ORF2 Region Research Articles

Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon

BackgroundPOLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF.ResultsUsing PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z.ConclusionsWe provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding.

Nine-Year Nationwide Environmental Surveillance of Hepatitis E Virus in Urban Wastewaters in Italy (2011-2019).

Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis worldwide. To provide insights into the epidemiology of HEV in Italy, a large-scale investigation was conducted into urban sewage over nine years (2011–2019), collecting 1374 sewage samples from 48 wastewater treatment plants located in all the 20 regions of Italy. Broadly reactive primers targeting the ORF1 and ORF2 regions were used for the detection and typing of HEV, followed by Sanger and next generation sequencing (NGS). Real-time RT-qPCR was also used to attempt quantification of positive samples. HEV RNA detection occurred in 74 urban sewage samples (5.4%), with a statistically significant higher frequency (7.1%) in central Italy. Fifty-six samples were characterized as G3 strains and 18 as G1. While the detection of G3 strains occurred in all the surveillance period, G1 strains were mainly detected in 2011–2012, and never in 2017–2019. Typing was achieved in 2 samples (3f subtype). Viral concentrations in quantifiable samples ranged from 1.2 × 103 g.c./L to 2.8 × 104 g.c./L. Our results suggest the considerable circulation of the virus in the Italian population, despite a relatively small number of notified cases, a higher occurrence in central Italy, and a noteworthy predominance of G3 strains.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV.HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e.This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

Hepatitis E Virus Infection among Asymptomatic Pregnant Women at the University College Hospital, Ibadan.

Introduction:The high mortality associated with fulminant Hepatitis E infection in pregnancy justifies the need to assess the epidemiologic proportion of this underestimated virus.Objectives:This study aimed to determine the burden of HEV infection among pregnant women attending antenatal Clinic in Ibadan.Methodology:HEV IgG and IgM serological surveys were carried out among 230 pregnant women attending antenatal clinic in Ibadan, Nigeria. Serum and stool samples from HEV IgM positive women were further analysed using two independent reverse transcriptase polymerase chain reactions (RT-PCR) assays, targeting ORF1 region of HEV genome. Socio-demographic variables associated with HEV in these women, were analyzed to estimate statistical significance (P < 0.05).Results:Eleven (4.8%) women had HEV IgM, while 39 (17.0%) women had HEV IgG. Three (27.3%) of the 11 anti-HEV IgM positive samples were positive for HEV RNA while all stool samples tested negative for HEV RNA. HEV infection among pregnant women was statistically associated with age (p = 0.044), and educational status (p = 0.005).Conclnusion:Recent HEV infection among this pregnant population is on the lower part of the scale, compared with other Sub-Saharan African countries. However, the HEV IgG seroprevalence rate suggests indirect evidence of past contact with HEV.

First Report of Banana Streak Virus Infecting Bananas (Musa spp.) in Georgia, U.S.A.

Banana streak virus (BSV) is a badnavirus containing a circular dsDNA genome of 7.4 kb that causes a viral leaf streak disease of banana (Musa spp.) worldwide (Dallot et al. 2001). On bananas, typical symptoms of BSV include broken or continuous chlorotic streaks or spindle-shaped patterns that are first chlorotic, become increasingly dark in color, and subsequently result in black streaking in older leaves (Dallot et al. 2001). In the summer of 2019, six symptomatic leaves from three different plants were collected from the University of Georgia Banana Research Plot, Tifton, GA. The cultivar is ‘Pisang Awak’, and it belongs to the tetraploid AABB genome group. Initial identification was performed by assessing disease symptoms including chlorotic streaks on leaves that became brown and black chlorotic streaks later, resembling those of banana streak disease (Lockhart 1986). BSV was verified via molecular and serological tests. Genomic DNA from leaf tissue of three different symptomatic plants and two healthy plants was extracted using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples were tested for the presence of BSV by two separate PCR amplification methods. One was targeting the aspartic protease (AP) and reverse transcription (RT) regions of ORF3 using primer pair BSV 4673 (5′-GGAATGAAAGAGCAGGCC-3′) and BSV 5317 (5′-AGTCATTGGGTYCAACCTCTGTCCC-3′) with an amplicon size of 644 bp (Manoranjitham et al. 2012). Another PCR analysis was targeting the coat protein (CP) region of ORF3 with primer pair BSV 156 (5′-TTACCTCCAGCACAAAGTCG-3′) and BSV 962 (5′-TTCTCCACAGGCATAACACC-3′) designed in this study with a product size of 806 bp. Both PCR reactions for all six symptomatic samples produced the expected sizes of the fragments for BSV; however, two samples from the healthy plants did not produce any bands. The amplified products were then sequenced and subjected to BLAST analysis for further identification. Sequence analysis revealed that the obtained partial sequence of the AP and RT region (MN567071) shared 100% identity to corresponding sequences from BSV OL virus isolate BSOLV-IN2 (accession no. JQ409540). Furthermore, the obtained CP gene sequence (MN567072) also showed 100% identity with BSV isolate BSOLV-IN2. Samples were also tested for the presence of BSV by real-time PCR with primers BSV 1075 (5′-CACATTGACGGAAAGGACAACG-3′) and BSV 1198 (5′-TGTGAAATACCTCCTCCACTGC-3′) targeting the RNase H gene (Delanoy et al. 2003), and the results confirmed its identity as BSV. In addition to the molecular tests, a DAS-ELISA test was carried out on the remaining symptomatic leaf tissues for further confirmation of BSV according to the manufacturer’s protocol (Nano Diagnostics, Fayetteville, AR). All three samples tested by ELISA were positive for BSV. To the best of our knowledge, this is the first report of BSV infecting Musa spp. in Georgia. This virus could pose an imminent threat to the newly growing banana industry in this area if it spread in banana fields under conditions in Georgia. Our findings will be helpful for banana growers in Georgia by taking appropriate control strategies to combat this viral disease. Further research is needed to elucidate epidemiology, symptomatology, vector transmission, and crop losses due to BSV in Georgia and the United States.

Increasing Recombinant Strains Emerged in Norovirus Outbreaks in Jiangsu, China: 2015\u20132018

From January 2015 to December 2018, 213 norovirus outbreaks with 3,951 patients were reported in Jiangsu, China. Based on viral RdRp and VP1 genes, eight genotypes, GII.2[P16] (144, 67.6%), GII.3[P12] (21, 9.9%), GII.6[P7] (5, 2.3%), GII.14[P7] (4, 1.9%), GII.4 Sydney[P31] (3, 1.4%), GII.1[P33] (1, 0.5%), GII.2[P2] (3, 1.4%), and GII.17[P17] (16, 7.5%) were identified throughout the study period. These genotypes were further regrouped as GII.R (Recombinant) and GII.Non-R (Non-recombinant) strains. In this report we showed that GII.R strains were responsible for at least 178 (83.6%) of 213 norovirus-positive outbreaks with a peak in 2017 and 2018. Most norovirus outbreaks occurred in primary schools and 94 of 109 (86.2%) outbreaks in primary schools were caused by GII.R, while GII.Non-R and GII.NT (not typed) strains accounted for 6 (5.5%) and 9 (8.3%) norovirus outbreaks, respectively. The SimPlot analysis showed recombination breakpoints near the ORF1/2 junction for all six recombinant strains. The recombination breakpoints were detected at positions varying from nucleotides 5009 to 5111, localized in the ORF1 region for four strains (GII.2[P16], GII.3[P12], GII.6[P7], and GII.14[P7]) and in the ORF2 region for the other (GII.4 Sydney[P31] and GII.1[P33]). We identified four clusters, Cluster I through IV, in the GII.P7 RdRp gene by phylogenetic analysis and the GII.14[P7] variants reported here belonged to Cluster IV in the RdRp tree. The HBGA binding site of all known GII.14 strains remained conserved with several point mutations found in the predicted conformational epitopes. In conclusion, gastroenteritis outbreaks caused by noroviruses increased rapidly in the last years and these viruses were classified into eight genotypes. Emerging recombinant noroviral strains have become a major concern and challenge to public health.

Food Safety Risks Associated with Hepatitis E Virus Detection in Wild Boar Liver

Hepatitis E virus (HEV) has significantly impacted humans due to its potential to cause acute viral hepatitis. Discovery of hepatitis E virus in domestic pigs and wild boars worldwide and the realization that it is highly prevalent, raised concerns of the implications for food-borne transmission of HEV in Europe. Present work focusses on molecular detection of hepatitis E virus in wild boar liver samples, underlining the possible role of wildlife as a source of HEV transmission to humans. During hunting season 2016-2017, liver samples were collected from 37 wild boars in Iași and Suceava County. All tissues samples were submitted for RNA isolation followed by nested RT-PCR. Genetic characterization of wild boar HEV targeted the structural gene in the ORF2 region of hepatitis E virus genome. After specific amplification by nested RT-PCR of a 348 nt fragment from HEV ORF2, five liver samples positive for hepatitis E virus genotype 3 RNA were identified. In the present study HEV detection in Romanian fresh liver from wild boars highlights the importance of swine as a possible source of foodborne transmission. Moreover, our results along with the reviewed literature data emphasize the necessity of efficient food safety control measures implementation.

The Neurospora RNA polymerase II kinase CTK negatively regulates catalase expression in a chromatin context-dependent manner.

Clearance and adaptation to reactive oxygen species (ROS) are crucial for cell survival. As in other eukaryotes, the Neurospora catalases are the main enzymes responsible for ROS clearance and their expression are tightly regulated by the growth and environmental conditions. The RNA polymerase II carboxyl terminal domain (RNAPII CTD) kinase complex (CTK complex) is known as a positive elongation factor for many inducible genes by releasing paused RNAPII near the transcription start site and promoting transcription elongation. However, here we show that deletion of CTK complex components in Neurospora led to high CAT-3 expression level and resistance to H2 O2 -induced ROS stress. The catalytic activity of CTK-1 is required for such a response. On the other hand, CTK-1 overexpression led to decreased expression of CAT-3. ChIP assays shows that CTK-1 phosphorylates the RNAPII CTD at Ser2 residues in the cat-3 ORF region during transcription elongation and deletion of CTK-1 led to dramatic decreases of SET-2 recruitment and H3K36me3 modification. As a result, histones at the cat-3 locus become hyperacetylated to promote its transcription. Together, these results demonstrate that the CTK complex is negative regulator of cat-3 expression by affecting its chromatin structure.

Association of Myroides odoratimimus in immunocompromized piglets with post weaning multisystemic wasting syndrome.

To study the association of opportunistic infection due to Myroides odoratimimus in piglets immunocompromised by porcine circovirus type 2 (PCV2) infection. The clinical samples (n=101) were analysed bacteriologically. The isolates were identified by their phenotypes and MALDI TOF-MS analysis as Myroides species. The phylogram constructed based on nucleotide sequences of the 16S rRNA gene showed identity (~99%) with the M. odoratimimus isolates. The minimum inhibitory concentration values for antibiotics revealed M. odoratimimus to be resistant against carbapenem, cephalosporins, aminoglycosides and fluoroquinolones. The presence of PCV2 in affected tissue samples was confirmed by amplification of the 565bp region of ORF2 of the PCV2 genome. The topology of the phylogenetic tree grouped the PCV2 with cluster-2d. PCV2 being immunosuppressive in nature might have impaired the immunity thereby increasing the susceptibility of immunocompromised piglets to opportunistic pathogens such as M. odoratimimus leading to disease severity and high mortality. The M. odoratimimus isolates were found to be multidrug resistant and evidenced for uncertain clinical relevance and hence could act as hidden source of public health hazard. Myroides odoratimimus is a rarely reported human pathogen. We reported the incidence of infection due to seemingly rare isolates of M. odoratimimus causing an outbreak of pneumonia in piglets. This appears, to the best of authors' knowledge, to be the first outbreak due to Myroides recorded in animal clinical cases described in the literature.

Currently Circulating Genotypes of Hepatitis E Virus in India, 2014–2018

AbstractPurpose: Hepatitis E virus (HEV) is an emerging pathogen causing acute viral hepatitis worldwide. Clinical manifestations often occur in young adults with an increased mortality rate among pregnant women. HEV genotypes 1 and 4 are mainly reported among humans and swines, respectively. Aims: The aim was to study the currently circulating genotypes of HEV in India. Materials and Methods: A retrospective cross-sectional study was carried out at Manipal Institute of Virology to know the circulating genotypes of hepatitis E, spanning over 5 years from August 2014 to September 2018. The serum samples screened serologically positive and confirmed positive for active infection by real-time reverse transcriptase-polymerase chain reaction (PCR) (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were further subjected to nested conventional PCR targeting the RdRp gene of non-structural ORF1 region. The purified PCR product was sequenced in BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA). The chromatograms obtained by sequencing were analysed using Sequencher 5.4.6, and HEV FASTA sequences were compared with reference sequences for HEV in GenBank Nucleotide Blast. Results: During the study period, there were 317 cases of laboratory-confirmed cases of acute viral hepatitis comprising 202, 70, 43 and 2 cases of hepatitis A, E, B and C, respectively. Serum samples of 70 acute hepatitis cases were positive for anti-hepatitis E IgM. According to the clinical case classification, there were 66 cases of acute viral hepatitis and four cases of fulminant hepatic liver failure. The mean age of the patients was 30.3 years (standard deviation = 12.5). The samples from various parts of India were genotyped as 1a. Conclusion: The HEV genotypes 1a was observed to be the currently circulating strain in the regions studied.

Hepatitis E virus in Tibetan pigs on the Qinghai-Tibetan Plateau: detection, molecular characterization and infection of BALB/c mice.

This study was performed to investigate the prevalence and genetic variation of hepatitis E virus (HEV) in Tibetan pigs and to determine its ability to infect mice. A total of 38 out of 229 (16.59%; 95% CI = 12.00%-22.10%) fecal samples from Tibetan pigs from the Qinghai-Tibetan Plateau in 2018 were positive for HEV RNA, which was detected by RT-nPCR. Significantly different detection rates were observed between samples from diarrheic and clinically healthy animals (OR = 9.56; 95% CI, 2.84-32.14; p < 0.001), suggesting a potential association between HEV infection and diarrhea in Tibetan pigs. Phylogenetic analysis showed that the HEV isolates were clustered into subtypes 4a (31 samples), 4b (1), 4d (2), and 4j (4). HEV-4a was the predominant subtype, indicating that it might be circulating in Tibetan pigs. Nine complete HEV genome sequences obtained from Tibetan pigs were found by phylogenetic analysis to be closely related to those of genotype 4 HEV isolates from humans. Two recombinant events were identified in both HEV-4a strains; a novel recombination breakpoint was first identified at the 3' end of the ORF2 region in the SWU/L9/2018 strain, and a common recombination region was found at the junction of the ORF1 and ORF2 regions in the SWU/31-12/2018 strain. Furthermore, HEV-4a could be detected in all BALB/c mice that were experimentally infected by gavage and contact exposure. The information presented here about the prevalence and genotype diversity of HEV from Tibetan pigs provides important insights into the epidemic features of HEV on the Qinghai-Tibetan Plateau.

Updated classification of norovirus genogroups and genotypes.

Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically, they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1, respectively. In recent years, several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters, we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 48 (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1, GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region, noroviruses can be divided into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.

Expression of short hairpin RNA (shRNA) targeting AC2 gene of Mungbean yellow mosaic India virus (MYMIV) reduces the viral titre in soybean

Mungbean yellow mosaic India virus (MYMIV) belonging to the family Geminiviridae and the genus Begomovirus is a severe pathogen of tropical legumes including soybean. The absence of genetically mapped loci conferring resistance together with the genetic diversity of begomoviruses infecting soybean warrants the utilization of RNA interference (RNAi) technology to develop virus resistance. However, viral suppressors of RNAi (VSRs) reduce the effectiveness of RNA silencing. Here, we report the effectiveness of Agrobacterium-mediated transient expression of shRNA, targeting a conserved region of AC2 ORF (a VSR) of MYMIV, in conferring virus resistance in soybean. Transient expression of shRNA showed progressive reduction of the viral titre estimated by the MYMIV-derived AC2 gene copy numbers from the initial inoculum by approximately 80-fold 20days post-application. In addition, the newly emerging leaves exhibited symptom recovery. Thus, this study proves that AC2 of MYMIV is a potent target gene for obtaining RNAi-mediated virus resistance in soybean. Agro-infiltration-based delivery of shRNA was an efficient means of gene silencing and could pave way for the development of transgenic virus-resistant soybean genotype.

A26 Molecular epidemiology of hepatitis E virus in Ireland 2016

Foodborne viruses such as hepatitis E virus (HEV) pose an increasing risk to public health and to confidence in Irish food. Hepatitis E has been acknowledged as a significant pathogen of likely zoonotic transmission, with pork products and shellfish being implicated as potential sources. The European Food Safety Authority has recommended that systematic strain typing of viruses in humans, animals, and food commodities is needed to improve understanding of etiological agents and foodborne transmission pathways, in particular for HEV. The dominant autochthonous genotype of HEV in Europe is genotype 3, thought to be associated with consumption of contaminated food, specifically pork products. However, little is known about the epidemiology of HEV in Ireland. In 2016, HEV became a notifiable disease in Ireland. Following this, as part of the Department of Agriculture, Food and the Marine-funded FoVIRA study, the molecular epidemiology of HEV in Irish clinical samples has been characterized for the first time. HEV RNA-positive clinical specimens from 2016 were genetically characterized (n = 14). A 450 nucleotide fragment of the ORF2 region of the HEV genome was amplified, with contiguous sequence assembly performed using DNA Lasergene v14. Sequences were aligned with ClustalW implemented in Bioedit v7.1.9 and compared to reference strains from GenBank. A maximum likelihood phylogenetic tree was constructed using the Hasegawa–Kishino–Yano model and a discrete gamma distribution to model evolutionary rate distances between sites. Evolutionary analyses were conducted in MEGA7. Statistical support was provided by bootstrapping with 1,000 replicates. Fourteen strains belonged to genotype 3 and were classified as the following subtypes: 3c (n = 7), 3e (n = 4), 3f (n = 1), 3 untyped (n = 1), and 1 untyped. Phylogenetic analysis showed the formation of two distinct clusters of genotype 3:3abchij and 3efg, with strong bootstrap support. A genotype 1 was detected and found to be associated with travel. Data generated from this research will contribute to a risk exposure assessment and will be used to identify potential control points and risk mitigation measures for viral foodborne pathogens. This study will provide a unique opportunity to build national capability in the area of food testing within Irish public laboratories.

Phylogeographic and genetic characterization of porcine circovirus type 2 in Taiwan from 2001\u20132017

Porcine circovirus type 2 (PCV2) is an important pathogen that causes significant economic losses in the swine industry worldwide. Five major PCV2 genotypes have been identified, including PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. To investigate the prevalence and phylodynamics of the different PCV2 genotypes in Taiwan, 214 PCV2 ORF2 sequences from Taiwan and other countries were analyzed. Genotypic differences were observed among PCV2a, 2b, and 2d at amino acid position 89 in ORF2, with isoleucine (I), arginine (R), and leucine (L), respectively. Similar to other countries, a genotypic shift was also observed in Taiwan, where the predominant genotype shifted from PCV2b to 2d after 2010. The estimated nucleotide substitution rate of Taiwanese strains in the ORF2 region was 8.467 × 10−4 substitutions per site per year. This rapid evolution rate of PCV2 may lead to the genotypic shift observed in Taiwan. The times to the most recent common ancestor (TMRCA) for PCV2a, -2b, and -2d-2 was dated to 1970, 1992 and 2004, respectively. Thus, the PCV2a, -2b, and -2d genotypes were already present in Taiwan before the introduction of the PCV2 vaccine.

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

BackgroundPorcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G710 to A710) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively.ResultsThough all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID50), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal.ConclusionsThese findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

A novel double-stranded RNA mycovirus that infects Macrophomina phaseolina.

Macrophomina phaseolina is a pathogenic fungus of the family Botryosphaeriaceae that causes stem rot or leaf blight in many economically important plants. Mycoviruses exist widely in fungi, but there are only a limited number of reports on mycovirus infection in M. phaseolina. A novel dsRNA virus, tentatively named "Macrophomina phaseolina fusagravirus 1" (MpFV1), was isolated from strain 2012-19 of M. phaseolina, and its molecular features were examined. The full-length cDNA of MpFV1 comprises 9,289 nucleotides with a predicted GC content of 48.1% and two discontinuous open reading frames (ORF 1 and 2). A-1 frameshift region with two typical factors, including a shifty heptamer (GGAAAAC) and an H-type pseudoknot, was predicted in the junction region of ORF1 and ORF2. The protein encoded by ORF1 shows significant similarity to a hypothetical protein, whereas ORF2 encodes an RNA-dependent RNA polymerase (RdRp) via a ribosomal frameshifting mechanism. Homology searches and phylogenetic analysis based on the RdRp sequence suggested that MpFV1 is a new member of the proposed family "Fusagraviridae".

Screening and Molecular Characterization of Hepatitis E Virus in Slaughter Pigs in Serbia.

Hepatitis E virus (HEV) is a zoonotic virus that can cause acute hepatitis in humans. Besides the fecal-oral route, transmission can occur by consumption of undercooked pig liver. Genotype 3 is the most frequent genotype found in Europe. Studies on HEV in slaughter-age pigs have not been conducted in Serbia so far. Pork meat production and consumption in Serbia is on average, higher than in the rest of Europe. With the aim to identify the circulating HEV genotypes, pig livers and swab samples from three pig slaughterhouses located in three different sub-regions of Serbia were collected. A nested RT-PCR was used to amplify the hypervariable HEV ORF-1 region (334bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several other countries, was performed. The average prevalence of HEV genotype 3 in 3-month-old pigs was 34%. Phylogenetic analysis revealed the majority of HEV amplification fragments from Serbia were grouped in four clades within sub-genotype 3a and were also genetically related to German, Italian, Slovenian, and American HEV sequences. Sub-genotypes 3b and 3j were also found in a single pig each. This study provides the first analysis of the genetic diversity and circulation dynamics of HEV in pigs at slaughterhouses in Serbia.

Human Papillomavirus Genome based Detection and Typing: A Holistic Molecular Approach.

Human Papillomavirus (HPV) is a species specific double-stranded DNA virus infecting human cutaneous or mucosal tissues. The genome structure of HPV is extremely polymorphic hence making it difficult to discriminate between them. HPV exhibits numerous dissimilar types that can be subdivided into high-risk (HR), probably high-risk and low-risk (LR), causing numerous types of cancers and warts around the genital organs in humans. Several screening methods are performed in order to detect cytological abnormalities and presence or absence of HPV genome. Currently available commercial kits and methods are designed to detect only a few HR/LR-HPV types, which are expensive adding to the economic burden of the affected individual and are not freely available. These gaps could be minimized through Polymerase Chain reaction (PCR) method, which is a gold standard and a cost-effective technique for the detection of most HPV (both known and unknown) types by using specific consensus primers in minimal lab setup. In this context, numerous studies have validated the effectiveness of different sets of consensus primers in the screening of HPVs. Numerous consensus primers, such as E6, E6/E7, GP-E6/E7, MY09/11, GP5+/GP6+, SPF10, and PGMY09/11 have been developed to detect the presence of HPV DNA. In addition, HPV detection sensitivity could be achieved through consensus primer sets targeting specific ORF regions like L1 and E6, which may finally assist in better diagnosis of several unknown HR-HPVs. The present review, provides a summary of the available methods, kits and consensus primer sets for HPV genome based detection, their advantages and limitations along with future goals to be set for HPV detection.

HPV18 Utilizes Two Alternative Branch Sites for E6*I Splicing to Produce E7 Protein

Human papillomavirus 18 (HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6E7 pre-mRNA. The E6 ORF region in the bicistronic E6E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences (BPS) upstream of its 3′ splice site, with an identical heptamer AACUAAC, for E6*I splicing. One heptamer has a branch site adenosine (underlined) at nt 384 and the other at nt 388. E6*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6*I splicing prefers the 3′ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.