Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Automatic detection and morphological delineation of bacteriophages in electron microscopy images.

Automatic detection, recognition and geometric characterization of bacteriophages in electron microscopy images was the main objective of this work. A novel technique, combining phase congruency-based image enhancement, Hough transform-, Radon transform- and open active contours with free boundary conditions-based object detection was developed to detect and recognize the bacteriophages associated with infection and lysis of cyanobacteria Aphanizomenon flos-aquae. A random forest classifier designed to recognize phage capsids provided higher than 99% accuracy, while measurable phage tails were detected and associated with a correct capsid with 81.35% accuracy. Automatically derived morphometric measurements of phage capsids and tails exhibited lower variability than the ones obtained manually. The technique allows performing precise and accurate quantitative (e.g. abundance estimation) and qualitative (e.g. diversity and capsid size) measurements for studying the interactions between host population and different phages that infect the same host.

Principles of virus structural organization.

Viruses, the molecular nanomachines infecting hosts ranging from prokaryotes to eukaryotes, come in different sizes, shapes, and symmetries. Questions such as what principles govern their structural organization, what factors guide their assembly, how these viruses integrate multifarious functions into one unique structure have enamored researchers for years. In the last five decades, following Caspar and Klug's elegant conceptualization of how viruses are constructed, high-resolution structural studies using X-ray crystallography and more recently cryo-EM techniques have provided a wealth of information on structures of a variety of viruses. These studies have significantly -furthered our understanding of the principles that underlie structural organization in viruses. Such an understanding has practical impact in providing a rational basis for the design and development of antiviral strategies. In this chapter, we review principles underlying capsid formation in a variety of viruses, emphasizing the recent developments along with some historical perspective.

The potyviruses of Australia.

Many potyviruses have been found in Australia. We analyzed a selected region of the coat protein genes of 37 of them to determine their relationships, and found that they fall into two groups. Half were isolated from cultivated plants and crops, and are also found in other parts of the world. Sequence comparisons show that the Australian populations of these viruses are closely related to, but less variable than, those in other parts of the world, and they represent many different potyvirus lineages. The other half of the potyviruses have only been found in Australia, and most were isolated from native plants. The sequences of these potyviruses, which are probably endemic, are on average five times more variable than those of the crop potyviruses, but surprisingly, most of the endemic potyviruses belong to one potyvirus lineage, the bean common mosaic virus lineage. We conclude that the crop potyviruses entered Australia after agriculture was established by European migrants two centuries ago, whereas the endemic plant potyviruses probably entered Australia before the Europeans. Australia, like the U.K., seems recently to have had c. one incursion of a significant crop potyvirus every decade. Our analysis suggests it is likely that potyviruses are transmitted in seed more frequently than experimental evidence indicates, and shows that understanding the sources of emerging pathogens and the frequency with which they 'emerge' is essential for proper national biosecurity planning.

Identification and classification of human cytomegalovirus capsids in textured electron micrographs using deformed template matching.

BACKGROUND: Characterization of the structural morphology of virus particles in electron micrographs is a complex task, but desirable in connection with investigation of the maturation process and detection of changes in viral particle morphology in response to the effect of a mutation or antiviral drugs being applied. Therefore, we have here developed a procedure for describing and classifying virus particle forms in electron micrographs, based on determination of the invariant characteristics of the projection of a given virus structure. The template for the virus particle is created on the basis of information obtained from a small training set of electron micrographs and is then employed to classify and quantify similar structures of interest in an unlimited number of electron micrographs by a process of correlation. RESULTS: Practical application of the method is demonstrated by the ability to locate three diverse classes of virus particles in transmission electron micrographs of fibroblasts infected with human cytomegalovirus. These results show that fast screening of the total number of viral structures at different stages of maturation in a large set of electron micrographs, a task that is otherwise both time-consuming and tedious for the expert, can be accomplished rapidly and reliably with our automated procedure. Using linear deformation analysis, this novel algorithm described here can handle capsid variations such as ellipticity and furthermore allows evaluation of properties such as the size and orientation of a virus particle. CONCLUSION: Our methodological procedure represents a promising objective tool for comparative studies of the intracellular assembly processes of virus particles using electron microscopy in combination with our digitized image analysis tool. An automated method for sorting and classifying virus particles at different stages of maturation will enable us to quantify virus production in all stages of the virus maturation process, not only count the number of infectious particles released from un infected cell.

Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain.

Recombinant adeno-associated viral (AAV) vectors can transduce cells of the CNS, resulting in long-term expression. AAV vector transduction varies depending on the serotype used and the region of the brain injected. AAV serotypes 7, 8, 9, and Rh10 have recently become available, but the transduction capabilities of these serotypes within the CNS have not been determined. We show that AAV 7, 8, 9, and Rh10 vectors expressing cDNA for a lysosomal enzyme transduce neurons, but not astrocytes or oligodendrocytes, in the cortex, striatum, hippocampus, and thalamus. Although all of the vectors contained the same genome, there were markedly different transduction patterns that could be due only to the differences in capsid proteins. The AAV 9 vector was found to undergo vector genome transport to distal neuronal cell bodies via known axonal pathways. This facilitated the distribution of enzyme, resulting in correction of lysosomal storage lesions in regions of a diseased brain that would not be corrected if the genome were not transported.

Epidemic spread of recombinant noroviruses with four capsid types in Hungary.

BACKGROUND: Noroviruses are common pathogens in gastro-enteritis outbreaks in humans worldwide. Noroviruses are genetically diverse group of viruses with multiple genogroups (GG) and genotypes. More recently, naturally occurring recombinant noroviruses were described. These viruses had a distinct polymerase gene sequence (designated GGIIb/Hilversum) and were disseminated through waterborne and food-borne transmission in Europe. OBJECTIVES: Our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in Hungary. STUDY DESIGN: From January 2001 to May 2004, samples containing "GGIIb/Hilversum polymerase" (GGIIb-pol) were selected for analysis of the viral capsid region (ORF2) by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. RESULTS: Thirty-four (14.4%) of 236 confirmed norovirus outbreaks were caused by the variant lineage with the GGIIb-pol. Four different recombinants were detected with capsids of Hu/NLV/GGII/Mexico/1989 (n=9, 43%), Hu/NLV/GGII/Snow Mountain/1976 (n=6, 28%), Hu/NLV/GGII/Hawaii/1971 (n=4, 19%) and Hu/NLV/GGII/Lordsdale/1993 (n=1, 5%). CONCLUSIONS: In Hungary, emerging recombinant noroviruses became the second most common norovirus variants-next to GGII-4/Lordsdale virus-causing epidemics of gastroenteritis in the last 4 years.

A refined circular template matching method for classification of human cytomegalovirus capsids in TEM images.

An automatic image analysis method for describing, segmenting, and classifying human cytomegalovirus capsids in transmission electron micrograph (TEM) images of host cell nuclei has been developed. Three stages of the capsid assembly process in the host cell nucleus have been investigated. Each class is described by a radial density profile, which is the average grey-level at each radial distance from the center. A template, constructed from the profile, is used to find possible capsid locations by correlation based matching. The matching results are further refined by size and distortion analysis of each possible capsid, resulting in a final segmentation and classification.

Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses.

The single-stranded genomic RNA of Taura syndrome virus (TSV) is 10205 nucleotides in length, excluding the 3' poly(A) tail, and contains two large open reading frames (ORFs) that are separated by an intergenic region of 207 nucleotides. The ORFs are flanked by a 377 nucleotide 5' untranslated region (UTR) and a 226 nucleotide 3' UTR followed by a poly(A) tail. The predicted amino acid sequence of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of several plant and animal RNA viruses. In addition, a short amino acid sequence located in the N-terminal region of ORF1 presented a significant similarity with a baculovirus IAP repeat (BIR) domain of inhibitor of apoptosis proteins from double-stranded DNA viruses and from animals. The presence of this BIR-like sequence is the first reported in a single-stranded RNA virus, but its function is unknown. The N-terminal amino acid sequence of three TSV capsid proteins (55, 40 and 24 kDa) were mapped in ORF2, which is not in the same reading frame as ORF1 and possesses an AUG codon upstream of the structural genes. However, the intergenic region shows nucleotide sequence similarity with those of the genus Cricket paralysis-like viruses, suggesting a similar non-AUG-mediated translation mechanism. The structure of the TSV genome [5' UTR-non-structural proteins-intergenic UTR-structural proteins-3' UTR-poly(A) tail] is similar to those of small insect-infecting RNA viruses, which were recently regrouped into a new virus genus, Cricket paralysis-like viruses.

Molecular phylogeny and proposed classification of the simian picornaviruses.

The simian picornaviruses were isolated from various primate tissues during the development of general tissue culture methods in the 1950s to 1970s or from specimens derived from primates used in biomedical research. Twenty simian picornavirus serotypes are recognized, and all are presently classified within the Enterovirus genus. To determine the phylogenetic relationships among all of the simian picornaviruses and to evaluate their classification, we have determined complete VP1 sequences for 19 of the 20 serotypes. Phylogenetic analysis showed that A13, SV19, SV26, SV35, SV43, and SV46 are members of human enterovirus species A, a group that contains enterovirus 71 and 11 of the coxsackie A viruses. SA5 is a member of human enterovirus species B, which contains the echoviruses, coxsackie B viruses, coxsackievirus A9, and enterovirus 69. SV6, N125, and N203 are related to one another and, more distantly, to species A human enteroviruses, but could not be definitely assigned to a species. SV4 and SV28 are closely related to one another and to A-2 plaque virus, but distinct from other enteroviruses, suggesting that these simian viruses are members of a new enterovirus species. SV2, SV16, SV18, SV42, SV44, SV45, and SV49 are related to one another but distinct from viruses in all other picornavirus genera, suggesting that they may comprise a previously unknown genus in Picornaviridae. Several simian virus VP1 sequences (N125 and N203; SV4 and SV28; SV19, SV26, and SV35; SV18 and SV44; SV16, SV42, and SV45) are greater than 75% identical to one another (and/or greater than 85% amino acid identity), suggesting that the true number of distinct serotypes among the viruses surveyed is less than 20.

Primary structure and phylogenetic analysis of the coat protein of a Toyama isolate of tobacco necrosis virus.

The amino acid sequence of the coat protein (CP) of a tobacco necrosis virus (TNV) strain, Toyama isolate, was determined by a combination of peptide and cDNA sequencing. The deduced sequence of 276 residues was compared with CPs of other TNV isolates and other plant virus isolates of Tombusviridae. It showed the highest similarity to the TNV Nebraska isolate with 92% identity and moderate similarity to the TNV strain A with 51% identity, confirming the previous serological analysis. It also showed overall similarity with CPs of mostly genera Necrovirus and Sobemovirus, and partial similarity with CPs of genera Tombusvirus and Carmovirus. Among 13 CPs that showed overall similarity, there were 10 completely conserved residues. These included three residues that participate in Ca2+ ligation at the interfaces of virion subunits in TNV crystal structure, suggesting that similar metal binding occur in the viruses of genera Necrovirus and Sobemovirus.

Re-analysis of human immunodeficiency virus type 1 isolates from Cyprus and Greece, initially designated 'subtype I', reveals a unique complex A/G/H/K/? mosaic pattern.

Human immunodeficiency virus type 1 (HIV-1) has been classified into three main groups and 11 distinct subtypes. Moreover, several circulating recombinant forms (CRFs) of HIV-1 have been recently documented to have spread widely causing extensive HIV-1 epidemics. A subtype, initially designated I (CRF04_cpx), was documented in Cyprus and Greece and was found to comprise regions of sequence derived from subtypes A and G as well as regions of unclassified sequence. Re-analysis of the three full-length CRF04_cpx sequences that were available revealed a mosaic genomic organization of unique complexity comprising regions of sequence from at least five distinct subtypes, A, G, H, K and unclassified regions. These strains account for approximately 2% of the total HIV-1-infected population in Greece, thus providing evidence of the great capability of HIV-1 to recombine and produce highly divergent strains which can be spread successfully through different infection routes.

Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes).

Serotype O is the most prevalent of the seven serotypes of foot-and-mouth disease (FMD) virus and occurs in many parts of the world. The UPGMA method was used to construct a phylogenetic tree based on nucleotide sequences at the 3' end of the VP1 gene from 105 FMD type O viruses obtained from samples submitted to the OIE/FAO World Reference Laboratory for FMD. This analysis identified eight major genotypes when a value of 15% nucleotide difference was used as a cut-off. The validity of these groupings was tested on the complete VP1 gene sequences of 23 of these viruses by bootstrap resampling and construction of a neighbour-joining tree. These eight genetic lineages fell within geographical boundaries and we have used the term topotype to describe them. Using a large sequence database, the distribution of viruses belonging to each of the eight topotypes has been determined. These phylogenetically based epidemiological studies have also been used to identify viruses that have transgressed their normal ecological niches. Despite the high rate of mutation during replication of the FMD virus genome, the topotypes appear to represent evolutionary cul-de-sacs.

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

[Inter-species transmission of the influenza virus]. / Transmission inter-espèces du virus de la grippe.

Influenza is an infection of human beings and several animal species. It is caused by influenza viruses which belong to the Orthomyxoviridae family. Type A influenza viruses are the most important as they cause severe epidemics and are responsible of important pathological troubles. Type A influenza viruses are classified in different sub-types depending of the nature of their surface glycoproteins: haemagglutinin (H) and neuraminidase (N). The nature of the genome and the mode of replication of influenza viruses account for the high variability of these two proteins which are responsible for the immunity to the virus. The continuous appearance of point mutations in the gene coding for the H protein, leads to the progressive emergence of new viral strains. This event which is called antigenic drift makes it necessary to annually assess the composition of the human flue vaccine. Genetic reassortment is another mechanism of antigenic variation. When the gene coding for the H protein, or when both genes coding for H and N proteins are involved in genetic reassortment, a new viral sub-type occurs which replace the precedent. This event, which is termed antigenic shift, occurs occasionally every 10 to 30 years, and it is responsible of the great human pandemics. The role of the animals and particularly the importance of pigs and poultry in the emergence of these new viruses is discussed.

Characterization of the coat protein gene of mite-transmitted blackcurrant reversion associated nepovirus.

The nucleotide sequence of the 3' terminal 3105 nucleotides (nt) of RNA2 of blackcurrant reversion associated virus (BRAV), the first mite-transmitted member of the nepovirus group, has been determined. The sequence contains an open reading frame of 1744 nt in the virus-sense strand, a 3' untranslated region of 1360 nt and a 3' poly(A) tail. Analysis of the amino-terminal residues of purified coat protein (CP) suggests that the CP gene is located between nts 1361 and 2959 (from the 3' terminus) in the RNA2, and that Asp/Ser is the proteolytic cleavage site of CP in the RNA2 encoded polyprotein. The predicted translation product from the CP gene is a polypeptide of 533 amino acids with a calculated Mr of 57 561. The amino acid sequence of BRAV CP showed highest similarity to blueberry leaf mottle virus (BLMV), and tomato ringspot virus (ToRSV), two members of the proposed sub-group three of nepoviruses possessing large RNA2 components. Nucleic and amino acid sequence comparisons between BRAV CP and the CPs of other nepoviruses indicate that specific conserved nepovirus CP domains occur in the BRAV CP thus confirming that BRAV is a member of the subgroup three of nepoviruses. reserved.

Bovine rotavirus strain 678 possesses VP4 of serotype P7[5] specificity.

Bovine rotavirus strain 678 is the first G8 strain of bovine origin but the literature is confusing as to its P type. In this study, two-way cross neutralization between 678 and 0510, a prototype G6P7[5] virus, was shown by plaque-reduction neutralization assays, establishing the P type of 678 as being P7[5]. The P7[5] specificity of 678 VP4 was reinforced by the finding that the VP8* portion of 678 VP4 had the highest amino acid identity with those of P7[5] bovine rotaviruses. Apparent contradiction with previous serological studies relates to intricacy of antigenicity and immunogenicity of UK VP4 in reassortants.

Distribution of VP7 serotypes and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea.

108 rotavirus strains obtained from children with diarrhea hospitalized in Palermo, Italy, in the years 1990-1994, were examined by seminested PCR to study the relative frequency and distribution of the four most common alleles of the gene 4. Such strains were selected from 344 human rotavirus strains recovered in palermo during those years after characterization by electropherotyping, subgrouping and G serotyping. One hundred and seven of the 108 strains could be classified into P types, the P[8], G1 (38.3%) and the P[8], G4 (52.3%) types being predominant. The unique strain whose P genotype could not be identified showed an unusual combination of long migration electrophoretic pattern and subgroup I specificity.

Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein.

VP7 genes of rotavirus (RV) 993/83 isolated from a German calf with diarrhea and of RV PO-13 isolated from a Japanese pigeon were sequenced. Alignment of the deduced VP7 amino acid sequence showed 98.8% sequence identity, while only 70% and 84% identity was seen with VP7 from chicken RV Ch-2 and turkey RV Ty-1, respectively. Over the antigenic regions A, B, and C mammalian RV 993/83 showed more aa identity with mammalian G3 RVs than with chicken RV Ch-2, which could explain the strong one-way cross-neutralization observed between RV 993/83 and G3 RVs. Despite marked VP7 sequence diversity avian RVs could not be differentiated into distinct G types.

Sequence analysis of the gene encoding the capsid protein of the Snow Mountain human calicivirus.

Snow Mountain virus (SMV) is the reference strain for serotype 3 as determined by immune electron microscopy of the human caliciviruses that are associated with epidemic gastroenteritis. In order to establish the genetic relationship of its capsid protein with those from other human caliciviriuses, the sequence of the open reading frame 2 (ORF2) encoding the SMV capsid protein was determined. The SMV ORF2 sequence was 1626 nucleotides in length and the deduced protein of 542 amino acids had a calculated molecular weight of 59.2 kD. The SMV capsid sequence showed approximately 48 and 77% amino acid sequence identity with the capsid proteins of the Norwalk (serotype 1) and Hawaii (serotype 2) human calicivirus reference strains, respectively, a finding consistent with its serotypic distinctiveness. Furthermore, the predicted amino acid sequence of the SMV capsid was found to share highest sequence identity (98%) with the Melksham human calicivirus in database searches.