Complete Polyprotein Research Articles

A novel flavivirus detected in two Aedes spp. collected near the demilitarized zone of the Republic of Korea.

Flaviviruses comprise a large and diverse group of positive-stranded RNA viruses, including tick-, mosquito- and unknown-vector-borne flaviviruses. A novel flavivirus was detected in pools of Aedes vexans nipponii (n=1) and Aedes esoensis (n=3) collected in 2012 and 2013 near the demilitarized zone (DMZ), Republic of Korea (ROK). Phylogenetic analyses of the NS5, E gene and complete polyprotein coding sequence (CDS) showed that the novel virus fell within the Aedes-borne flaviviruses (ABFVs), with nucleotide identity ranging from 57.8-75.1 %, 46.1-74.2 % and 51.1-76.2 %, respectively. While the novel ABFV was distant from other flaviviruses within the group, it formed a clade with Ilomantsi virus (ILOV). Sequence alignments of the partial NS5 gene, full-length E gene and polyprotein CDS between the novel virus and ILOV showed approximately 76.2 % nucleotide identity and 90 % amino acid identity, respectively. The ABFV identified in Aedes mosquitoes from the ROK is a novel ABFV based on the sequence analyses and is designated as Panmunjeom flavivirus (PANFV).

Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R.

ABSTRACTMany DNA tumor viruses promote cellular transformation by inactivating the critically important tumor suppressor protein p53. In contrast, it is not known whether p53 function is disrupted by hepatitis C virus (HCV), a unique, oncogenic RNA virus that is the leading infectious cause of liver cancer in many regions of the world. Here we show that HCV-permissive, liver-derived HepG2 cells engineered to constitutively express microRNA-122 (HepG2/miR-122 cells) have normal p53-mediated responses to DNA damage and that HCV replication in these cells potently suppresses p53 responses to etoposide, an inducer of DNA damage, or nutlin-3, an inhibitor of p53 degradation pathways. Upregulation of p53-dependent targets is consequently repressed within HCV-infected cells, with potential consequences for cell survival. Despite this, p53 function is not disrupted by overexpression of the complete HCV polyprotein, suggesting that altered p53 function may result from the host response to viral RNA replication intermediates. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting HCV replication, showing that p53 inhibition results directly from viral activation of PKR. The hepatocellular abundance of phosphorylated PKR is elevated in HCV-infected chimpanzees, suggesting that PKR activation and consequent p53 inhibition accompany HCV infection in vivo. These findings reveal a feature of the host response to HCV infection that may contribute to hepatocellular carcinogenesis.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins.

Porcine sapelovirus among diarrhoeic piglets in India.

Porcine sapelovirus (PSV) A belongs to the genus Sapelovirus, family Picornaviridae. PSV infections in pigs have been reported from European countries, United States, Japan, China, Korea and Brazil. The virus has been isolated/detected from faeces of healthy pigs as well as those affected with diarrhoea, respiratory signs, encephalitis, skin lesions and fertility disorders. This study was planned to investigate whether PSV is prevalent among pigs in India and to characterize PSV encountered in the study population. The study revealed that five of 70 (7.14%) faecal samples were found positive for PSV using RT-PCR. Three viruses were successfully isolated from faecal samples using IB-RS-2 cell line. Complete genome sequencing and analysis of one Indian PSV isolate revealed highest homology (88%) with V13 strain from England. Phylogenetic analysis of the complete polyprotein nucleotide sequences of 14 strains of PSV classified the viruses into four distinct clades. This first report from India adds to our knowledge on genetic diversity of PSV detected so far among pigs in different countries. A large-scale surveillance of the virus is required to understand its genomic diversity and economic impact.

Molecular characterization of two sugarcane streak mosaic virus isolates from Iran with emphasis on its population structure.

Sugarcane streak mosaic virus (SCSMV; the genus Poacevirus, the family Potyviridae) is an economically important causal agent of sugarcane mosaic disease in Asia. In this study, for the first time, we determined the complete genomic sequences of two Iranian SCSMV isolates, IR-Khuz6 and IR-Khuz57 from sugarcane. The sequences of both isolates were 9,782 nucleotides (nt) long, excluding the 3' poly(A) tail. Both of them contained a 5'-untranslated region (UTR) of 199 nt, an open reading frame of 9,393 nt encoding a polyprotein of 3,130 amino acids (aa), and 3'-UTR of 190 nt. SCSMV-IR-Khuz6 and IR-Khuz57 genome nucleotide sequences were in 97.7% identical and shared identities of 81-92.4% with 10 other SCSMV isolates available in the GenBank. The highest identity was shared with the isolate PAK (NC_014037) from Pakistan. When separate genes were compared, most of the genes shared the highest identities with Pakistani isolate. Phylogenetic analysis of the complete genomic nucleotide and polyprotein amino acid sequences reveals that all SCSMV isolates clustered into two main groups. Both IR-Khuz6 and IR-Khuz57 clustered with isolates from Pakistan (PAK) and India (IND671) in group II but formed a separate subgroup. Population genetic analysis revealed greater between-group than within-group evolutionary divergence values, further supporting the results of the phylogenetic analysis. The results indicate that gene flow and selection pressure are important evolutionary factors shaping the genetic structure of SCSMV populations with implications for global exchange of sugarcane germplasm.

Complete genome sequence of a novel sub-subgenotype 2.1g isolate of classical swine fever virus from China.

Current subgenotype 2.1 isolates of classical swine fever virus (CSFV) play a dominant role in CSF outbreaks in China, and a novel sub-subgenotype 2.1g of CSFV was recently identified, but the complete genome sequence of this new sub-subgenotype has not been reported. In this study, complete genome of 2.1g isolate GD19/2011 collected from Guangdong province of China in 2011 was sequenced. It was found to be 12,298 nucleotides (nt) in length, including a 375-nt 5'UTR, a 11,697-nt opening reading frame (ORF), and a 227-nt 3'UTR. GD19/2011 shared 91.0-93.7% and 95.6-97.5% nt and amino acid sequence identity, respectively, with other subgenotype 2.1 isolates. The topology of a phylogenetic tree constructed based on complete genome sequences of GD19/2011 and other CSFV isolates was identical to that obtained with full-length E2 gene sequences, but it was significantly different from those obtained with the 5'UTR and core sequences. Serial passages of GD9/2011 in PK-15 cells generated a highly cell-adapted virus stock with an infectious titer of 10(7.8) TCID50/ml at the 12(th) passage in which two amino acid substitutions, S476R and N2494S, were observed in comparison with the complete polyprotein sequence of the original isolate from kidney tissue, GD19/2011. This is the first report of the complete genome sequence of a 2.1g isolate, and the GD19/2011 isolate will be useful for further analysis of the evolution and virulence of CSFV isolates.

Presence of atypical porcine pestivirus (APPV) genomes in newborn piglets correlates with congenital tremor.

Pestiviruses are highly variable RNA viruses belonging to the continuously growing family Flaviviridae. A genetically very distinct pestivirus was recently discovered in the USA, designated atypical porcine pestivirus (APPV). Here, a screening of 369 sera from apparently healthy adult pigs demonstrated the existence of APPV in Germany with an estimated individual prevalence of 2.4% and ~10% at farm level. Additionally, APPV genomes were detected in newborn piglets affected by congenital tremor (CT), but genomes were absent in unaffected piglets. High loads of genomes were identified in glandular epithelial cells, follicular centers of lymphoid organs, the inner granular cell layer of the cerebellum, as well as in the trigeminal and spinal ganglia. Retrospective analysis of cerebellum samples from 2007 demonstrated that APPV can be found in piglets with CT of unsolved aetiology. Determination of the first European APPV complete polyprotein coding sequence revealed 88.2% nucleotide identity to the APPV sequence from the USA. APPV sequences derived from different regions in Germany demonstrated to be highly variable. Taken together, the results of this study strongly suggest that the presence of APPV genomes in newborn piglets correlates with CT, while no association with clinical disease could be observed in viremic adult pigs.

Complete genome sequence of yam chlorotic necrotic mosaic virus from Dioscorea parviflora.

The complete genome sequence of yam chlorotic necrotic mosaic virus (YCNMV) was determined. It is a monopartite ssRNA 8208 nucleotides in length (excluding the poly(A) tail) and encoding a polyprotein of 2622 amino acids. Sequence analysis showed that the P1 region and some conserved motifs, such as the typical potyvirus aphid-transmission motifs DAG, PTK and KITC, are absent. Phylogenetic analysis based on the complete polyprotein sequences of YCNMV and selected members of the family Potyviridae clearly showed that this virus should be assigned to the genus Macluravirus and suggest that YCNMV is a new member of the genus Macluravirus.

Genomic variability and molecular evolution of Asian isolates of sugarcane streak mosaic virus.

Sugarcane streak mosaic virus (SCSMV), an economically important causal agent of mosaic disease of sugarcane, is a member of the newly created genus Poacevirus in the family Potyviridae. In this study, we report the molecular characterization of three new SCSMV isolates from China (YN-YZ211 and HN-YZ49) and Myanmar (MYA-Formosa) and their genetic variation and phylogenetic relationship to SCSMV isolates from Asia and the type members of the family Potyviridae. The complete genome of each of the three isolates was determined to be 9781 nucleotides (nt) in size, excluding the 3' poly(A) tail. Phylogenetic analysis of the complete polyprotein amino acid (aa) sequences (3130 aa) revealed that all SCSMV isolates clustered into a phylogroup specific to the genus Poacevirus and formed two distinct clades designated as group I and group II. Isolates YN-YZ211, HN-YZ49 and MYA-Formosa clustered into group I, sharing 96.8-99.5% and 98.9-99.6% nt (at the complete genomic level) and aa (at the polyprotein level) identity, respectively, among themselves and 81.2-98.8% and 94.0-99.6% nt (at the complete genomic level) and aa (at the polyprotein level) identity, respectively, with the corresponding sequences of seven Asian SCSMV isolates. Population genetic analysis revealed greater between-group (0.190±0.004) than within-group (group I=0.025±0.001 and group II=0.071±0.003) evolutionary divergence values, further supporting the results of the phylogenetic analysis. Further analysis indicated that natural selection might have contributed to the evolution of isolates belonging to the two identified SCSMV clades, with infrequent genetic exchanges occurring between them over time. These findings provide a comprehensive analysis of the population genetic structure and driving forces for the evolution of SCSMV with implications for global exchange of sugarcane germplasm.

Comparative genomic analysis of pre-epidemic and epidemic Zika virus strains for virological factors potentially associated with the rapidly expanding epidemic

Less than 20 sporadic cases of human Zika virus (ZIKV) infection were reported in Africa and Asia before 2007, but large outbreaks involving up to 73% of the populations on the Pacific islands have started since 2007, and spread to the Americas in 2014. Moreover, the clinical manifestation of ZIKV infection has apparently changed, as evident by increasing reports of neurological complications, such as Guillain–Barré syndrome in adults and congenital anomalies in neonates. We comprehensively compared the genome sequences of pre-epidemic and epidemic ZIKV strains with complete genome or complete polyprotein sequences available in GenBank. Besides the reported phylogenetic clustering of the epidemic strains with the Asian lineage, we found that the topology of phylogenetic tree of all coding regions is the same except that of the non-structural 2B (NS2B) coding region. This finding was confirmed by bootscan analysis and multiple sequence alignment, which suggested the presence of a fragment of genetic recombination at NS2B with that of Spondweni virus. Moreover, the representative epidemic strain possesses one large bulge of nine bases instead of an external loop on the first stem-loop structure at the 3′-untranslated region just distal to the stop codon of the NS5 in the 1947 pre-epidemic prototype strain. Fifteen amino acid substitutions are found in the epidemic strains when compared with the pre-epidemic strains. As mutations in other flaviviruses can be associated with changes in virulence, replication efficiency, antigenic epitopes and host tropism, further studies would be important to ascertain the biological significance of these genomic changes.Emerging Microbes and Infections (2016) 5, e22; doi:10.1038/emi.2016.48; published online 16 March 2016

Complete Genome Sequence of an Atypical Dengue Virus Serotype 2 Lineage Isolated in Brazil.

Here, we report the complete polyprotein sequence of a dengue virus 2 strain isolated in Brazil. This virus belongs to the American genotype and has the ability to cause neurovirulence in immunocompetent adult mice. The data presented here may help understand the genetic determinants responsible for neurovirulence.

Genome sequence of a virus isolate from tamarillo (Solanum betaceum) in Colombia: evidence for a new potyvirus.

Based on the results of a deep sequencing transcriptome study of tamarillo (Solanum betaceum), we report the genome sequence of a virus from this host plant. Since this probably represents a new member of the genus Potyvirus, the name tamarillo leaf malformation virus (TaLMV) has been proposed. Phylogenetic analysis reveals that TaLMV is the closest relative of Colombian datura virus (CDV), followed by three other potyviruses: tobacco etch virus, potato virus A and tobacco vein mottling virus. This is the first sequence of a potyvirus infecting Solanum betaceum containing the complete polyprotein coding region.

Putative New West Nile Virus Lineage inUranotaenia unguiculataMosquitoes, Austria, 2013

West Nile virus (WNV) is becoming more widespread and markedly effecting public health. We sequenced the complete polyprotein gene of a divergent WNV strain newly detected in a pool of Uranotaenia unguiculata mosquitoes in Austria. Phylogenetic analyses suggest that the new strain constitutes a ninth WNV lineage or a sublineage of WNV lineage 4.

Isolation and molecular characterization of a novel picornavirus from baitfish in the USA.

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼30–32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

Tall oatgrass mosaic virus (TOgMV): a novel member of the genus Tritimovirus infecting Arrhenatherum elatius

A novel tritimovirus of the family Potyviridae was isolated from tall oatgrass, Arrhenatherum elatius, exhibiting mosaic symptoms. The virus, for which the name tall oatgrass mosaic virus (TOgMV) is coined, has a filamentous particle of 720 nm and is associated with pinwheel inclusion bodies characteristic of members of the family Potyviridae. The virus was mechanically transmitted to tall oatgrass seedlings, which subsequently exhibited mosaic symptoms. The experimental host range was limited to a few monocot species. The complete genome sequence of TOgMV was determined to be 9359 nucleotides, excluding the 3' polyadenylated tail. The viral RNA encodes one large putative open reading frame of 3029 amino acids with a genome organization typical of monopartite potyvirids. Pairwise comparison of putative mature proteins and proteinase cleavage sites indicated that TOgMV is most closely related to members of the genus Tritimovirus. Phylogenetic analysis of the complete polyprotein and CP sequences of representative members of the family Potyviridae indicate that TOgMV is a distinct tritimovirus naturally infecting tall oatgrass.

Barkedji virus, a novel mosquito-borne flavivirus identified in Culex perexiguus mosquitoes, Israel, 2011

Barkedji virus, named after the area of its first identification in Senegal, is a newly discovered flavivirus (FV), for which we propose the abbreviation BJV. In the present study, we report the first-time detection of BJV in Culex perexiguus mosquitoes in Israel in 2011 and determination of its almost complete polyprotein gene sequence. We characterized the BJV genome and defined putative mature proteins, conserved structural elements and potential enzyme motifs along the polyprotein precursor. By comparing polyproteins and individual proteins of BJV with several other FVs, a distant relationship of BJV to Nounane virus (NOUV), a recently described African FV, is demonstrated. Phylogenetic analysis of 55 selected flaviviral polyprotein gene sequences exhibits two major clusters, one made up of the classical three clades of FVs: mosquito-borne, tick-borne and those without known vectors. The other cluster exclusively contains so-called 'insect-specific' FVs, which do not replicate in vertebrate cells. Based on our phylogenetic analysis, BJV is related to other members of the mosquito-borne clade with yet unknown vertebrate hosts, such as NOUV, Donggang virus, Chaoyang virus and Lammi virus. However, with a maximum identity of only 54 % to NOUV, BJV represents a distinct new virus species.

Comparative Genomic and Phylogenetic Analysis of the First Usutu Virus Isolate from a Human Patient Presenting with Neurological Symptoms

Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5′ and 3′ untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.

Comparison of complete polyprotein sequences of two isolates of salmon alphavirus (SAV) type I and their behaviour in a salmonid cell line

Salmon pancreas disease virus is an alphavirus (family Togaviridae) affecting mainly Atlantic salmon (Salmo salar L.). Both polyprotein sequences of the Scottish isolate (SAV4640) were determined and compared with those of Irish isolate SAVF93-125. High amino acid sequence similarity (99.4%) was found. Six amino acid deletions were found in the E2 gene of SAV4640. SAVF93-125 demonstrated a high viral load in culture despite high Mx expression. Approximately 50% of cells infected with SAVF93-125 exhibited a cytopathic effect by day 8. SAV4640 successfully entered the cells, inducing 10,500-fold higher Mx expression at day 2 compared to SAVF93-25; however, no replication was observed based on results of the nsP1 qRT-PCR.

Complete genome sequence of a South Korean isolate of Brugmansia mosaic virus

We determined the complete genome sequence of a South Korean (SK) isolate of Brugmansia mosaic virus (BruMV), which has recently been proposed to be a member of a new species in the genus Potyvirus. The genomic RNA of BruMV isolate SK is 9781 nucleotides in length (excluding the 3'-terminal poly (A)) and shares complete nucleotide and polyprotein amino acid sequence identities of 85.6% and 93.1%, respectively, with the type isolate (D-437) of BruMV described in the USA. To our knowledge, this is the first report providing evidence of considerable sequence variation in BruMV.

Genetic variation of the Turnip mosaic virus population of Vietnam: A case study of founder, regional and local influences

Turnip mosaic virus (TuMV) is one of the most important viruses infecting a wide range of plant species, primarily from the family Brassicaceae. Thirty TuMV isolates were collected from Brassica and Raphanus plants in Vietnam during 2006–2008. Host reaction studies showed that many of the isolates belonged to Brassica/Raphanus (BR) host-infecting type. Sequence-based phylogenetic and population genetic analyses were made of the complete polyprotein gene sequences, and of four non-recombinogenic regions of those sequences (i.e. genes of the helper-component proteinase protein, protein 3, nuclear inclusion b protein and coat protein). These were used to assess the subpopulation differentiation and divergence between Vietnamese TuMV populations and those of nearby Asian countries. Nine inter- and intralineage recombination type patterns were identified in the genomes of the Vietnamese isolates, of which seven were novel. All the Vietnamese non-recombinant isolates fell into the world-B group of TuMV and clustered with Chinese isolates. The estimates of genetic differentiation and gene flow reveal that the TuMV populations of Vietnam, China and Japan are genetically linked but have clear local founder effects. This, the first population genetic study of a TuMV population in Southeast Asia, indicates the importance of such studies for providing the scientific basis of control strategies.