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Objectives In latest years GII.4[P16] and GII.2[P16] noroviruses have become predominant in some temporal/geographical settings. In parallel with the emergence of the GII.P16 polymerase type, norovirus surveillance activity in Italy experienced increasing difficulties in generating sequence data on the RNA polymerase genomic region A, using the widely adopted JV12A/JV13B primer set. Two sets of modified primers (Deg1 and Deg2) were tested in order to improve amplification and typing of the polymerase gene. Methods Amplification and typing performance of region A primers was assessed in RT-PCR on 452 GII norovirus positive samples obtained from 2194 stool samples collected in 2016–2019 from children hospitalized with acute gastroenteritis. Results The use of Deg1 increased the rate of samples types in region A from 49.5% to 81.4% and from 21.9% to 69.7% in 2016 and 2017, respectively. The rate of Deg1 typed samples remained high in 2018 (90.1%), but sharply decreased to 11.8% in 2019. The second primers set, Deg2, was able to increase to 64.9% the rate of 2019 samples typed in region A, while typing efficiently 73.2%, 69%, and 86.4% of samples collected in 2016, 2017 and 2018, respectively. Conclusions The plasticity of norovirus genomes requires continuous updates of the primers used for strain characterization.

Background Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. The aim of the study was to characterise HIV-1 Pol CTL epitopes in mostly sample pairs obtained during early and chronic stages of infection. Methods Illumina deep sequencing was performed for all samples while Sanger sequencing was only performed on baseline samples. Codons under immune selection pressure were assessed by computing nonsynonymous to synonymous mutation ratios using MEGA. Minority CTL epitope variants occurring at $$\ge$$ ≥ 5% were detected using low-frequency variant tool in CLC Genomics. Los Alamos HIV database was used for mapping mutations to known HIV-1 CTL epitopes. Results Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of sequencing method. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and Conclusion Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes.

Coronaviruses (CoV) are widely distributed pathogens of human and animals and can cause mild or severe respiratory and gastrointestinal disease. Antigenic and genetic similarity of some CoVs within the Betacoronavirus genus is evident. Therefore, for the first time in Slovenia, we investigated the genetic diversity of partial 390-nucleotides of RNA-dependent-RNA polymerase gene (RdRp) for 66 human (HCoV) and 24 bovine CoV (BCoV) positive samples, collected between 2010 and 2016 from human patients and cattle with respiratory disease. The characterized CoV strains belong to four different clusters, in three separate human clusters HCoV-HKU1 (n = 34), HCoV-OC43 (n = 31) and HCoV 229E (n = 1) and bovine grouping only as BCoVs (n = 24). BCoVs from cattle and HCoV-OC43 were genetically the most closely related and share 96.4–97.1% nucleotide and 96.9–98.5% amino acid identity.

The aim of the study was directed to investigate the prevalence of Escherichia coli in raw milk, cream, large and small scale yoghurt, Kareish cheese, and large and small scale ice cream by conventional bacteriological methods as well as detection of the wzy gene (O-antigen polymerase gene) using polymerase chain reaction for further identification of recovered isolates. A total of 200 random samples including 40 raw milk, 40 fresh cream, 20 of each large and small scale yoghurt, 40 Kareish cheese and 20 of each large and small scale ice cream were obtained from different localities in Beni-Suef governorate, Egypt. The present study could provide useful information for the prevalence of E. coli in the examined samples: 75%, 62.5%, 0%, 25%, 80%, 10% and 25% respectively. E. coli isolate were serologically identified as: O26, O111:H4, O121, O125:H21, O169, O126:H7 and O158 in raw milk, O6:H16, O55:H7, O119:H6, O125:H6 and O146:H21 in fresh cream, O55:H7, O125:H21 in small scale yoghurt and O18, O55:H7, O114:H21, O158, O125:H21and O153:H45 in Kareish cheese. Interestingly PCR successfully amplified the wzy gene (O-antigen polymerase gene) in all E. coli isolates which is associated with LPS biosynthesis and bacterial pathogenicity and increases the ability of E. coli to withstand the anti-bacterial defense mechanism.

Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus “mixing vessels.” In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels., Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as “mixing vessels,” being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host. IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus “mixing vessels.” In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as ‘mixing vessels’, being susceptible to both avian and human origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports some, albeit limited, activity of avian origin influenza virus polymerases. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the super pro-viral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the LRR and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the ‘mixing vessel’ trait of swine and further our understanding of the evolution and ecology of viruses in this host.

The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.

Infection caused by hepatitis B virus (HBV) is still a major global health problem and can cause liver cirrhosis and hepatocellularcarcinoma as well. Telbivudine is one among the drugs used to treat the disease routinely. However, using this drug in a long term therapymight cause mutations in HBV polymerase gene that decreases the effectiveness of the therapy. Here with the researchers report the geneticvariations of the gene isolated from telbivudine-which is used treated chronic hepatitis B patients in Surabaya, Indonesia. The blood serawere collected at Dr. Soetomo hospital from 10 telbivudine-treated and 10 untreated chronic hepatitis B patients. The DNA viral wasisolated and purified from each serum. Sequence polymerase gene at nucleotides 455 to 796 was amplified by PCR, and then analyzedbio informatically to determine their mutation profile. This study revealed a point mutation in HBV25 sample at nucleotide A1525G thatgives rise to I509V modification. Such mutation is also observed in a sequence that is available in Gen Bank with an accession numberAY641562. Additionally, the researchers found point mutations A1554G, T1593C, and C1629T in HBV25 sample and a point mutationA1554G in HBV20 sample. However, these mutations are silent. To conclude, the mutation in HBV polymerase gene among telbivudinetreatedchronic hepatitis B patients in Surabaya is known as A1525G.

Of the zoonotic genotypes of hepatitis E virus, the genotype 3 (HEV3) has emerged as the most pathogenic strain causing chronic hepatitis in immunosuppressed patients in industrialized nations. The epidemiology of HEV3 is rather complex because of its hitherto well recognized sources and routes of transmission. Currently, ribavirin is the only effective drug that however, induces mutations in viral polymerase gene leading to drug-resistance or -nonresponse, and teratogenic effects in pregnant women.

The prevalence of Herpes simplex virus 2 (HSV2) is increasing at an alarming rate in the world. Most of the HSV2 cases are not diagnosed properly, although a range of molecular and serological diagnoses exist. Herein, we have reported a very rapid detection method specific for HSV2 using real-time PCR. The primers specific for HSV2 were designed using the Primer-BLAST tool and 120 base pairs of the polymerase gene were amplified using real-time PCR with SYBR Green dye. The designed primer pair was found highly efficient in detecting only HSV2 DNA, but not HSV1. The threshold cycle (Ct) value for HSV2 reactions by designed primers was found to be an average of 22.55 for a standard copy number of viral DNA that may denote the efficiency of the primers. The melting temperature (Tm) of the amplicon using designed primers (82.60C) was also higher than that using reference primers (about 780C), indicating the high GC content of the amplified template. The designed primer pair will help clinicians to detect the HSV2 DNA specifically and diagnose the associated disease rapidly.

Background Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. Methods HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Results Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Conclusions Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy.

Highly pathogenic avian influenza virus H5N1 infects a wide range of host species, with a few cases of sporadic pigeon infections reported in the Middle East and Asia. However, the role of pigeons in the ecology and evolution of H5N1 viruses remains unclear. We previously reported two H5N1 virus strains, isolated from naturally infected pigeons in Egypt, that have several unique mutations in their viral polymerase genes. Here, we investigated the effect of these mutations on H5N1 polymerase activity and viral growth and identified three mutations that affected viral polymerase activity. The results showed that the PB1-V3D mutation significantly decreased polymerase activity and viral growth in both mammalian and avian cells. In contrast, the PB2-K627E and PA-K158R mutations had moderate effects: PB2-K627E decreased and PA-K158R increased polymerase activity. Structural homology modelling indicated that the PB1-V3D residue was located in the PB1 core region that interacts with PA, predicting that the PB1 mutation would produce a stronger interaction between PB1 and PA that results in decreased replication of pigeon-derived H5N1 viruses. Our results identified several unique mutations responsible for changes in polymerase activity in H5N1 virus strains isolated from infected pigeons, emphasizing the importance of avian influenza surveillance in pigeons and in studying the possible role of pigeon-derived H5N1 viruses in avian influenza virus evolution.

Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.

The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008–2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment.

A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian influenza viruses, and bears some amino acids associated with mammalian receptor binding, raising concern over the possibility of a new influenza pandemic. Besides HA and NA, the mutation of the polymerase is known to have an important role in virulence, host adaptation and transmissibility in mammalians. In this article, the annotation of the polymerase protein domain associated with molecular function has been highlighted, suggesting the combination of RNA polymerase of H7N9 viruses is still not stable for host adaptation. In addition, the mutation hallmarks in polymerase gene of H7N9 are compared, providing the potential determinants of the evolution in the H7N9 influenza A virus.

A major cause of anti-retroviral therapy (ART) failure is the drug resistance-associated mutations in polymerase gene of HIV-1. Paucity of data regarding potential drug resistance to protease inhibitors (PIs) prompted us to carry out this study. Drug resistance (DR) genotyping of the entire protease gene was performed in 104 HIV-1 ART-naive and first-line ART-experienced patients. The DR pattern was analyzed using the Stanford HIV-DR database, International AIDS Society-USA mutation list and REGA algorithm version 8.0. Subtyping was done using Mega 4 and REGA HIV-1 subtyping tool-v2.01. Majority of our sequences 98 (96 %) were subtype C and remaining four (3.92 %) were subtype A1. In three (2.9 %) DE patients, major DR-associated mutation at D30 N and M46I positions were detected. Approximately 70 % polymorphisms as minor mutations were observed in protease gene, of which 14 distinct amino acids changes were linked to partial DR such as G16E, K20R, M36I, D60E, I62V, L63P, I64M, H69K, T74A/S, V77I, V82I, I85V, L89M, and I93L. The two major and several minor mutations detected in this study confer low/intermediate levels of resistance to most PIs independently or together. Our results conclude that resistance testing in HIV-1-infected patients should be performed before the initiation of PI therapy for better therapeutic outcome in this region. This information not only will shed light on the extent of current DR in HIV strains but also will aid in patient treatment and drug designing.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation., Author Summary Avian influenza (AI) virus H5N1 subtype strains have been sporadically transmitted to humans with high mortality (>60%), presenting a serious global health threat. In particular, 63% of recent human H5N1 infection cases worldwide have been reported in Egypt, which is now regarded as a hot spot for H5N1 virus evolution. H5N1 clade 2.2.1 viruses are unique to Egypt and probably have the greatest evolutionary potential for adaptation from avian to human hosts. Here, using a comprehensive database approach, we identified various novel polymerase mutations in clade 2.2.1 virus strains, isolated from patients, that enabled enhanced viral replication in both human airway epithelial cells and mouse lungs. Interestingly, the mutations identified acted cooperatively with the PB2-E627K mutation, the most well-known human adaptation mutation, to produce a further increase in viral replication in human hosts. These results provide the first broad-spectrum data on the polymerase characteristics of AI viruses that have been selected in infected patients, and also give new insight into the human adaptation mechanisms of AI viruses.

Accurate and timely detection of drug-associated viral mutants is important during antiviral therapy. Combining Smart Amplification Process (SMAP) with competition probe, an assay specifically designed to detect point mutation at codon 204 of the hepatitis B virus (HBV) polymerase gene was developed. This assay was sensitive to detect 20 copies of mutant/reaction and recognize as little as 1% of minor mutants in the viral population. The comparison of direct sequencing and SMAP method on 35 clinical specimens showed the concordance in 88% of the cases. This method provides an efficient alternative for rapid identification of HBV mutation associated with lamivudine resistance.

Summary. For phylogenetic comparison of hepatitis B virus (HBV) isolates, often a region of the HBV surface gene is analysed. Because the surface gene completely overlaps the polymerase gene, its evolution is constrained, and it may not be the best choice for genetic comparison of HBV isolates. Analysing serial sample pairs of 33 chronically HBV-infected, untreated patients, with a cumulative follow-up of 184 years, the synonymous and nonsynonymous substitution rates of a part of the overlapping HBV surface and polymerase genes were compared to those of a nonoverlapping part of the HBV core gene. The substitution rate of the HBV core gene was higher (8.15 × 10−4vs 4.57 × 10−4 substitutions/site/year) than that of the surface gene. The difference was mainly due to a significantly lower synonymous substitution rate in the surface gene, with dN/dS ratios of 0.412 in the core gene and 0.986 in the surface gene. Contrary to the core gene, the number of substitutions in the surface gene was higher in low viraemic hosts, who control HBV infection by suppressing replication. The number of substitutions in the core gene correlated more strongly with the duration of follow-up. The overlapping HBV surface and polymerase genes experience strong negative selection, which limits the number of substitutions. Because the HBV core gene reflects the duration of infection more accurately, it is more suitable for the analysis of short-term viral evolution and of hepatitis B transmission chains.

Cytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility.Validate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations.Bacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays.Sixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded.Results add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.

Previously, a virulent avian metapneumovirus, farm isolate Italy 309/04, was shown to have been derived from a live vaccine. Virulence due to the five nucleotide mutations associated with the reversion to virulence was investigated by their addition to the genome of the vaccine strain using reverse genetics. Virulence of these recombinant viruses was determined by infection of 1-day-old turkeys. Disease levels resulting from the combined two matrix mutations was indistinguishable from that produced by the recombinant vaccine, whereas the combined three L gene mutations increased disease to a level (P

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli. Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.

Objective:To elucidate the evolutionary characteristics of polymerase PA,PB1,and PB2 genes of the novel influenza virus A/H1N1 in 2009 pandemic.Methods:The sequences of the PA,PB1,and PB2 genes of the novel H1N1 strains in 2009 pandemic,and the reference sequences of human,swine,and avian influenza viruses isolated during different years at different locations were retrieved from NCBI.Molecular Evolutionary Genetics Analysis version 4.0(MEGA4.0)software was employed to align and blunt nucleotide sequences,construct phylogenetic tree,deduce and align PB2 protein sequences,and the results were compared between the novel A/H1N1 and each of the reference strains.Results:The sequences of the PA,PB1,and PB2 genes of 2009 novel A/H1N1 strains isolated from different locations shared a high homology and clustered in a unique new clade,and were close to the swine influenza viruses.The PA,PB1,and PB2 genes of the novel H1N1 viruses had a high similarity with the corresponding sequences of a human H1N1 strain isolated in Iowa State of USA in 2005(A/Iowa/CEID23/2005/H1N1).Alignments of the deduced protein sequences showed that the 627th amino acid of PB2 of the novel H1N1 strains and A/Iowa/CEID23/2005/H1N1 were glutamic acid(Glu),which was the same as that in the avian influenza virus in Iowa State of USA in 2005(DQ889682),and was different from those of the reference sequences of human A/H1N1 strains isolated from 1918 to 2008,which were lysine(Lys).Conclusion:The 2009 novel A/H1N1 virus might be originated from the human A/H1N1 strains isolated in 2005 in Iowa State of America(A/Iowa/CEID23/2005/H1N1),and the polymerase gene of the novel H1N1 virus might re-assort with avian A influenza virus.

We report a novel and simple method for mutation detection in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) in nanostructured molecular assemblies, called reverse micelles. The intercalation of SYBR Green I into the duplex DNA exhibits fluorescent emission in a CTAB/isooctane reverse micellar system as well as in an aqueous solution. We found marked differences in the fluorescence intensity between perfectly matched and mismatched 52-mer synthetic oligonucleotides, which were designed to contain the YMDD motif of the hepatitis B virus (HBV) polymerase gene, in a reverse micellar solution. Using this method, we successfully detected a mutation in PCR-amplified oligonucleotides of the HBV polymerase gene in sera of four patients with chronic hepatitis B. This detection method does not require DNA immobilization, chemical modification of DNA, or any special apparatus; it only needs a normal fluorescence spectrophotometer, an inexpensive dye, and just 10 pmol of sample DNA.

Phylogenetic relationships among hepatitis B virus (HBV) genotypes were investigated using different regions across the genome. The phylogenetic analysis in conjunction with graphical examination of phylogenetic distance matrices and distance frequency distribution plotting suggest the clustering of HBV genotypes into three higher-order hierarchical groups: group I, comprising genotypes A–E and G; group II, comprising genotypes F and H; and a hypothetical group III. Present-day genotype G is postulated to be a recombinant with the non-polymerase region of group III virus and the polymerase gene of an ancestral virus belonging to group I.

Noroviruses are recognized as the most common cause of nonbacterial gastroenteritis worldwide. In this study, we investigated the molecular epidemiology of noroviral isolates in Canada from 2004 to 2005 by sequencing the RNA polymerase gene and capsid N-terminal/shell (N/S) domain. Norovirus genogroups I and II were thus found to have co-circulated in Canada during the studied period, with a higher incidence of genogroup II (95.7%). The GII-4 or Lordsdale subgroup was the predominant genotype, suggesting that norovirus genogroup II is the major cause of viral gastroenteritis in Canada, as it is in many other countries. Phylogenetic analyses of the RNA polymerase gene and the capsid N/S domain indicated different genotypes for 2 strains, suggesting probable genetic recombination. Sequencing of the norovirus polymerase gene may reflect actual classification but should be supported by sequence information obtained from the capsid gene.

The genome of hepatitis B virus (HBV) provides a striking example of gene overlapping. In particular, the surface protein gene S is overlapped completely by the polymerase gene P. Evolutionary constraints in overlapping genes have been demonstrated for many viruses, with one of the two overlapping genes being subjected to positive selection (adaptive evolution), while the other one is subjected to purifying selection. Yet, for HBV to persist successfully, adaptive evolution of both the P and S genes is essential. We propose that HBV employs a mechanism that allows the independent adaptive evolution of both genes. We hypothesize that (i) the adaptive evolution of P occurs via p1/s3 non-synonymous substitutions, which are synonymous in S, (ii) the adaptive evolution of S occurs via p3/s2 non-synonymous substitutions, which are synonymous in P, and (iii) p2/s1 substitutions are rare. Analysis of 450 HBV sequences demonstrated that this mechanism is operational in HBV evolution both within and among genotypes. Positions were identified in both genes where adaptive evolution is operational. Whilst significant parts of the P and S genes were subjected to positive selection, with the K a/K s ratio for either the P or the S gene being >1, there were only a few regions where the K a/K s ratios in both genes were >1. This mechanism of independent evolution of the overlapping regions could also apply to other viruses, taking into account the increased frequency of amino acids with a high level of degeneracy in the proteins encoded by overlapping genes of viruses.

The incorporation of the eight gene segments of influenza A virus in virions, either through a random or a specific mechanism, has been under debate for many years. Using reverse genetics techniques and trans-complementation of viral proteins, we showed that the production of viruses containing seven gene segments is very inefficient, which can be overcome by providing the 8th gene segment in a non-functional form. We conclude that sequences in the 5′ and 3′ ends of the polymerase gene segments contain packaging signals. Our methods will facilitate the further mapping of the packaging signals of influenza A virus.

Real-time quantitative PCR systems (Q-PCR) for the rapid detection and quantification of microorganisms in clinical specimens employ oligodeoxyribonucleotide primers and probes for specificity, which makes them vulnerable to false negatives caused by sequence diversity in the template. Schaade et al. (J. Clin. Microbiol. 39 :3809, 2001) reported a sequence variant (C630T) in the cytomegalovirus (CMV) glycoprotein B (gB) gene that, although detectable in their Q-PCR assay, could not be accurately quantified. In an effort to evaluate the impact of CMV sequence variants in our patient population by use of a similar Q-PCR assay, we surveyed 54 isolates of CMV, each from a different patient. We detected evidence for the C630T variant in 4 of 54 (7.4%) patients. Furthermore, isolates from two additional patients were completely negative in the test. Sequencing of these false-negative isolates revealed multiple mutations within the probe hybridization sites. A Q-PCR that targeted the CMV polymerase gene instead of gB detected all 54 isolates. We suggest that Q-PCR assays for viral load be rigorously tested on large panels of viral isolates to assess the impact of sequence diversity on detection as well as quantification.

Treatment of chronic hepatitis B has been greatly improved by the use of lamivudine, but mutations occur in the polymerase region of hepatitis B virus (HBV) and lamivudine-resistant mutants frequently develop. The emergence of lamivudine-resistant strains of HBV is a problem for treating chronic hepatitis B using lamivudine. We observed biochemical and virological changes in 15 patients with chronic hepatitis B for a median period of 29 months (range: 4-42 months) after the emergence of lamivudine-resistant mutants of HBV. Patterns of mutation of the polymerase gene were examined by sequencing the LLAQ motif in domain B and the YMDD motif in domain C. Exacerbation of liver dysfunction occurred in 14 (93.3%) of the 15 patients at a median of 4 months after the emergence of mutations. However, exacerbation of liver dysfunction was observed only in four patients (26.7%) at the time of appearance of the first mutations and in 80.0% of the patients at the time of appearance of the second mutations. Increase in serum alanine aminotransferase (ALT) levels was significantly greater at the time of appearance of second mutations (P = 0.0096). In most cases, wild-type HBV was mutated with the substitution of only rtM204I at first, and rtL180M/M204I mutations and then rtL180M/M204V mutations subsequently appeared. Further mutations of the polymerase region caused clinical deterioration. Thus as mutations emerge in the polymerase region, the clinical outcome deteriorates. Thus, monitoring the patterns of mutation of the polymerase gene is useful when using lamivudine for treating HBV.

A small round virus (SRV) was isolated in 1988 from droppings of enteritis-affected turkeys in North Carolina and tentatively identified as an enterovirus on the basis of size (18-24 nm in diameter), intracytoplasmic morphogenesis, and a single-stranded RNA genome of approximately 7.5 kb. Additional characterization studies based on antigenic and genomic analyses were done to determine the relationship of this turkey enterovirus-like virus (TELV) to turkey astrovirus 2 (TAstV2), a recently characterized SRV of turkeys. Cross-immunofluorescence studies with TELV- and TAstV2-specific antisera indicated a close antigenic relationship between these viruses. TELV RNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with oligonucleotide primers specific for TAstV2 polymerase gene (open reading frame [ORF] 1b) and capsid protein gene (ORF 2). Subsequent sequence analyses of these TELV-derived RT-PCR products indicated a high degree of similarity with polymerase gene (98.8%) and capsid gene (96.9%) of TAstV2. These studies definitively identify TELV (North Carolina, 1988 isolate) as TAstV2.

Recently, a benzo-1,2,4-thiadiazine antiviral agent (C 21 H 21 N 3 O 4 S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 μM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

Comment on : Enrichment of deleterious variants of mitochondrial DNA polymerase gene (POLG1) in bipolar disorder. [Psychiatry Clin Neurosci. 2017]

Human infection by H7N9 influenza virus was first identified in China in March 2013. As of 12 August 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses possess several polymerase gene mutations previously associated with human adaptation and potential pandemic capabilities. However, the function of these mutations in the emergence and pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K mutation, which occurs in over 70 % of the H7N9 patient isolates, promotes the replication of H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and in the pathogenicity of the H7N9 virus.

Seven independent laboratory mutants were derived from seven distinct wild-type varicella-zoster virus (VZV) isolates after exposure to increasing concentrations of foscarnet (PFA) and were found to be resistant to this drug. Single base changes resulting in amino acid substitutions were observed in the nucleotide sequence of the DNA polymerase gene of each PFA-resistant mutant. The mutations were found to occur within the domain II (Arg-665 Gly for strains vrMOR and vrVER; Val-666 Leu for vrLEB; Gln-692 Arg for vrOLI) and domain III (Arg-806 Ser for vrABD; Leu-809 Ser for vrALI and vrCHA) of DNA polymerase gene. In addition, the PFA-resistant mutants exhibited a phenotype characterized by slow growth, the strains showing a marked delay in immediate-early antigen plaque formation compared with the wild-type VZV from which they were derived. These results may have implications for successful isolation and characterization of PFA-resistant strains from clinical samples containing mixed viral populations. J. Med. Virol. 59:84–90, 1999. © 1999 Wiley-Liss, Inc.

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses--infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)--in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583-590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.

Prolonged therapy for chronic hepatitis B (HBV) with nucleoside analogs may result in the emergence of HBV mutants resistant to antivirals. Here, we describe the transient selection of an HBV polymerase gene mutant that was associated with viral persistence in an immune competent patient treated with famciclovir. Viral polymerase gene sequence was analyzed directly on polymerase chain reaction (PCR) products and also after cloning. The results showed the transient selection of a V542I mutant in the C domain of the viral polymerase. This mutation was associated with a stop codon at amino acid position 199 in the overlapping S gene. The mutated sequence was subcloned in a vector expressing the entire HBV pregenome to study its replication capacity after transient transfection in cultured hepatoma cells. The results showed that the V542I mutant has a decreased replication capacity compared with wild type virus and does not produce HBsAg. The sensitivity of the V542I mutant to penciclovir, the active metabolite of famciclovir, was further studied in tissue culture. This mutant was shown to be resistant to penciclovir, but remained sensitive to lamivudine, as was subsequently observed in vivo. These findings indicate that a prolonged administration of famciclovir may allow for the selection of HBV polymerase gene mutants in immune competent patients. The impaired replication capacity of this V542I mutant may have contributed to the absence of outgrowth of this viral strain in vivo. The study of the in vitro sensitivity of HBV polymerase mutants to nucleoside analogs will be important to design new anti-HBV strategies.

The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.

To the Editor: Sapovirus and norovirus are causative agents of gastroenteritis in children and adults. Norovirus is the most important cause of outbreaks of gastroenteritis, whereas only a few outbreaks of sapovirus have been reported (1,2). On the basis of complete capsid gene sequences, sapovirus can be divided into 5 genogroups, among which GI, GII, GIV, and GV infect humans, whereas sapovirus GIII infects porcine species. We report 2 outbreaks of gastroenteritis in Hokkaido, Japan. The first outbreak (A) occurred at a college from May 29 to June 2, 2000. A total of 12 persons (11 students and 1 teacher) reported symptoms of gastroenteritis (nausea, vomiting, stomachache, diarrhea, and fever); 11 stool specimens were collected from days 1 to 7 after onset of illness (Table). These specimens were negative for norovirus (data not shown), but 5 were positive for sapoviruslike viruses by electron microscopy (Table). Table Analysis of 18 stool specimens for sapovirus during 2 outbreaks of gastroenteritis, Japan* The 11 specimens were then examined for sapovirus by using nested reverse transcription–PCR (RT-PCR) as described (3). A total of 9 (82%) of 11 specimens were positive for sapovirus. Sequence analysis showed that these 9 viruses had 100% nucleotide identity and likely represented the same sapovirus strain (termed Yak2 strain, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB046353","term_id":"148717098","term_text":"AB046353"}}AB046353). To determine the number of cDNA copies per gram of stool, we performed real-time RT-PCR as described (4). The number of sapovirus cDNA copies ranged from 5.36 × 105 to 7.47 × 109/g stool (median 5.49 × 109 copies/g stool) (Table). The second outbreak (B) occurred at a kindergarten from February 1 to 22, 2005. A total of 23 persons (15 children and 8 adults) reported symptoms of gastroenteritis (nausea, vomiting, stomachache, diarrhea, and fever); 7 stool specimens were collected (Table). These specimens were negative for norovirus (data not shown), but all were positive for sapovirus by nested RT-PCR. The 7 sequences from this outbreak had 100% nucleotide identity and likely represented the same sapovirus strain (termed Nay1 strain, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF213768","term_id":"126565998","term_text":"EF213768"}}EF213768). The number of sapovirus cDNA copies ranged from 1.14 × 109 to 5.41 × 1010/g stool (median 2.50 × 1010 copies/g stool) (Table). One positive sapovirus specimen from each outbreak was subjected to further sequence analysis in which a single overlapping PCR fragment covering the partial polymerase gene and capsid gene was amplified. The Yak2 and Nay1 sequences shared ≈71% nucleotide identity for this fragment and likely represented different sapovirus strains. The Yak2 sequence closely matched sapovirus GIV Ehime1107 and SW278 sequences (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"DQ058829","term_id":"70799531","term_text":"DQ058829"}}DQ058829 and {"type":"entrez-nucleotide","attrs":{"text":"AY237420","term_id":"45545440","term_text":"AY237420"}}AY237420, respectively) and had 98% and 97% nucleotide identity for the entire fragment, respectively (5). The Nay1 sequence closely matched the sapovirus GII C12 sequence ({"type":"entrez-nucleotide","attrs":{"text":"AY603425","term_id":"51243518","term_text":"AY603425"}}AY603425) and had 91% nucleotide identity for the entire fragment. The Nay1 sequence closely matched the C12 sequence, which was detected in Osaka, Japan, in 2001 (6), whereas the Yak2 sequence closely matched the Ehime1107 sequence, which was detected in Matsuyama, Japan, in 2002 (5) and the SW278 sequence, which was detected in Sweden in 2003 (1). We recently described the C12 strain as intragenogroup recombinant sapovirus strain (6), whereas the Ehime1107 and SW278 strains were described as intergenogroup recombinant sapovirus strains (5). Our results indicate that recombination sites in intragenogroup and intergenogroup recombinant sapovirus strains were at the polymerase and capsid junction (5,6). Sapovirus Sydney53 ({"type":"entrez-nucleotide","attrs":{"text":"DQ104360","term_id":"308196023","term_text":"DQ104360"}}DQ104360) and Sydney3 strains ({"type":"entrez-nucleotide","attrs":{"text":"DQ104357","term_id":"308196021","term_text":"DQ104357"}}DQ104357), which were detected in Australia from August 2001 to August 2004 (7), closely matched C12 and Ehime1107/SW278 sequences, respectively. These results showed that recombinant sapovirus strains are stable in the environment and may be globally distributed. Our findings also suggest a changing distribution of sapovirus-associated gastroenteritis in Hokkaido because different sapovirus GI strains were predominant sapovirus strains that caused causing outbreaks of gastroenteritis in Hokkaido (8,9). In a recent study, the number of norovirus cDNA copies per gram of stool specimen was analyzed and a discrepancy was found between the different norovirus genogroups (10). Chan et al. found that noroviruses GI and GII showed medians of 8.4 × 105 and 3.0 × 108 copies/g of stool specimen, respectively, and speculated that increased viral loads were caused by higher transmissibility of norovirus GII strains (10). Our results showed that sapovirus GII Nay1 and GIV Yak2 strains showed higher viral loads than norovirus GII strains. These results suggest that a high degree of shedding of sapovirus GII Nay1 and GIV Yak2 strains may have caused the outbreak of gastroenteritis. However, to elucidate this suggestion, further studies are needed with other sapovirus strains.

Two herpes simplex virus mutants containing mutated residues within the conserved Exo III motif of the polymerase gene were previously shown to be defective in 3′–5′ exonuclease activity and exhibited extremely high mutation frequencies. In this study, we have shown that these mutants also exhibited higher resistance to phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs tested, including acyclovir and gancicolvir, compared to wild-type virus. Marker transfer experiments and sequencing analyses demonstrated that these altered phenotypes were the result of mutations within the Exo III motif. The data indicate that, aside from leading to exonuclease deficiency, mutations in the Exo III motif may also affect interaction of nucleoside triphosphates with the catalytic sites of polymerase activity.

Resistance to infection in cowpea by strains of cucumber mosaic virus (CMV) involves a local, hypersensitive response (HR) and a localization of infection. These responses can be separated by mutation at two sites (nucleotides 1978 and 2007, in codons 631 and 641) in the CMV 2a polymerase gene. Changes to both sites of a restricted strain allow systemic infection without an HR and increase the accumulation of both the 2a protein and viral RNA in protoplasts, while changing position 1978 alone results in a systemic infection, a systemic HR, and an increase in viral RNA accumulation in protoplasts. It is suggested that the inhibition response observed in protoplasts, where an HR does not occur, leads to localization of infection in whole plants and that different plant genes are involved in eliciting the HR and the localization response.

Expression of the mosquito larvicidal genes coding for 51 and 42kDa proteins from Bacillus sphaericus cloned into E. coli, under the control of T7RNA polymerase promoter system, resulted in a high level of larvicidal activity of the recombinant. The clone MS16 expressed the binary toxins at high level even under uninduced conditions. This indicates that the ‘leaky’ regulation of the T7RNA polymerase gene was responsible for the constitutive expression of mosquito larvicidal proteins (MLP). The economic advantage of producing the MLP in a rapidly-growing and sugar-utilising organism such as E. coli was demonstrated in a batch cultivation where production of the MLP was growth-associated.

We previously reported that influenza A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory-droplet-transmissible virus (CT-Sw/1204) had acquired simultaneous hemagglutinin (HA D225G ) and neuraminidase (NA S315N ) mutations. Incorporating these mutations into the nonpathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or noticeable disease. We therefore exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did expression of individual polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA 225G and NA 315N modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.

We generated reassorted PR8 viruses containing six different combinations of avian influenza virus (AIV) polymerase genes from A/chicken/Korea/01310/2001 (H9N2) (01310) and A/chicken/Korea/KBNP-0028/2000 (H9N2) (0028) to examine the effects of the AIV polymerase genes PB1, PB2, and PA on replication efficiency in different host cells and pathogenicity in mice. The virus titers of the reassorted viruses possessing 01310 [rPR8-PB2(01310)] and 0028 [rPR8-PB2(0028)] PB2 genes were significantly higher than those of the others except the rPR8 virus in embryonated chicken eggs at 37°C, and those of avian polymerase reassorted viruses were significantly less than rPR8 in MDCK cells at 32 and 37°C. rPR8-PB2(01310), rPR8-PB2(0028), and rPR8-PA(0028) caused no body weight loss in BALB/c mice but rPR8-PA(01310), rPR8-PB1(01310), and rPR8-PB1(0028) caused mortality and significantly different body weight loss compared to those in the mock treatment. In contrast to rPR8-PB2(0028) and rPR8-PA(0028), rPR8-PB2(01310) was not isolated from infected mice, and rPR8-PB1(0028) was less pathogenic than rPR8-PB1(01310). We determined the amino acid residues that were specific to the less pathogenic polymerases. A comparison with those of pandemic 2009 H1N1, human fatal H5N1 and H7N9, and pathogenic AIVs to mice without adaptation revealed that they possessed the mammalian pathogenic constellation of polymerases. Thus, the novel polymerase genes and amino acid residues may be useful to understand the host-barrier overcome of AIVs in mice and to develop safer and efficacious vaccines.

Noroviruses (NoVs) are one of the major etiological agents of acute gastroenteritis in all age groups. In this study, we identified an intergenotype NoV recombinant strain in the fecal specimens of two male infants with acute diarrhea in Bangladesh. Phylogenetic analysis showed that the identified strains were recombinant NoV strains with a GII.3 capsid and a GII.16 polymerase gene. The recombination breakpoint was located in the ORF1/ORF2 overlap region. To the best of our knowledge this is the first report of a NoV recombinant GII.16/GII.3 strain worldwide.

In order to determine the overall molecular heterogeneity of echoviruses (EVs) we performed a genetic analysis of the prototype strains. Nucleotide and derived amino acid sequences from different genomic regions (5'UTR, capsid protein-coding and 3D polymerase genes) were used for molecular comparisons. On the basis of a comparison of partial amino acid sequences from the capsid protein VP2, all the sequenced EVs excluding EV22 and EV23 form a single cluster which is genetically homogeneous. All previously sequenced coxsackie B viruses (CBVs) and coxsackievirus A9 also belong to this same genetic cluster. Similar results were obtained when the 5'UTR or 3D polymerase gene sequences were used in comparisons. When amino acid sequences of the major capsid proteins of EV1 and EV16 were compared to those of previously sequenced enteroviruses, the length of the loops connecting the beta-sheets appeared to be relatively constant in the EV/CBV cluster. It can be concluded that EVs and CBVs have diverged relatively late in evolution.

A cold-passaged (cp) temperature-sensitive (ts) mutant of human respiratory syncytial virus designated RSV cpts-248 was previously derived by random chemical mutagenesis of the non-ts mutant cp-RSV that possesses one or more host range mutations. We previously demonstrated in rodents and seronegative chimpanzees that the cpts-248 virus is more attenuated than cp-RSV and is more stable genetically than previously isolated RSV ts mutants. In the present study, we determined that the acquisition of the ts phenotype and the increased attenuation of the cpts-248 virus are associated with a single nucleotide substitution at nucleotide 10,989 that results in a change in the coding region (amino acid position 831) of the polymerase gene. The identification of this attenuating ts mutation is important because cpts-248 was used as the parent virus for the generation of a number of further attenuated mutants that are currently being evaluated as candidate vaccine strains in clinical trials in infants. Furthermore, technology now exists to rationally design new vaccine candidates by incorporating multiple attenuating mutations, such as the one identified here, into infectious viruses that are genetically stable and appropriately attenuated.

The nucleotide sequence (8396 nucleotides) was determined, from the 3′-end of the putative polymerase gene to the poly(A) tail, for a Taiwanese virulent isolate, TFI, of transmissible gastroenteritis virus (TGEV). The TFI nucleotide sequence had very high identity to the British virulent field isolate FS772/70 (98.3%), the attenuated Purdue 115 (96.7%) and from the S gene to ORF-4 gene region, to the low passaged virulent Miller (98.3%) strains of TGEV. Comparison of the TFI S protein sequence with those determined from other TGEV strains and those of the TGEV variant, porcine respiratory coronavirus, isolated from Europe and North America showed that they had changed very little over a period of 4 decades. The two extra amino acids found to be present in the spike proteins of the virulent FS772/70 and Miller strains when compared to the avirulent Purdue strain were found to be present in the TFI strain. The genomic organisation of the TFI strain was the same as that of the other TGEV viruses.

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro . Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.