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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.

We developed a loop-mediated isothermal amplification (LAMP)–fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5’-FAM–labeled backward loop primer (FLB) and 3’-dabcyl–labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.

Endogenous retroviruses (ERVs) arise from the infection and integration of past retroviruses into animal hosts. We performed large-scale genomic mining of 101 avian genomes for discovery of ERVs having none-avian origin and investigated the cross-species transmission events. Phylogenetic analysis of the reverse transcriptase (RT) of polymerase gene (pol) and the transmembrane subunit (TM) of the envelope gene (env) supported that avian ERVs with a mammalian env gene existed in at least 15 avian species and can be divided into two major groups: Group-1 were of recombinant ERVs with an alpha-like pol gene and a gamma-like env gene, and Group-2 included ERVs with both gamma-like pol and env genes. Group-1 represented the avian alpharetroviral/mammalian gammaretroviral recombinant while Group-2 documented viral jump from mammals to birds. Molecular dating analysis suggested that Group-1 ERVs had integrated into avian genomes continuously, until recent past. We have expanded the knowledge of ERVs with cross-order transmission.

Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 101.2 copies/mu l and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.

RNA viruses encoding a polymerase gene (riboviruses) dominate the known eukaryotic virome. Next-generation sequencing is revealing a wealth of new riboviruses with uncharacterised phenotypes, precluding classification by traditional taxonomic methods. These are often classified on the basis of polymerase sequence identity, but standardised methods to support this approach are currently lacking. To address this need, we describe the polymerase palmprint, a well-defined segment of the palm sub-domain delineated by well-conserved catalytic motifs. We present a novel algorithm, Palmscan, which identifies palmprints in nucleotide and amino acid sequences. We describe PALMdb, a reference database of palmprints derived from public sequence databases. Palmscan source code and PALMdb data are deposited at https://github.com/rcedgar/palmscan and https://github.com/rcedgar/palmdb, respectively.

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.

With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. KEY POINTS: • Promoter driving T7-RNA polymerase was optimized. • Up to 60-fold dynamic range in expression, depending on CO2 conditions. • Two-thirds of total protein is T7-RNA polymerase dependent.

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.

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.

Infection with equid alphaherpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism (A2254/G2254) in the genome region of open reading frame 30 which results in an amino acid variation (N752/D752) of the EHV-1 DNA polymerase, is significantly associated with the neuropathogenic potential of naturally occurring strains. In recent years, an increase in the number of cases of equine neurological disease caused by neuropathogenic variants of EHV-1 has been observed in numerous countries. The purpose of this study was to detect the presence of the viral genome of EHV-1 and equid herpesvirus 4 (EHV-4) in the bronchopulmonary lymph nodes of 47 horses from various locations in Uruguay, obtained from a slaughterhouse, and to determine whether the EHV-1 genomes possessed the mutation associated with neuropathogenesis (G2254/D752). The genes encoding glycoprotein H (gH) of EHV-1 and glycoprotein B (gB) of EHV-4 were amplified by a semi-nested polymerase chain reaction. Of the samples analysed, 28% and 6% of lymph nodes contained the genes for gH and gB, respectively. The viral DNA polymerase gene was amplified and sequenced. Twelve of the 13 genomes sequenced presented the nucleotide G2254, while the remaining 1 showed both nucleotides, A2254 and G2254. The results confirm the presence of EHV-1 in Uruguay. Furthermore, there is evidence for the first time of the detection of EHV-4, and high-frequency detection of the neuropathogenic variant (G2254/D752) of EHV-1 in Uruguay. These findings provide new insights into the epidemiological situation of EHV-1 and EHV-4 in that country.

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.

Human cytomegalovirus UL54 DNA polymerase gene mutations that confer foscarnet resistance in clinical practice typically cluster in the amino terminal 2, palm and finger domains. Exposure to foscarnet in cell culture selected for mutations elsewhere in UL54, including amino acid substitutions S290R in the amino terminal 1 domain and E951D in the palm 2 domain. These are newly confirmed to confer foscarnet resistance and slightly decreased ganciclovir susceptibility. Other emergent substitutions N495K, T552N and T838A are known to confer foscarnet resistance, while additional ones Q783R and V798A only slightly affected susceptibility. An expanded set of domains is involved in foscarnet resistance and its genotypic diagnosis.

Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID50/ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1.

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.

Up to now, only a single adenovirus (AdV) isolate seemingly specific for pigeons, hence named pigeon AdV-1 (PiAdV-1), has been characterised at DNA sequence level. In the present work, the prevalence and diversity of AdVs occurring in domestic pigeon were examined by a survey performed on randomly collected samples using a very efficient, consensus nested PCR targeting the viral DNA polymerase gene. The newly detected viruses were characterised by sequencing and phylogeny analysis. Amplification of additional genome fragments was attempted by the use of several other PCR methods aiming at the hexon gene. During a 4-year survey, samples from dead or live, healthy pigeons originating from 27 lofts were examined in Hungary. Almost 50% of the samples (48 out of 97) proved to be positive for AdV. Sequence analysis revealed the presence of four hitherto unknown pigeon AdV types. PiAdV-1 was also identified in one sample. Two novel viruses named PiAdV-2 and -3 were found to belong to the genus Aviadenovirus, and two other novel types (PiAdV-4 and -5) to the genus Siadenovirus. This is the first report on the occurrence of siadenoviruses in birds belonging to the order Columbiformes. Approximately two-thirds of the PiAdV-2 genome was sequenced and analysed.

Among all known retroviruses, foamy viruses (FVs) have the most stable virus–host co-speciation history, co-diverging in concert with their vertebrate hosts for hundreds of millions of years. However, detailed molecular analyses indicate that different parts of their genome might have different evolutionary histories. While their polymerase gene displays a robust and straightforward virus–host co-speciation pattern, the evolutionary history of their envelope (env) gene, is much more complicated. Here, we report eleven new FV env sequences in two mandrill populations in Central Africa, geographically separated by the Ogooué River into the North and the South populations. Phylogenetic reconstruction of the polymerase gene shows that the two virus populations are distinct, and each contains two variants of env genes co-existing with one another. The distinction between the two env variants can be mapped to the surface domain, flanked by two recombination hotspots, as previously reported for chimpanzee and gorilla FVs. Our analyses suggest that the two env variants originated during the diversification of Old World monkeys and apes, ∼30 million years ago. We also show that this env gene region forms two phylogenetically distinct clades, each displaying a host co-divergence and geographical separation pattern, while the rest of the genome of the two strains is phylogenetically indistinguishable in each of the host-specific groups. We propose possible evolutionary mechanisms to explain the modular nature of the FV genome.

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.

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream – in the city of Caxias do Sul, Rio Grande do Sul, Brazil – was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.

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.

Background & objectives: Bats are recognized as important reservoirs for emerging infectious disease and some unknown viral diseases. Two novel viruses, Malsoor virus (family Bunyaviridae , genus, Phlebovirus ) and a novel adenovirus (AdV) (family, Adenoviridae genus, Mastadenovirus ), were identified from Rousettus bats in the Maharashtra State of India. This study was done to develop and optimize real time reverse transcription - polymerase chain reaction (RT-PCR) assays for Malsoor virus and real time and nested PCR for adenovirus from Rousettus bats. Methods: For rapid and accurate screening of Malsoor virus and adenovirus a nested polymerase chain reaction and TaqMan-based real-time PCR were developed. Highly conserved region of nucleoprotein gene of phleboviruses and polymerase gene sequence from the Indian bat AdV isolate polyprotein gene were selected respectively for diagnostic assay development of Malsoor virus and AdV. Sensitivity and specificity of assays were calculated and optimized assays were used to screen bat samples. Results: Molecular diagnostic assays were developed for screening of Malsoor virus and AdV and those were found to be specific. Based on the experiments performed with different parameters, nested PCR was found to be more sensitive than real-time PCR; however, for rapid screening, real-time PCR can be used and further nested PCR can be used for final confirmation or in those laboratories where real-time facility/expertise is not existing. Interpretation & conclusions: This study reports the development and optimization of nested RT-PCR and a TaqMan-based real-time PCR for Malsoor virus and AdV. The diagnostic assays can be used for rapid detection of these novel viruses to understand their prevalence among bat population.

Arising from J. E. Kravchenko, I. B. Rogozin, E. V. Koonin & P. M. Chumakov , 735–739 (2005); doi:10.1038/nature0384810.1038/nature03848 Mitochondria are involved in a variety of metabolic processes and one of their main functions is to perform oxidative phosphorylation1,2, which requires a crosstalk between the mitochondrial and nuclear genomes to accomplish coordinated gene expression3,4. Splice variants of the mitochondrial RNA polymerase gene (Polrmt) have been reported to encode a nuclear RNA polymerase isoform (spRNAP-IV), which is thought to facilitate this coordination by transcribing a specific subset of nuclear genes5,6,7. Here we report that analysis of Polrmt gene expression, subcellular fractionation and fluorescence microscopy do not support the existence of a nuclear POLRMT isoform in mouse and human cells, and that conditional knockout of Polrmt does not affect expression of the nuclear genes previously reported to be transcribed by spRNAP-IV. We thus conclude that POLRMT has an exclusive mitochondrial role and that it is absolutely required for expression of mitochondrial DNA (mtDNA) in mammalian mitochondria.

Nipah virus (NiV) was identified in Malaysia in late September 1998 as the etiological agent of an outbreak of acute encephalitis with the high mortality rate in human. NiV polymerase gene (L) encodes RNA-dependent RNA polymerase (RdRp) which is an enzyme required for viral replication. The aim of this study is to investigate the role of the N terminal of polymerase protein in replication of NiV. Based on the extensive deduced amino acid sequence analyses of a number of L proteins of non-segmented negative-strand (NNS) RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. The functional characterization of the NiV L polymerase was addressed in this research by generating a series of progressive N- deletions in the cloned gene and testing them in a minigenome assay. Five mutations that delete increasing amounts of the amino terminus of the L protein (20 amino acids) were generated at the amino-terminal. The ability of new recombinants plasmids in viral replication were tested by using minigenome system which is based on an intracellular and plasmid-based replication assay. This system contains an inserted chloramphenicol acetyltransferase (CAT) gene, for readout of the replication of the mini genome directed by wild type or mutant L protein. The results demonstrated that the first 100 amino acids of the NH2-terminal domain spanning a highly conserved motif are directly involved in transcription of the genome RNA. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed.

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature ( T m ). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.

Endogenous bornavirus-like L (EBLL) elements are inheritable sequences derived from ancient bornavirus L genes that encode a viral RNA-dependent RNA polymerase (RdRp) in many eukaryotic genomes. Here, we demonstrate that bats of the genus Eptesicus have preserved for more than 11.8 million years an EBLL element named eEBLL-1, which has an intact open reading frame of 1,718 codons. The eEBLL-1 coding sequence revealed that functional motifs essential for mononegaviral RdRp activity are well conserved in the EBLL-1 genes. Genetic analyses showed that natural selection operated on eEBLL-1 during the evolution of Eptesicus. Notably, we detected efficient transcription of eEBLL-1 in tissues from Eptesicus bats. To the best of our knowledge, this study is the first report showing that the eukaryotic genome has gained a riboviral polymerase gene from an ancient virus that has the potential to encode a functional RdRp.

Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification. To be able to select for mezophilic enzymes we have employed multiple freezing-thawing cycles of emulsion as a bacterial cell wall disruption step instead of high temperature incubation. Subsequently WGA like plasmid DNA amplification could be performed by phi29 DNA polymerase applying different selection pressure conditions (temperature, buffer composition, modified dNTP, time, etc.). In our case the library of random phi29 DNA polymerase mutants was subjected to seven selection rounds of isothermal CSR (iCSR). After the selection polymerase variant containing the most frequent mutations was constructed and characterized. The mutant phi29 DNA polymerase can perform WGA at elevated temperatures (40-42°C), generate two to five times more of DNA amplification products, and has significantly increased half-life at 30 and 40°C, both in the presence or the absence of DNA substrate.

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.

Adenovirus-associated enteritis was diagnosed by histopathology of small intestine in a 2-year-old alpaca ( Vicugna pacos). Electron microscopy confirmed intracytoplasmic and intranuclear adenoviral particles within enterocytes. Nucleic acid was extracted from paraffin-embedded tissue sections, and a pan-adenovirus nested polymerase chain reaction (PCR) assay was employed to target a partial sequence of the polymerase gene. The PCR product (321 bp) was cloned and sequenced. Comparison of the nucleotide sequence against the National Center for Biotechnology Information (NCBI) nucleotide database demonstrated 68% identity with the isolates Canine adenovirus 1 and Bovine adenovirus 3. Comparison of the predicted amino acid sequence against the NCBI database demonstrated 75% identity with Bovine adenovirus 3. Phylogenetic analysis supported the relatively close relationship of this isolate to Bovine adenovirus 3, but the alpaca isolate was sufficiently distant to be considered a potentially novel adenovirus for this species.

Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.

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

Bearded dragons (Pogona vitticeps) are popular pets in the United States. Agamid Adenovirus 1 (AgAdV1) is an important infectious agent of bearded dragons. The only AgAdV1 sequences available to date are from a highly conserved region of the DNA polymerase gene. Degenerate primers were designed to amplify a variable region of the AgAdV1 hexon gene for sequencing. Genetic differences were identified within the hexon gene of 17 bearded dragons from 4 collections. Much less diversity was present in the polymerase gene. Bayesian analysis of the hexon nucleotide alignment identified two larger groups and two isolates that did not tightly cluster with these two groups. Multiple genotypes were identified within collections, and individual genotypes were seen in different collections. Three bearded dragons appeared to be infected by multiple strains. These findings show that this hexon region is useful for AgAdV1 genotyping, which can be used epidemiologically as well as in future investigations of AgAdV1 evolution and clinical implications of strain differences.

The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms “pre-P” by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.