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Chimeric alphaviruses in which the 6K and glycoprotein E1 moieties of Sindbis virus are replaced with those of Ross River virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. We have sequenced the entire domain encoding the E2, 6K, and E1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. In three independent passage series, amino acid 380 of E2, which is in the transmembrane domain, was mutated from the original isoleucine to serine in two instances and to valine once. We have now changed this residue to seven others by site-directed mutagenesis and tested the effects of these mutations on the growth of both the chimera [SIN(RRE1)] and of parental Sindbis. These results indicate that the transmembrane domains of glycoproteins E2 and E1 of alphaviruses interact in a sequence-dependent manner and that this interaction is required for efficient budding and assembly of infectious virions.

Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).

The potyvirus Papaya ringspot virus (PRSV) is found throughout the tropics and subtropics. Its P biotype is a devastating pathogen of papaya crops and its W biotype of cucurbits. PRSV-P is thought to arise by mutation from PRSV-W. However, the relative impact of mutation and movement on the structure of PRSV populations is not well characterized. To investigate this, we have determined the coat protein sequences of isolates of both biotypes of PRSV from Vietnam (50), Thailand (13), India (1) and the Philippines (1), and analysed them together with 28 PRSV sequences already published, so that we can better understand the molecular epidemiology and evolution of PRSV. In Thailand, variation was greater among PRSV-W isolates (mean nucleotide divergence 7.6%) than PRSV-P isolates (mean 2.6%), but in Vietnamese populations the P and W biotypes were more but similarly diverse. Phylogenetic analyses of PRSV also involving its closest known relative, Moroccan watermelon mosaic virus, indicate that PRSV may have originated in Asia, particularly in the Indian subcontinent, as PRSV populations there are most diverse and hence have probably been present longest. Our analyses show that mutation, together with local and long-distance movement, contributes to population variation, and also confirms an earlier conclusion that populations of the PRSV-P biotype have evolved on several occasions from PRSV-W populations.

The Hepatitis A virus (HAV) has been classified in seven different genotypes, which include human (I, II, III, and VII) and simian (IV, V, and VI) groups. The sequence analysis of HAV strains contributes to the molecular epidemiology of the virus. Although the infection with HAV is endemic in Argentina and vaccination is being implemented in this country, using both IA and IB strains, there are very few data on the genotypes of the circulating viruses. On the basis of the sequences of 20 isolates collected in Buenos Aires during a 2-year period (extended to 3 years by two additional specimens), we observed the presence of a single sub-genotype, IA, but with a high genetic diversity. We analyzed the VP1-2A junction and also the VP3-VP1 region. Most of the Argentine isolates grouped in at least two clusters. One of these was related to South American strains, thus suggesting a co-circulation of related isolates in neighbor countries. The other cluster was composed only of Argentine specimens. Other sequences were more scattered along the phylogenetic tree. However, we demonstrated that a consistent genetic relatedness of sequences could only be inferred on the basis of a more extensive sequencing of each isolate., (Copyright 2002 Wiley-Liss, Inc.)

Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus ATADENOVIRUS: In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions. These characteristics are in accordance with the genus-defining markers of atadenoviruses. Comparison of the cleavage sites of the viral protease in core protein pVII also confirmed SnAdV-1 as a candidate member of the genus ATADENOVIRUS: Thus, the hypothesis on the possible reptilian origin of atadenoviruses (Harrach, Acta Veterinaria Hungarica 48, 484-490, 2000) seems to be supported. However, the base composition of DNA sequence (>18 kb) determined from the SnAdV-1 genome showed an equilibrated GC content of 51%, which is unusual for an atadenovirus.

The modes of evolution of the proteins of Potato virus Y were investigated with a maximum-likelihood method based on estimation of the ratio between non-synonymous and synonymous substitution rates. Evidence for diversifying selection was obtained for the 6K2 protein (one amino acid position) and coat protein (24 amino acid positions). Amino acid sites in the coat proteins of other potyviruses (Bean yellow mosaic virus, Yam mosaic virus) were also found to be under diversifying selection. Most of the sites belonged to the N-terminal domain, which is exposed to the exterior of the virion particle. Several of these amino acid positions in the coat proteins were shared between some of these three potyviruses. Identification of diversifying selection events in these different proteins will help to unravel their biological functions and is essential to an understanding of the evolutionary constraints exerted on the potyvirus genome. The hypothesis of a link between evolutionary constraints due to host plants and occurrence of diversifying selection is discussed.

Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis. Two approaches were used to identify immunodominant antigens of LETV, the LETD-associated herpesvirus. The first approach targeted glycoprotein B, which is known to be immunogenic and neutralizing in other species. The second strategy identified LETV proteins recognized on Western blots by antibodies in immune green turtle plasma. A 38-kDa protein was resolved by two-dimensional gel electrophoresis, sequenced, and identified as a scaffolding protein encoded by the overlapping open reading frames of UL26 and UL26.5. Glycoprotein B and the scaffolding protein were cloned and expressed in Escherichia coli. The expressed proteins were recognized on Western blots by antibodies in immune green turtle plasma. Phylogenetic studies based on UL26, DNA polymerase, and glycoprotein B revealed that LETV clusters with the alphaherpesviruses.

Transmission electron micrographs of Escherichia coli cells induced by cloned lysis genes from RNA bacteriophages GA (group A-II) and SP (group B-IV) revealed various morphological aspects of intermediates of lysing cells. Cells induced by the SP lysis gene became stretched and also tapered in shape and fragmentation of parts of the cells had also occurred. Cells induced by the GA lysis gene showed many ballooning structures on the cell surfaces and others leaked material through the cell wall. Some balloon-like structures also appeared on the surfaces of cells induced by the cloned lysis gene of RNA phage SP and material also appeared to be leaking through the cell wall in the photographs. The lysing cells observed by transmission electron microscopy showed various morphological aspects of intermediates of the lysing process.

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein. Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.

The genetic trajectory leading to viral attenuation was studied in a canine parvovirus (CPV) strain grown on dog kidney cells for 115 transfers. Consensus sequences of viral populations at passages 0, 3, 30, 50, 80, and 115 were obtained from PCR products covering 86% of the genome; clones from each of the 80th and 115th passages were also sequenced, covering 69% of the genome. Sixteen changes were fixed in the 115th-passage virus sample. Levels of polymorphism were strikingly different over time, in part because of a plaque-cloning step at passage 112 that reduced variation: passage 80 had 19 variants common among the clones, but passage 115 had only a single common variant. Several mutations increased in the culture at the same time, with most reaching fixation only after the 80th passage. The pattern of evolution was consistent with recombination and not with separate selective sweeps of individual mutations. Thirteen of the changes observed were identical to or at the same positions as changes observed in other isolates of CPV or feline panleukopenia virus.

The mechanisms employed by nonenveloped animal viruses to penetrate the membranes of their host cells remain enigmatic. Membrane penetration by the nonenveloped mammalian reoviruses is believed to deliver a partially uncoated, but still large ( approximately 70-nm), particle with active transcriptases for viral mRNA synthesis directly into the cytoplasm. This process is likely initiated by a particle form that resembles infectious subvirion particles (ISVPs), disassembly intermediates produced from virions by proteolytic uncoating. Consistent with that idea, ISVPs, but not virions, can induce disruption of membranes in vitro. Both activities ascribed to ISVP-like particles, membrane disruption in vitro and membrane penetration within cells, are linked to N-myristoylated outer-capsid protein micro 1, present in 600 copies at the surfaces of ISVPs. To understand how micro 1 fulfills its role as the reovirus penetration protein, we monitored changes in ISVPs during the permeabilization of red blood cells induced by these particles. Hemolysis was preceded by a major structural transition in ISVPs, characterized by conformational change in micro 1 and elution of fibrous attachment protein sigma 1. The altered conformer of micro 1 was required for hemolysis and was markedly hydrophobic. The structural transition in ISVPs was further accompanied by derepression of genome-dependent mRNA synthesis by the particle-associated transcriptases. We propose a model for reovirus entry in which (i) primed and triggered conformational changes, analogous to those in enveloped-virus fusion proteins, generate a hydrophobic micro 1 conformer capable of inserting into and disrupting cell membranes and (ii) activation of the viral particles for membrane interaction and mRNA synthesis are concurrent events. Reoviruses provide an opportune system for defining the molecular details of membrane penetration by a large nonenveloped animal virus.

Recombination is one of the factors that contribute to genetic diversity in foot-and-mouth disease virus (FMDV). Similarity and bootscan analyses have provided evidence of recombination in the capsid-coding (P1) region of the virus. In the present study, of the 14 subtype A22 field isolates that were distributed in three previously described genotypes (IV, VI and VII) based on the 1D (VP1-encoding) gene sequence (Tosh et al., 2002 ), one isolate (IND 170/88) was found to be a hybrid of genotypes VI and VII in the P1 region. VP1, VP4, the 5' region of VP2 and the 3' region of VP3 of this virus were characteristic of genotype VI, whereas the remaining 3' region of VP2 and the 5' region of VP3 were characteristic of genotype VII. No insertion or deletion was observed in the recombinant virus. Recombination in the P1 region may provide an escape mechanism for the virus.

Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.

The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.

Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

Here we report on the conformational changes that are responsible for the appearance of the Dicentrarchus labrax encephalitis virus (DlEV) coat protein as a doublet in SDS-PAGE. Wild-type and mutated forms of the coat protein cDNA were expressed in E. coli. The study of the resulting recombinant molecules excluded the possibility of the involvement of a precursor autocatalysis mechanism or a ribosomal frameshifting event in the doublet formation. The appearance of the coat protein doublet was found to be beta-mercaptoethanol sensitive. Based on this observation, we carried out substitution of all cysteine residues. The obtained results demonstrated the importance of intramolecular disulfide bonding between cysteines 187 and 201 on coat protein conformational changes.

Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3' half and 5' halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

The requirements for intercellular movement of Potato virus X (PVX) 12K, 8K, and coat proteins (CP) differed in two Nicotiana spp. Plasmids containing the green fluorescent protein (GFP) gene fused to PVX 12K, 8K, or CP genes were bombarded to tobacco leaves. Protein movement was observed in N. benthamiana but not N. tabacum leaves. GFP:12K and GFP:8K moved cell-to-cell in 25K-expressing transgenic N. tabacum source but not sink leaves. In N. tabacum, GFP:12K and GFP:8K intercellular movement depends on the 25K and leaf developmental stage. Leaves were bombarded using two biolistic delivery methods and the results were similar indicating that movement of GFP:12K or GFP:8K is independent of the delivery system. Mutations in 12K, 8K, and CP genes within the PVX genome inhibited viral intercellular movement in both Nicotiana spp. Thus plasmodesmata gating is not an essential function of these proteins for virus cell-to-cell movement. These proteins likely provide additional activities for virus cell-to-cell movement.

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.

We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly (HS) syndrome in the USA. We showed that avian HEV is genetically related to swine and human HEVs. Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human HEVs and the Australian chicken big liver and spleen disease virus (BLSV). The region encoding the C-terminal 268 amino acid residues of avian HEV ORF2 was cloned into expression vector pRSET-C. The truncated ORF2 protein was expressed in E. coli as a fusion protein and purified by affinity chromatography. Western blot analysis revealed that the avian HEV ORF2 protein reacted with antisera against the Sar-55 strain of human HEV and with convalescent antisera against swine HEV and the US2 strain of human HEV, as well as with antiserum against BLSV. Convalescent sera from specific-pathogen-free chickens experimentally infected with avian HEV also reacted with the recombinant capsid proteins of swine HEV and Sar-55 human HEV. Antisera against the US2 human HEV also reacted with recombinant ORF2 proteins of both swine HEV and Sar-55 human HEV. The antigenic cross-reactivity of the avian HEV putative capsid protein with those of swine and human HEVs was further confirmed, for the most part, by ELISA assays. The data indicate that avian HEV shares certain antigenic epitopes in its putative capsid protein with swine and human HEVs, as well as with BLSV. The results have implications for HEV diagnosis and taxonomy.

Rice ragged stunt oryzavirus (RRSV) is a member of the genus oryzavirus within the family Reoviridae. Its genome consists of ten segments of dsRNA. The functions of all products encoded by these viral genome segments, except one encoded by S9, have not yet been elucidated. In the present study, the ORF of S 8 of RRSV-Philippines isolate was sequenced and expressed in E. coli. The 67 kD product of S8 could be self-cleaved into two fragments with molecular weights of 43 kD and 26 kD. Western blotting indicated that both 67 kD and 43 kD products were major structural proteins of the virus. It was also found that the 67 kD protein could self aggregate into aggregates with higher sedimentation rate in sucrose gradients during centrifugation. Moreover, the self-aggregation process could be accelerated by the complex of S6 product and genome dsRNAs of RRSV. These results suggest that the S8 products, 67 kD or 43 kD, may be the structural components of the viral inner-capsid.

Emiliania huxleyi-specific viruses ( EhV) were isolated from E. huxleyi blooms off the coast of Plymouth, UK, in July 1999 and July/August 2001, and from an E. huxleyi bloom induced during a mesocosm experiment in a fjord off Bergen, Norway, during June 2000. Transmission electron microscopy revealed that all 10 virus isolates are 170-200 nm in diameter with an icosahedral symmetry. Their density is approximately 1.2 in CsCl gradients and they have large double stranded DNA genomes approximately 410 kb in size. Phylogenetic analysis of the DNA polymerase genes of these viruses suggests that EhV belongs to a new genus within the family of algal viruses, Phycodnaviridae. We propose to name this new virus genus Coccolithovirus. Differences within members of the Coccolithovirus were elucidated by host range analysis of the virus isolates and sequence analysis of a gene fragment encoding part of their putative major capsid protein. All 10 virus isolates within this new genus only infected E. huxleyi strains that have previously been shown to exhibit low dimethylsulphoniopropionate lyase (DMSP-lyase) activity (CCMP1516, CCMP374 and L), while E. huxleyi strains with high DMSP-lyase activity (CCMP373 and CCMP379) were resistant to infection.

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2- and VP3- viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr- viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.

For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin alpha 2/beta heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.

LINE-1 are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. LINE-1 elements transpose by reverse transcription of their own transcript. Transposition requires synthesis of a full-length, sense-strand transcripts and proteins encoded by open reading frame (ORF) 1 and ORF2. Although severely repressed in most normal tissues, LINE-1 occasionally leads to disease by insertional mutagenesis. In the present study, Northern blot and in situ hybridization analyses revealed a template-strand transcription of LINE-1 ORF2 (encoding reverse transcriptase, RT) in lymphoid organs and the liver from MRL-+/+ and Fas-deficient MRL/lpr strains and their normal ancestors. While these sense transcripts are restricted to the nucleus in hepatocytes, they are also found in the cytoplasm in splenocytes. In contrast to transcription, ORF2 translation was detected only in MRL strains, as shown by the cytoplasmic labelling of splenic cells obtained with a monoclonal antibody recognizing the LINE-1 RT. This antibody coprecipitated two proteins of 45 and 12 kDa from MRL/lpr lymphoid organ lysates that were removed by pretreatment with anti-beta2-microglobulin antiserum, suggesting a structural association between a LINE-1 product and a major histocompatibility complex class I or class I-like molecule.

The LINE-1 (L1) retrotransposon, the most important human mobile element, shapes the genome in many ways. Now two groups provide evidence that L1 retrotransposition is associated with large genomic deletions and inversions in transformed cells. If these events occur at a similar frequency in vivo, they have had a substantial effect on human genome evolution.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

As invading viruses do not harbor functional ribosomes in their virions, successful amplification of the viral genomes requires that viral mRNAs compete with cellular mRNAs for the host cell translation apparatus. Several RNA viruses have evolved remarkable strategies to recruit the host translation initiation factors required for the first steps in translation initiation by host cell mRNAs. This review describes the ways that three families of RNA viruses effectively usurp limiting translation initiation factors from the host.

The trans-splicing reaction involves the association of 5' and 3' splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3' acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5' splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.

Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.

Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene delivery into the central nervous system (CNS). However, host inflammatory and immune responses may play a critical role in limiting the use of rAAV vectors for gene therapy and functional genomic studies in vivo. Here, we evaluated the effect of repeated injections of five rAAV vectors expressing different genetic sequences (coding or noncoding) in a range of combinations into the rat brain. Specifically, we wished to determine whether a specific immune or inflammatory response appeared in response to the vector and/or the transgene protein after repeated injections under conditions of mannitol coinjection. We show that readministration of the same rAAV to the CNS is possible if the interval between the first and second injection is more than 4 weeks. Furthermore, our data demonstrate that rAAV vectors carrying different genetic sequences can be administered at intervals of 2 weeks. Our data therefore suggest that the AAV capsid structure is altered by the vector genetic sequence, such that secondary structures of the single-stranded genome have an impact on the antigenicity of the virus. This study provides guidelines for more rational design of gene transfer studies in the rodent brain and, in addition, suggests the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

Genomic variability within the sequences of VP1/2A junction among polioviruses from across the globe has revealed the existence of several endemic genotypes and their epidemiological inter-relationships; but such data on Indian isolates are scanty. The present work was intended to ascertain the persistence and transmission pattern of different genotypes of wild type 1 polioviruses circulating in India. Forty-eight wild type 1 poliovirus isolates obtained from different parts of India during 1996-8 were subjected to RT-PCR and nucleotide sequencing using M13 tailed primers. A 293 base pair region was amplified and sequenced for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, the isolates from six different states of India confirmed a single dominant genotype 4. Phylogenetic analysis revealed the circulation and active inter-state transmission of many genetically distinct strains of wild poliovirus type 1 belonging to genotype 4. This warrants the need for insisting on more efficient surveillance mechanisms so as to assess the impact of an extensive pulse polio immunization programme in India.

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse. The different strains of TMEV are divided into two subgroups according to the pathology they provoke. The neurovirulent strains GDVII and FA induce an acute fatal encephalitis, while persistent strains, like DA and BeAn, cause a chronic demyelinating disease associated with viral persistence in the central nervous system. Different receptor usage was proposed to account for most of the phenotype difference between neurovirulent and persistent strains. Persistent but not neurovirulent strains were shown to bind sialic acid. We characterized DA and GDVII derivatives adapted to grow on CHO-K1 cells. Expression of glycosaminoglycans did not influence infection of CHO-K1 cells by parental and adapted viruses. Mutations resulting from adaptation of DA and GDVII to CHO-K1 cells notably mapped to the well-characterized VP1 CD and VP2 EF loops of the capsid. Adaptation of the DA virus to CHO-K1 cells correlated with decreased sialic acid usage for entry. In contrast, adaptation of the GDVII virus to CHO-K1 cells correlated with the appearance of a weak sialic acid usage for entry. The sialic acid binding capacity of the GDVII variant resulted from a single amino acid mutation (VP1-51, Asn-->Ser) located out of the sialic acid binding region defined for virus DA. Mutations affecting tropism in vitro and sialic acid binding dramatically affected the persistence and neurovirulence of the viruses.

Three viral gastroenteritis (VGE) outbreaks that occurred in 1998-1999, in Hungary were investigated for the presence of human caliciviruses (HuCVs). HuCVs in stool specimens were detected by reverse transcription-polymerase chain reaction (RT-PCR) using primer pair 289/290, which was designed based on the RNA-dependent RNA polymerase (RdRp) sequence. RT-PCR results were confirmed by sequencing showing that all three outbreak strains belonged to genogroup II of "Norwalk-like viruses" (NLVs). Two strains had high sequence identity with strains in known genetic clusters (Hawaii and Lordsdale clusters). The third strain (MOH) had distinct RdRp sequence, sharing 77/86% (nt/aa) identity with Snow Mountain virus (SMV), the closest genogroup II virus. To characterize MOH further, we cloned, sequenced, and expressed in baculovirus its capsid gene. It had 75/79% (nt/aa) identity with SMV, but 97/98% (nt/aa) identity with NLV/Hillingdon/90/UK, a recently identified genetic cluster of HuCVs. The recombinant MOH (rMOH) capsid protein self-assembled into virus-like particles (VLPs), which is antigenically distinct from other recombinant HuCV capsid antigens available in our laboratory. Further study of this VLP will have important applications in antigenic characterization and diagnosis of HuCVs., (Copyright 2002 Wiley-Liss, Inc.)

Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.

The coat protein (CP) and movement protein (MP) sequences of a new tobamovirus infecting Hibiscus rosa-sinensis L were determined. The CP gene encodes 163 amino acid (aa) residues and with a theoretical molecular weight of 18.19 kDa. The MP gene encodes 282 amino acids and its theoretical molecular weight is 30.36 kDa. The nucleotide (nt) and aa sequences of the CP were 46.88 % to 51.63 % and 45.34 % to 57.06 % identical to other tobamoviruses, respectively. The nt and aa sequence identities of MP ranged from 38.81 % to 43.90 % and 30.85 % to 37.88 %, respectively. The predicted virion origin of assembly (OAS) was located in the CP gene. Phylogenetic trees generated based on the nt and aa sequences of both CP and MP genes indicate that this new virus clusters with members of subgroup II of tobamoviruses. Although this hibiscus virus shared a high nt and aa sequence identity with Sunn-hemp mosaic virus (SHMV), Western analysis showed that it is serologically unrelated to SHMV. We propose the name Hibiscus virus S (HVS) for this Singapore isolate. This is the first report on partial nt sequence of a tobamovirus that infects hibiscus.

DNA sequences for the coat protein (CP) and part of the cytoplasmic inclusion (CI) protein genes after reverse transcriptase-polymerase chain reaction (RT-PCR) were reported for the first time for isolates of zucchini yellow mosaic virus (ZYMV) from Austria, Germany, Italy and Slovenia. Comparison of the DNA sequences of 30 isolates from different geographical regions worldwide revealed highest similarities of the Austrian isolates to those from Slovenia and Hungary. Isolates from Germany and Italy were only distantly related and clustered with isolates from other parts of the world. Our results indicate that a specific isolate can spread rapidly to geographically adjacent areas but may not be directly related to isolates found in other neighboring countries.

Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.

Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.

Epidemiological and experimental studies have clearly shown that high-risk HPV infection is the main etiologic factor for cervical cancer. Recent studies have indicated that the E6 and E7 gene products play a critical role in cervical carcinogenesis. The E6 and E7 products interfere with the p53 and pRB functions, respectively, and deregulate the cell cycle. The HPV DNA is integrated into the host's chromosomes with disruption of the E2 gene. This disruption promotes the expression of E6 and E7, leading to the accumulation of DNA damage and the development of cervical cancer. The study of the immune response against HPV has been hampered by the lack of a cell culture system for the virus. A breakthrough was made by the discovery that a major capsid protein L1 self-assembles into virus-like particles (VLP) when expressed in eukaryotic systems. Clinical trials of VLP-based vaccines are in progress, and DNA vaccines for the HPV surface protein genes are under development. The E7 and E6 oncoproteins are attractive targets for cancer immunotherapy because their expression is required to maintain the oncogenicity of cervical cancer cells. Cancer immunotherapy for cervical cancer with vaccinations of E7 peptides or dendritic cell-based immunotherapy is moving toward clinical trials.

Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains. Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C. perfringens. We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.

After attachment to receptors, reovirus virions are internalized by endocytosis and exposed to acid-dependent proteases that catalyze viral disassembly. Previous studies using the cysteine protease inhibitor E64 and a mutant cell line that does not support reovirus disassembly suggest a requirement for specific endocytic proteases in reovirus entry. This study identifies the endocytic proteases that mediate reovirus disassembly in murine fibroblast cells. Infection of both L929 cells treated with the cathepsin L inhibitor Z-Phe-Tyr(t-Bu)-diazomethyl ketone and cathepsin L-deficient mouse embryo fibroblasts resulted in inefficient proteolytic disassembly of viral outer-capsid proteins and decreased viral yields. In contrast, both L929 cells treated with the cathepsin B inhibitor CA-074Me and cathepsin B-deficient mouse embryo fibroblasts support reovirus disassembly and growth. However, removal of both cathepsin B and cathepsin L activity completely abrogates disassembly and growth of reovirus. Concordantly, cathepsin L mediates reovirus disassembly more efficiently than cathepsin B in vitro. These results demonstrate that either cathepsin L or cathepsin B is required for reovirus entry into murine fibroblasts and indicate that cathepsin L is the primary mediator of reovirus disassembly. Moreover, these findings suggest that specific endocytic proteases can determine host cell susceptibility to infection by intracellular pathogens.

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion. Numerous recent studies have focused on either the soluble form of CA or the polymer end product of in vitro CA assembly. The CA polymer, in particular, has been used to study CA-CA interactions because it is a good model for the CA interactions within the virion core. However, analysis of the process of in vitro CA assembly can yield valuable insights into CA-CA interactions and the mechanism of core assembly. We describe here a method for the analysis of CA assembly kinetics wherein the progress of assembly is monitored by using turbidity. At pH 7.0 the addition of either of the isolated CA domains (i.e., the N or the C domain) to an assembly reaction caused a decrease in the assembly rate by competing for binding to the full-length CA protein. At pH 8.0 the addition of the isolated C domain had a similar inhibitory affect on CA assembly. However, at pH 8.0 the isolated N domain had no affect on the rate of CA assembly but, when mixed with the C domain, it alleviated the C-domain inhibition. These data provide biochemical evidence for a pH-sensitive homotypic N-domain interaction, as well as for an N- and C-domain interaction.

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

A reverse transcription-dependent polymerase chain reaction (RT-PCR) is described that amplifies the genes encoding the capsid proteins VP1-3 of at least three evolutionary lineages each of the foot-and-mouth disease (FMD) virus types A, Asia1 and O. Most of these lineages are circulating at present in Asia and Africa. The method is not only suitable to confirm suspected outbreaks of FMD, but also describes the modulation of major and minor antigenic sites in the course of an epizootic by nucleotide sequence determination of the obtained RT-PCR products. Such knowledge helps to choose suitable vaccines for disease control.