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PubMed1987

The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avian or human influenza virus internal genes. Twenty-four reassortant viruses were isolated and first evaluated for infectivity in avian (primary chick kidney [PCK]) and mammalian (Madin-Darby canine kidney [MDCK]) tissue culture lines. Reassortant viruses with two specific constellations of viral polymerase genes exhibited a significant host range restriction of replication in mammalian (MDCK) tissue culture compared with that in avian (PCK) tissue culture. The viral polymerase genotype PB2-avian (A) virus, PB1-A virus, and PA-human (H) virus was associated with a 900-fold restriction, while the viral polymerase genotype PB2-H, PB1-A, and PA-H was associated with an 80,000-fold restriction of replication in MDCK compared with that in PCK. Fifteen reassortant viruses were subsequently evaluated for their level of replication in the respiratory tract of squirrel monkeys, and two genetic determinants of attenuation were identified. First, reassortant viruses which possessed the avian influenza virus nucleoprotein gene were as restricted in replication as a virus which possessed all six internal genes of the avian influenza A virus parent, indicating that the nucleoprotein gene is the major determinant of attenuation of avian-human A/Pintail/79 reassortant viruses for monkeys. Second, reassortant viruses which possessed the viral polymerase gene constellation of PB2-H, PB1-A, and PA-H, which was associated with the greater degree of host range restriction in vitro, were highly restricted in replication in monkeys. Since the avian-human influenza reassortant viruses which expressed either mode of attenuation in monkeys replicated to high titer in eggs and in PCK tissue culture, their failure to replicate efficiently in the respiratory epithelium of primates must be due to the failure of viral factors to interact with primate host cell factors. The implications of these findings for the development of live-virus vaccines and for the evolution of influenza A viruses in nature are discussed.

Animals↗
PubMed1989

Glycoprotein evolution of vesicular stomatitis virus New Jersey.

A T1 ribonuclease fingerprinting study of a large number of virus isolates had previously demonstrated that considerable genetic variability existed among natural isolates of the vesicular stomatitis virus (VSV) New Jersey (NJ) serotype [S.T. Nichol (1988) J. Virol. 62, 572-579]. Based on these results, 34 virus isolates were chosen as representing the extent of genetic diversity within the VSV NJ serotype. We report the entire glycoprotein (G) gene nucleotide sequence and the deduced amino acid sequence for each of these viruses. Up to 19.8% G gene sequence differences could be seen among NJ serotype isolates. Analysis of the distribution of nucleotide substitutions relative to nucleotide codon position revealed that third position changes were distributed randomly throughout the gene. Third base changes constituted 84% of the observed nucleotide substitutions and affected 89% of the third base positions located in the G gene. Only three short oligonucleotide stretches of complete sequence conservation were observed. The remaining nucleotide changes located in the first and second positions were not distributed randomly, indicating that most of the amino acids coded by the G gene cannot be altered without reducing the fitness of the VSV NJ serotype viruses. Despite these constraints, up to 8.5% amino acid differences were observed between virus isolates. These differences were located throughout the G protein including regions adjacent to defined major antibody neutralization epitopes. Apparent clusters of amino acid substitutions were present in the hydrophobic signal sequence, transmembrane domain, and within the cytoplasmic domain of the G protein. A maximum parsimony analysis of the G gene nucleotide sequences allowed construction of a phylogram indicating the evolutionary relationship of these viruses. The VSV NJ serotype appears to contain at least three distinct lineages or subtypes. All recent virus isolates from the United States and Mexico are within subtype I and appear to have evolved from an ancestor more closely related to the Hazelhurst historic strain than other older strains. The implications of these findings for the evolution, epizootiology, and classification of these viruses are discussed.

Amino Acid Sequence↗
PubMed1996

A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis.

Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.

Base Sequence↗
PubMed2026

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗
PubMed2006

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family.

The approximately 30-kb coronavirus (+)RNA genome is replicated and transcribed by a membrane-bound replicase complex made up of 16 viral nonstructural proteins (nsp) with multiple enzymatic activities. The complex includes an RNA endonuclease, NendoU, that is conserved among nidoviruses but no other RNA virus, making it a genetic marker of this virus order. NendoU (nsp15) is a Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Neither biochemical nor sequence homology criteria allow a classification of nsp15 into existing endonuclease families. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus nsp15 at 2.6-A resolution. Nsp15 exhibits a unique fold and assembles into a toric hexamer with six potentially active, peripheric catalytic sites. The structure and the spatial arrangement of the catalytic residues into an RNase A-like active site define a separate endonuclease family, endoU, and represent another spectacular example of convergent evolution toward an enzymatic function that is critically involved in the coronavirus replication cycle.

Amino Acid Sequence↗
PubMed2005

A test of Chargaff's second rule.

In 1968, Chargaff and his colleagues discovered a rule in Bacillus subtilis: in single stranded DNA, A=T and C=G. This rule has since been confirmed many times in other bacterial and eukaryotic genomes. To the best of our knowledge, this rule has not been tested before in either single stranded DNA or RNA genomes. Over 3400 genomic sequences were examined here and included for the first time both double and single stranded DNA and RNA genomes. We found that: (1) with the exception of the organellar DNA, this parity rule holds for all types of double stranded DNA genomes and (2) that this rule fails to hold for other types of genomes. The parity rule appears to be a selective force on genome evolution and codon use.

Archaea↗
PubMed2019

A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.

barcoding↗
PubMed2001

Evolution of coinfection with human immunodeficiency virus and hepatitis C virus in patients treated with highly active antiretroviral therapy.

A retrospective analysis of data from a cohort of patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) who were treated with highly active antiretroviral therapy (HAART) at 3 infectious diseases units in northern Italy was performed. While the patients were receiving HAART, CD4(+) cell counts significantly increased and HIV RNA serum levels decreased. However, no significant overall changes in alanine aminotransferase (ALT) levels and HCV RNA serum levels were observed. Fifteen (4.6%) of 323 patients died within 3 years of follow-up; death was related to cirrhosis in 5 patients (1.6%). No significant difference was observed between cirrhosis-related mortality and mortality related to other causes. Patients with ALT levels >4 times the normal values at initiation of HAART showed a significant decrease in ALT levels, whereas patients with normal ALT levels at initiation of HAART showed a significant increase over time, suggesting that HAART may have long-term beneficial or detrimental effects, depending on patient characteristics.

Alanine Transaminase↗
PubMed2026

Characterization of yam virus X isolates from Dioscorea trifida in Brazil.

OBJECTIVE: Yam virus X (YVX; Potexvirus ecsdioscoreae) is a positive-sense, flexuous RNA virus belonging to the family Alphaflexiviridae. It has been first reported from Guadeloupe, a French archipelago located in the Caribbean Sea. In this study, we investigated the virome in yam (Dioscorea spp.) plant material collected in the state of Bahia (Brazil) by high-throughput sequencing (HTS) on Illumina platform. The objective of the investigation was to explore the occurrence of YVX in yam from South America, and to study its genetic diversity compared to the only one YVX genome sequence available in the GenBank public database. RESULTS: An initial investigation by HTS of bulked RNA extracts (n=23, combined into 4 pools) revealed occurrence of YVX only in samples collected in the region of Valença. Subsequent screening by RT-PCR of the individual samples composing the pool uncovered infection with YVX only in Discorea trifida. Total RNA extracts from three infected plants were individually sequenced, resulting in the assembly of three complete genome sequences of YVX, showing ~84% nucleotide identity to the reference sequence from Guadeloupe. Our results contribute to expanding the pool of sequences available for YVX, supporting detection purposes and stimulating additional investigations for future studies on YVX diversity and evolution.

Brazil↗
PubMed2006

Dynamics of dsRNA mycoviruses in black Aspergillus populations.

Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes ranging from 0.8 to 4.4 kb in size. In one strain the virus shows clear visible effects on its host with non-sporulating sectors. We quantified the fitness costs of these and more 'cryptic' virus infections on mycelial growth rate and spore production, and on competitive ability with respect to other strains under different growth conditions. Mycovirus infection proved detrimental in all these measures. The reduced success in interference competition due to mycovirus infection belies co-evolution of mycovirus and host to a mutually beneficial symbiosis, like in killer virus systems in yeast and smut and agrees more to recent infections. For a stable virus infection frequency in the black Aspergillus population, fitness costs and spontaneous loss should be balanced with new infections. Implications of even small viral fitness effects combined with the observed transmission limits for host and mycovirus are discussed.

Aspergillus↗
PubMed1988

Rates and dates of divergence between AIDS virus nucleotide sequences.

The acquired immune deficiency syndrome (AIDS), caused by a retrovirus called human immunodeficiency virus (HIV), has become a pandemic. A knowledge of the rate of nucleotide substitution in HIV and of the history and pattern of spread of the virus is important for understanding the epidemiology and pathogenesis of AIDS and for developing therapies and vaccine strategies. A new model has been developed and used to estimate the substitution rates in various regions in the HIV genome. The rate of nonsynonymous (amino acid-changing) substitution is lowest in the regions coding for the capsid proteins and the reverse transcriptase, being approximately 1.7 X 10(-3) nucleotide substitutions/site/year. The nonsynonymous rate is extremely high (14 X 10(-3] in the hypervariable regions of the envelope gene, suggesting extremely rapid change in viral antigenicity. The nonsynonymous rates in the other coding regions are between 3 X 10(-3) and 7 X 10(-3). The average synonymous rate for the HIV genome is 10 X 10(-3). These rates are 10(6) times greater than the rates in DNA genomes and at least as high as the rates in other RNA viruses. Evidence is provided for a case of recombination between different HIV strains. Our analysis suggests that the AIDS virus had existed in central Africa before 1960 and spread to North America before the mid 1970s. The evolutionary relationships among HIV isolates are inferred from nucleotide sequence data, and the result is consistent with the view that AIDS spread from Haiti to the United States.

Base Sequence↗
PubMed2006

Changes in viral loads of lamivudine-resistant mutants and evolution of HBV sequences during adefovir dipivoxil therapy.

The addition of adefovir dipivoxil (ADV) to ongoing lamivudine therapy is effective against lamivudine-resistant virus in patients with hepatitis B virus (HBV) infection. We studied 39 patients who received ADV added to lamivudine for breakthrough hepatitis. We determined early viral changes (12 weeks) in YMDD mutants (rtM204I [YIDD sequence], rtM204V [YVDD]) and rtL180M in all 39 patients as well as amino acid changes in the polymerase reverse transcriptase (rt) region and precore/core promoter mutations in 15 patients who received long-term treatment (more than 1 year). Changes in rtM204I and rtL180M viral loads were greater than that of the rtM204V, albeit statistically insignificant. Moreover, the greatest change in viral load was seen for rtM204I without hepatitis B e antigen (HBeAg). The precore mutant was replaced with wild-type virus in three of eight patients after 1 year of added ADV therapy. Compared to baseline with lamivudine therapy only, new amino acid mutations were seen in the rt region at baseline with ADV in seven patients. At 1 year after ADV coadministration, the YMDD motif was replaced with wild-type (rt204M) in two patients, in whom mutations were fewer and of a different type. We conclude that the rtM204I may be more sensitive to ADV in vivo. ADV tended to select wild-type virus from precore mutants. Moreover, viruses that were wild-type in the rt region reappeared after 1 year of ADV coadministration in some patients.

Adenine↗
PubMed1995

Activation of expression of multiple subfamilies of human Alu elements by adenovirus type 5 and herpes simplex virus type 1.

The nearly one million Alu repetitive elements in the human genome can be grouped into a number of subfamilies. Comparisons between subfamily consensus sequences suggest that Alu evolution is characterized by the sequential amplification and dispersal of a limited number of Alu founder sequences. The S, Sb and Sb1 subfamilies provide an example of such a related series of Alu subfamilies. We have previously demonstrated that adenovirus type 5 and herpes simplex virus type 1 activate RNA polymerase III transcription of endogenous Alu elements in HeLa cells. Here, we report that expression of Alu sequences belonging to the S, Sb and Sb1 subfamilies was activated following infection with these viruses. The data indicate that transpositionally inactive Alu elements can give rise to high levels of pol III transcripts in the presence of appropriate trans-acting factors and demonstrate that the class III promoters of a significant number and variety of Alu sequences are functional in vivo. Multiple subfamilies of Alu sequences were induced in transformed and non-transformed cell types, suggesting that induction of Alu expression may be part of the normal cellular response to viral infection.

Adenoviruses, Human↗
PubMed2002

The genetic diversity of European type PRRSV is similar to that of the North American type but is geographically skewed within Europe.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen. Two PRRSV genotypes exist, North American and European, which are only 55-70% identical at the nucleotide level. Previous studies have shown high nucleotide diversity in the North American genotype and low nucleotide diversity in the European genotype. Here, we analyzed the ORF5 and ORF7 genes for a large number of new European type PRRSV isolates in conjunction with existing database sequences. This new analysis showed that contrary to previous assumptions, genetic diversity is at least as high in the European genotype as in the North American genotype. Furthermore, we showed that genetic diversity of European type PRRSV has a marked geographical pattern, with exceptionally high genetic diversity among Italian sequences. The geographical pattern of diversity in relation to the epidemiology of PRRSV in Europe is discussed. Discrepancies between ORF5- and ORF7-based genealogies were observed, and further analysis of the data set confirmed the presence of recombination. We were therefore able to report the first observation of recombination in wild-type isolates of European genotype PRRSV.

Animals↗
PubMed1998

Evolutionary rate and genetic heterogeneity of human T-cell lymphotropic virus type II (HTLV-II) using isolates from European injecting drug users.

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 x 10(-4) and 2.7 x 10(-5) nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10(-2) and 10(-4).

Biological Evolution↗
PubMed1988

Nucleotide sequence of the fusion and phosphoprotein genes of human respiratory syncytial (RS) virus Long strain: evidence of subtype genetic heterogeneity.

The nucleotide and deduced amino acid sequences of the fusion (F) and phosphoprotein (P) genes of the Long strain of human respiratory syncytial (RS) virus have been determined from cDNA copies cloned into pBSV9 shuttle vector. Comparison of these sequences with their counterparts of other strains reveals genetic heterogeneity within the same subtype. The percentage of nucleotide and amino acid changes occurring in both proteins is similar. Thus, the Long F and P proteins share 97.9% and 98.3% amino acid identity, respectively, with their homologs of the A2 strain. Nevertheless the F2 subunit of the fusion protein accumulates 3.1 times more amino acid changes than the F1 subunit. In addition, the percentage of nucleotide changes in the 3' extracistronic sequences is 6 times higher in the P than in the F gene. These results are discussed in terms of selective pressures operating in the evolution of RS virus in nature.

Amino Acid Sequence↗
PubMed1997

Antigenic structure, function, and evolution of the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1.

Twenty-two monoclonal antibodies directed to the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1 (HPIV-1) were used in competition assays to create an antigenic map of neutralization sites. Eighty-seven clinical strains isolated over 35 years from multiple geographic regions were reacted in ELISA, hemagglutinin-inhibition, and microneutralization assays with these monoclonal antibodies. Together these assays revealed 21 epitopes on five nonoverlapping antigenic sites (I, III-VI) with a sixth (II) bridging site connecting sites I, III, and IV. Only 7 (33%) of these epitopes were conserved among all isolates. Previously described HPIV-1 genotypes were associated with the presence or absence of specific antigenic sites and evidence of probable immune selection within genotypes. Two sites were present on all isolates tested (III, V), and one (VI, genotype A) has not been found for 15 years. Forty hemagglutinin-neuraminidase nucleotide sequences were analyzed in terms of homology, structure, and evolution. These data may be useful in future epidemiologic, therapeutic, or vaccine-related work.

Antibodies, Monoclonal↗