- Volume 82, Issue 11, 2001

In order to obtain attenuated live vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV), a series of deletions was introduced at the 3′ end of the viral genome using an infectious cDNA clone of the Lelystad virus isolate. RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus. It is shown that C-terminal truncation of the nucleocapsid (N) protein, encoded by ORF7, was tolerated for up to six amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing viral RNA. Deletion analysis of the 3′ UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of seven nucleotides behind the stop codon of ORF7. Deletion of 32 nucleotides in this region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated that the viable deletion mutants were genetically stable at the site of mutation. In addition, the deletions did not affect the growth properties of the recombinant viruses in vitro, while their antigenic profiles were similar to that of wild-type virus. Immunoprecipitation experiments with the six-residue N protein-deletion mutant confirmed that the truncated protein was indeed smaller than the wild-type N protein. The deletion mutants produced in this study are interesting candidate vaccines to prevent PRRS disease in pigs.

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) exist, a North American type and a European type. The co-existence of both types in some countries, such as Denmark, Slovakia and Canada, creates a risk of inter-type recombination. To evaluate this risk, cell cultures were co-infected with either a North American and a European type of PRRSV or two diverse types of European isolate. Subsequently, an approximately 600 bp region of the PRRSV genome was tested for recombination by quantitative real-time RT–PCR. Between 0·1 and 2·5% RNA recombination was found between the European isolates, but no recombination was detected between the European and North American types. Calculation of the maximum theoretical risk of European–American recombination, based on the sensitivity of the RT–PCR system, revealed that RNA recombination between the European and North American types of PRRSV is at least 10000 times less likely to occur than RNA recombination between diverse European isolates.

The picornavirus membrane-associated polypeptide 2C is believed to be required for viral RNA synthesis. Hepatitis A virus (HAV)- and human rhinovirus (HRV)-encoded recombinant 2C proteins have been expressed, purified and examined for their ability to interact with the terminal sequences of viral positive- and negative-strand RNAs. The results demonstrate that both the HAV- and the HRV-encoded 2C polypeptide specifically interact with the 3′-terminal sequences of the negative-strand RNA, but not with the complementary sequences at the 5′ terminus of the positive-strand RNA. This interaction was detected by both mobility gel shift and UV cross-linking assays. Furthermore, complex formation exhibited dose-dependency and competition assays confirmed specificity. These results are consistent with our previous observation using the poliovirus 2C protein. The implication of the picornavirus 2C protein binding to the 3′-terminal sequence of the negative-strand untranslated region in viral RNA synthesis is discussed.

The complete nucleotide sequences are reported of two strains of echovirus 7, the prototype Wallace strain (Eo7-Wallace) and a recent Malaysian strain isolated from the cerebrospinal fluid of a child with fatal encephalomyelitis (Eo7-UMMC strain). The molecular findings corroborate the serological placement of the UMMC strain as echovirus 7. Both Eo7-Wallace and Eo7-UMMC belong to the species human enterovirus B and are most closely related to echovirus 11. Eo7-UMMC has undergone significant genetic drift from the prototype strain in the 47 years that separate the isolation of the two viruses. Phylogenetic analysis revealed that Eo7-UMMC did not arise from recombination with another enterovirus serotype. The molecular basis for the severely neurovirulent phenotype of Eo7-UMMC remains unknown. However, it is shown that mutations in the nucleotide sequence of the 5′ untranslated region (UTR) of Eo7-UMMC result in changes to the putative structure of the 5′ UTR. It is possible that these changes contribute to the neurovirulence of Eo7-UMMC.

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the family Picornaviridae. The sequence from the 5′ poly(C) tract to the 3′ poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genus Erbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. In order to gain insight into the genetic variability and mode of evolution of HAV in South America, an analysis was performed of sequence data obtained from the VP1 amino terminus and the VP1/2A region of HAV strains isolated over a short period of time in Uruguay, Argentina and Chile. Sequences obtained from 22 distinct HAV isolates were compared with published sequences from 21 different strains isolated all over the world. Phylogenetic analysis revealed that all strains isolated belong to a unique sub-genotype (IA). Strains isolated during an outbreak period showed a higher degree of heterogeneity than anticipated previously and the co-circulation of different isolates. The genetic variability among strains isolated in this region seems to be higher in comparison with strains isolated in other regions of the world.

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5′ cap. Both RNAs were largely resistant to 3′ polyadenylation and ligation, suggesting the presence of an interfering 3′ structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5′ and 3′ non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT–PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3′ terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0·4 kb) that was newly synthesized and corresponded to the 3′-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.

The pathology of respiratory syncytial virus (RSV) disease in bonnet monkeys parallels findings with human RSV disease. RSV-infected animals pre-immunized with a formalin-inactivated (FI) RSV vaccine develop inflammation in peribronchiolar, perivascular, interstitial and intra-alveolar sites with lung inflammation scores significantly higher than animals with a primary RSV infection and those pre-immunized with an FI-Vero cell control vaccine (P=0·05). Animals previously infected and re-exposed to RSV had significantly lower alveolar, interstitial and total lung inflammation scores than in primary infection (P=0·05). Immunization with two intra-muscular doses of 0·5 ml of the FI-RSV vaccine administered 21 days apart resulted in little serum-neutralizing and ELISA antibody, low levels of secretory IgA and a low lymphocyte proliferative response that was significantly lower than the response observed in animals that were previously infected with live RSV. Higher RSV virus titres were detected in the lungs and lung lavage fluid of monkeys immunized with the FI-RSV vaccine than in those with a primary infection (P=0·001). RSV was detected by in situ hybridization in pulmonary inflammatory infiltrates, where the single most abundant infiltrating cellular species was macrophages, so it may be these cells that support the enhanced virus replication that contributes to the enhanced pulmonary pathology of FI-RSV immunization.

Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene and the deduced 57 amino acid sequences were aligned with sequences of mumps virus strains published previously. Four neurovirulent genotypes of the SH protein gene, genotypes C, D, H and a new genotype, designated J, were found. There was a dynamic fluctuation of the different genotypes over the two decade period of time. Genotype J was found from 1981 to 1988; genotypes C and H exhibited a similar distribution in time. Genotype D was found between 1979 and 1982, it then disappeared and reappeared again in 1996. From 1996 onwards, genotype D was found to be the predominant genotype, which is in contrast to the situation seen in the neighbouring country of Sweden, where, since 1985, only genotype A has been found.

We determined the complete nucleotide sequences of two poorly characterized strains of Borna disease virus (BDV) and compared them to reference strains V and He/80. Strain H1766 was almost 98% and 95% identical to strains V and He/80, respectively, whereas strain No/98 was only about 81% identical to both reference strains. In contrast to earlier reports, we found an additional A residue at the extreme 3′-end of the single-stranded RNA genome in all four BDV strains. The exact numbers of nucleotides in the four BDV genomes could not be determined due to a micro-heterogeneity at the 5′-end. If our longest sequence is a correct copy of the viral RNA, the two ends of the BDV genome would show almost perfect complementarity. All three transcription start sites, all four termination sites, both splice donor sites and both major splice acceptor sites are highly conserved, whereas a minor alternative splice acceptor site is not. The L protein of No/98 differs at 7% of its amino acid positions from the polymerase in the other strains, with most differences mapping to the C-terminal moiety of the molecule. Re-evaluation of L protein sequences of strains V and He/80 revealed differences at several positions compared to published information, indicating that variant forms of the viral polymerase have previously been characterized. These results are important because correct structures of genome ends and of the polymerase gene are the most critical parameters for the future development of techniques that will permit the genetic manipulation of BDV.

The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence 139RSSEDKSTQTTGR151. Site-directed mutagenesis of residues D143 and Q147 to an A residue abolished binding to LC8. The P–LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140–150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.

The level of heterosubtypic immunity (Het-I) and the immune mechanisms stimulated by a heterosubtypic influenza virus infection were investigated in pigs. Pigs are natural hosts for influenza virus and, like humans, they host both subtypes H1N1 and H3N2. Marked Het-I was observed when pigs were infected with H1N1 and subsequently challenged with H3N2. After challenge with H3N2, pigs infected earlier with H1N1 did not develop fever and showed reduced virus excretion compared with non-immune control pigs. In addition, virus transmission to unchallenged group-mates could be shown by virus isolation in the non-immune control group but not in the group infected previously with H1N1. Pigs infected previously with homologous H3N2 virus were protected completely. After challenge with H3N2, pigs infected previously with H1N1 showed a considerable increase in serum IgG titre to the conserved extracellular domain of M2 but not to the conserved nucleoprotein. These results suggest that antibodies against external conserved epitopes can have an important role in broad-spectrum immunity. After primary infection with both H1N1 and H3N2, a long-lived increase was observed in the percentage of CD8+ T cells in the lungs and in the lymphoproliferation response in the blood. Upon challenge with H3N2, pigs infected previously with H1N1 again showed an increase in the percentage of CD8+ T cells in the lungs, whereas pigs infected previously with H3N2 did not, suggesting that CD8+ T cells also have a role in Het-I. To confer broad-spectrum immunity, future vaccines should induce antibodies and CD8+ T cells against conserved antigens.

Currently, three genotypes of hepatitis delta virus (HDV) are described. The most common, genotype I, has a worldwide distribution; in contrast, genotype II has been found previously only in Japan and Taiwan, while genotype III is found exclusively in South America. Considering the high prevalence of HDV in Northern Siberia (Russia), restriction fragment length polymorphism (RFLP) was used to analyse HDV genotypes from 29 infected patients living in Yakutia. Of these isolates, 11 were characterized by partial nucleotide sequencing and two isolates were completely sequenced. Phylogenetic inference methods included maximum parsimony, maximum likelihood and distance analyses. A restriction pattern consistent with HDV genotype I was found in 14 samples, while the remaining 15 showed a different restriction pattern, inconsistent with any known genotype. Five Yakutian HDV isolates with the type I restriction pattern were sequenced and confirmed to be affiliated with genotype I, although the phylogenetic results indicate that they were heterogeneous and did not cluster together. Sequencing of eight isolates with the new RFLP pattern revealed that these isolates were most closely related to HDV genotype II. In contrast to HDV Yakutian genotype I sequences, all of these type II sequences formed a well-defined clade on phylogenetic trees. Comparison of clinical presentations during hospitalization between patients infected with HDV type I (n=14) and type II (n=15) did not reveal any differences in the severity of infection. These data indicate that the distribution of genotype II is not restricted to Taiwan or Japan, but spreads over Northern Asia, appearing in the native population of Yakutia. Type II Yakutian strains appeared to form a well-defined subclade and could be associated with severe chronic hepatitis in this area.

Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag–Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Δp6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag–Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag–Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA–CA and CA–NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag–Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag–Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag–Pol precursor.

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G140E, V153L and E177G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4+ cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G177E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E140G and L153V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.

The nucleotide changes of the DNA polymerase gene and the susceptibility of acyclovir (ACV)-resistant varicella-zoster virus (VZV) mutants to anti-herpetic drugs were determined and compared to those of herpes simplex virus type 1 (HSV-1) mutants. The seven ACV-resistant VZV mutants were classified into three groups, N779S, G805C and V855M, according to the sequences of their DNA polymerase genes. The amino acid substitutions N779S and G805C were identical in position to the N815S and G814C mutations in the HSV-1 DNA polymerase mutants, respectively, and the V855M amino acid substitution was similar to the HSV-1 V892M mutation. All three groups of VZV mutants were susceptible to ACV, phosphonoacetic acid, vidarabine and aphidicolin, at levels similar to those seen with the respective HSV-1 mutants, except for subtle differences that were due possibly to the non-conserved regions in their sequences. Although both the HSV-1 and the VZV DNA polymerase genes show 53% sequence similarity, both viruses essentially show a similar biochemical behaviour.

After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC50 of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC50 in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A→G substitution of the U69 protein kinase (PK) gene resulted in an M318V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A961V amino acid substitution within the DNA polymerase. The M318V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M460V and M460I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M318V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M318V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.