considered an Amerindian genotype, subdivides into two clades and the precore mutation occurs in Central American F strains. To investigate the relationship between substitutions at position 1858 and these clades, the precore and small S genes of 48 strains of HBV genotype F were subjected to phylogenetic analyses. Isolates of one clade, formed mainly of Central American strains, all had T1858 and Thr45 in the S gene, whereas in the other clade, formed mainly of South American strains and one strain from Polynesia, all had C1858 and Leu45. The latter strain was related to strains from Venezuela and Colombia, supporting an Amerindian contribution to the Polynesian population. The position of the Polynesian strain in the phylogenetic tree indicates that the two clades have resulted from an early split, showing a high degree of genetic stability of the stem of the HBsAg encapsidation signal.

Human papillomavirus type 16 (HPV-16)-associated vulval intraepithelial neoplasia (VIN) is frequently a chronic, multifocal high-grade condition with an appreciable risk of progression to vulval cancer. The requirement to treat women with VIN has recently stimulated the use of immunotherapy with E6/E7 oncogene vaccines. Animal models have shown that E2 may also be a useful vaccine target for HPV-associated disease; however, little is known about E2 immunity in humans. This study investigated the prevalence of HPV-16 E2-specific serological and T-cell responses in 18 women with HPV-16-associated VIN and 17 healthy volunteers. E2 responses were determined by full-length E2–GST ELISA with ELISPOT and proliferation assays using E2 C-terminal protein. As positive controls, HPV-16 L1 responses were measured using virus-like particles (VLPs) and L1–GST ELISA with ELISPOT and proliferation using VLPs as antigen. The VIN patients all showed a strong serological response to L1 compared with the healthy volunteers by VLP (15/18 vs 1/17, P<0·001) and L1–GST ELISA (18/18 vs 1/17, P<0·001). In contrast, L1-specific cellular immune responses were detected in a significant proportion of controls but were more prevalent in the VIN patients by proliferation assay (9/17 vs 17/18, P<0·02) and interferon-γ ELISPOT (9/17 vs 13/18, P=not significant). Similar and low numbers of patients and controls were seropositive for E2-specific Ig (2/18 vs 1/17). In spite of previous studies showing the immunogenicity of E2 in eliciting primary T-cell responses in vitro, there was a low prevalence of E2 responses in the VIN patients and controls (2/18 vs 0/17).

The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric β-galactosidase–E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.

Human papillomavirus (HPV) type 16 is the major cause of cervical carcinoma, the incidence of which is decreasing in western countries. In Finland the incidence is, however, increasing in women aged <40 years, but possible underlying changes in HPV-16 epidemiology are unknown. To assess incidence trends of HPV infections, paired sera from a random sample of 8000 women with two pregnancies/sera within 5 years, taken from the serum bank of the Finnish Maternity Cohort (1983–98), were analysed for HPV-6, -11 and -16 antibodies. For 23–31-year-old women, HPV-16 incidence increased over the period 1983–97. HPV-16 seroprevalence increased from 17 % in 1983–85 to 24 % in 1995–97, but HPV-6 and HPV-11 prevalence was stable at 9–12 % throughout the study period. Epidemic spread of the oncogenic HPV-16, but not the non-oncogenic HPV-types, throughout the 1980s and 1990s preceded an increase in the incidence of cervical carcinoma in fertile-aged Finnish women.

The SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40ΔVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40ΔVp3 and SV40 ΔVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.

Fifteen 8-week-old conventional pigs were selected from a farm where pigs were suffering from postweaning multisystemic wasting syndrome (PMWS). Ten of the animals were diseased pigs showing typical signs of PMWS (wasting and respiratory disorders) and positive for infection with porcine circovirus type 2 (PCV2), and the other five animals selected as controls were pen-mate, apparently healthy pigs. Blood samples and lymphoid tissues were taken from each animal for haematological, serological and histopathological studies. Also, cytokine mRNA expression of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p40 and IFN-γ from inguinal and bronchial lymph nodes, tonsils, spleen and thymus was determined by semi-quantitative RT-PCR. Pigs suffering from PMWS showed severe alterations of haematological parameters such as anaemia, lymphopenia with decrease of CD8+ and IgM+ cells, monocytosis and neutrophilia. Also, extensive lymphocyte depletion and altered cytokine mRNA expression patterns were seen in most of the examined lymphoid organs. Those cytokine mRNA alterations were characterized by an overexpression of IL-10 mRNA in thymus and IFN-γ mRNA in tonsils, and by decreases in the mRNA expression of several cytokines as IL-2 and IL-12p40 in the spleen, IL-4 in tonsils, and IFN-γ, IL-10, IL-12p40 and IL-4 in inguinal lymph nodes. Also, the IL-10 mRNA overexpression was histologically associated with the thymic depletion and atrophy observed in PMWS pigs. In conclusion, the cytokine mRNA imbalance, specially the increased mRNA levels of IL-10 in the thymus, jointly with the histopathological and haematological disorders, are highly indicative of a T-cell immunosuppression, enhancing the notion that the immune system of PMWS-affected pigs is severely impaired.

Adeno-associated virus type 2 integrates preferentially into the AAVS1 locus on chromosome 19 of the human genome. It was reported previously that transfection with two plasmids, one for Rep and the other carrying a transgene flanked by inverted terminal repeats (ITRs), enables preferential integration of the latter into AAVS1. Aiming at increasing the frequency of AAVS1-specific integration, the Rep- to transgene-plasmid ratio necessary to achieve a higher frequency of site-specific integration was examined. 293 cells were co-transfected with the Rep78 plasmid and an ITR-flanked Neo gene at different ratios. G418-resistant clones were selected randomly. Extensive Southern blot analysis showed an optimum range of Rep78 expression. In that range, approximately 20 % of clones harboured the Neo gene at AAVS1. Excess Rep expression, however, resulted in ‘abortive’ integration of the Neo gene, a rearrangement of AAVS1 without transgene integration. Rep78 appeared to cause abortive integration more extensively than Rep68. Deleterious effects of the Rep protein on the AAVS1 locus should be considered to develop an improved AAVS1-targeted system.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP–MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a ‘double tag’ construct, the GST–PCP–MT cDNA was flanked by the 3′-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST–PCP and MT fragments. MT-His6 was purified on Ni–NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly588/Gly589 bond cleavage. The GST–PCP fragment purified on glutathione S–agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

Cowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the MP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified MP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the MP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.