Aa Changes Research Articles

Metabolic Profiling on Alternaria Toxins and Components of Xinjiang Jujubes Incubated with Pathogenic Alternaria alternata and Alternaria tenuissima via Orbitrap High-Resolution Mass Spectrometry.

Xinjiang jujubes (Zizyphus rhamnaceae) are important agro-economical foods with the highest planting area and yields in China; however, black spot disease and contaminated Alternaria toxins have unfortunately caused a decline or loss of jujube nutritional quality in recent years. In this study, we used ultrahigh-performance liquid chromatography coupled to Orbitrap high-resolution mass spectrometry to profile both Alternaria toxins and components in three representative Xinjiang jujubes, Hami Huang, Hetian Jun, and Ruoqiang Hui. Before liquid chromatography-mass spectrometry analysis, jujubes were inoculated with two main pathogens of Alternaria alternata (Aa) and Alternaria tenuissima (At). Different combinations of jujube varieties with pathogenic isolates display different metabolic profiles, as expected. Moreover, four major Alternaria toxins, alternariol, alternariol monomethyl ether, altenuene, and tenuazonic acid, were detected in all samples. The inoculation of both pathogens significantly decreased the levels of nutrients and metabolites in jujube, including four saponins, three organic acids, and three alkaloids, whereas it increased the level of several glycerol phosphates. The flavonoid profiles are diverse. Lastly, inoculation of Aa changes more metabolites in jujubes than At. Our data provide insights to better understand the detrimental contamination of Alternaria pathogens in Xinjiang jujubes and improve food safety of jujubes.

Genetic Features of MAPT, GRN, C9orf72 and CHCHD10 Gene Mutations in Chinese Patients with Frontotemporal Dementia.

Mutations in microtubule associated protein tau (MAPT), progranulin (GRN), chromosome 9 open-reading frame 72 (C9orf72) and CHCHD10 genes have been reported causing frontotemporal dementia (FTD) in different populations. However, collective analysis of mutations in these four genes in Chinese FTD patients has not been reported yet. The aim of this study was to investigate the genetic features of Chinese patients with MAPT, GRN, C9orf72 or CHCHD10 gene mutations in an FTD cohort recruited from multi clinical centers in Shanghai metropolitan areas, China. MAPT, GRN and CHCHD10 genes were analysed by direct sequencing, and C9orf72 hexanucleotide repeat expansion was analysed by repeat-primed PCR in 82 patients with sporadic FTD. The identified gene variants were screened in 400 age matched controls. We found one known pathogenic variant (rs63750959) and one novel mutation (NG_007398.1: g.120962C>T; H299Y) of MAPT gene, one novel variant (c.750C>A; D250E) of GRN gene and two novel mutations in CHCHD10 gene (c.63C>T, no AA change; c.71G>A, P24L). No abnormal C9orf72 gene hexanucleotide repeat expansion was identified in this cohort. Collectively, genetic testing could discover 4.9% sporadic FTD patients with genetic causes. In addition, MAPT and CHCHD10 might be more important genes affecting Chinese with FTD.

426 Ileal amino acid digestibility values vary depending on the exogenous indigestible indexes in pigs fed a soybean meal-based diet

The objective was to test a hypothesis that ileal AA digestibility values may vary depending on the indigestible index sources including chromium oxide, titanium dioxide, and Celite. Eight barrows with an initial BW of 58.1 kg (SD = 4.3) fitted with a T-cannula in the distal ileum were randomly allotted to a 2-period crossover design with 2 diets and 8 pigs in each period. A soybean meal (SBM)-based diet and an N-free diet were prepared. Both experimental diets contained 0.4% chromium oxide, 0.4% titanium dioxide, and 0.4% Celite as indigestible indexes. A washout diet with no index was provided ad libitum to the pigs for 7 d before each 9-d experimental period. The diets were provided at daily levels of 3 times the estimated maintenance requirement for energy. Equal meals were provided at 0800 and 2000. Ileal digesta were collected from 0800 to 1600 on each day. In the same experiment, we found that the minimum adaptation period before ileal digesta collection to have constant index concentrations was 3 to 4 d. Therefore, the digestibility and endogenous loss of AA data were pooled from d 5 to 9 to investigate the influence of indigestible index on AA digestibility and endogenous losses. The ileal digesta are often collected during d 6 and 7 in many experiments, and thus, the data from d 6 and 7 were also pooled for testing index effects on AA digestibility and endogenous losses. Based on the pooled data from d 5 to 9, the apparent ileal digestibility (AID) and the standardized ileal digestibility (SID) of all AA except Tyr calculated using Cr or Ti as an index were greater (P < 0.05) than the digestibility values calculated using AIA. During d 5 to 9, the basal endogenous losses (BEL) of Arg, His, Ile, Leu, Lys, Thr, Trp, Val, Ala, Asp, Glu, Gly, Pro, and Ser calculated using Cr as an index were less (P < 0.05) than the BEL values calculated using Ti but greater (P < 0.05) than the BEL values calculated using AIA. When the data from d 6 and 7 were pooled, the AID, SID, and BEL of AA changes by indigestible indexes had the same pattern as the data from d 5 and 9. Overall, AA digestibility calculated using Cr or Ti was greater than that calculated using AIA.

Теоретичне обгрунтування співвідношення треоніну і триптофану у раціонах ремонтних курчат

&lt;p&gt;We determined the optimal content and ratio of threonine and tryptophan in the mix fodder of replacement chicks at different periods of their growing. We studied the effect of different levels of threonine and tryptophan in mixed fodder on chicken growth and development. We registered that under optimal AA content and ratio the replacement chickens at the end of the growing period outweighed the chickens from control group by 140 g. We also studied the influence of different content of AA and temporal changes in their ratio on digestibility of feed nutrients by replacement chickens from market egg flock. It was found that the optimal content of threonine and tryptophan and their ratio at certain periods of chicken growth caused the increase in digestibility of protein by 2 %, fiber – by 1 %, and BEV – by 2 %. We also proved that the decrease or increase of AA ratio in mix fodder of replacement chickens leads to the manifestation of antagonism between amino acids and the deterioration of their function in bird organism. The changes in AA ratio had significant effects on chicken productivity, the digestibility of feed nutrients, the assimilation, and efficiency of amino acid use. This effects were considerably stronger than the effect of changes in AA content. The degree of influence of the ratio between threonine and tryptophan varies throughout the growing period, it has been established that the ratio of threonine to tryptophan in the period of limited chick feeding (9-6 weeks) has a lesser effect on the productivity of repair young (R&lt;sup&gt;2&lt;/sup&gt; = 0.89) the second one (R&lt;sup&gt;2&lt;/sup&gt; = 0.92) and the last growing periods (R&lt;sup&gt;2&lt;/sup&gt; = 0.92). We performed a mathematical model (polynomial trend line) to describe the growth of replacement chickens during all experiment periods, the pattern of dependences between chicken organism and AA content, AA ratio, age, and productivity. This nonlinear model is more relevant and reliable than linear dependence.&lt;/p&gt;

Evolutionary changes in the capsid P2 region of Australian strains of the norovirus GII.Pe_GII.4.

The protruding (P) 2 region of the norovirus capsid is thought to include hypervariable sites involved in receptor binding. This study examines the changes that occurred in the P2 region of GII.Pe_GII.4 norovirus in the course of its evolution from a precursor phase (2008-2009), to an intermediate phase (2010) and finally to an epidemic phase (2012-2015). Twenty-two P2 region amino acid (aa) sequences (166 aa long) from all phases of the evolution of the virus were compared and the changes analysed.Results/key findings. Twenty sites in the P2 region underwent aa change and of these, 10 corresponded to previously proposed hypervariable sites and 10 to novel hypervariable sites. It was notable that aa changes at two sites, X and Y, only emerged as the epidemic phase progressed. 3D computer modelling of the P2 region indicated that neither X nor Y were in the uppermost 'crown', but further down in the 'neck' portion. The location of X and Y and the nature of aa change at Y suggest these sites were important in enhancing the structural integrity of the capsid, which in turn may have facilitated the longer term viability of the virus. The current study helps establish the validity of previously proposed hypervariable sites in the P2 region as well as indicating new ones. It also provides quantitative and qualitative data on how these sites changed over the evolutionary history of a particular norovirus strain.

Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro

The HBV core protein represents an attractive target for new antiviral therapies due to its multiple functions within the viral life-cycle. Here, we report the antiviral activity of the capsid assembly modulator (CAM) BAY41-4109 and two nucleos(t)ide analogues (NAs) on a diverse panel of 54 HBV clinical isolates from genotype (GT) A-H and assessed the impact of core amino acid (aa) substitutions using site-directed mutants (SDMs).The median EC50 values of BAY41-4109 across genotypes ranged from 26 nM in GT G to 215 nM in GT F irrespective of the presence of NA resistance mutations compared to 43 nM for the GT D reference construct. Combined analyses of clinical isolates and SDMs identified aa changes at positions 29, 33 and 118 led to reduced antiviral activity of BAY41-4109 with fold changes in EC50 values of 6, 46, and 9 for D29G, T33N, and Y118F, respectively. These aa substitutions are located within the CAM binding pocket, and are expected to have an effect on CAM binding based on structural modeling. Importantly aa variations at these positions were rarely (<0.3%) observed as naturally occurring in public sequence databases. NA's remained fully active against these variants.Our study demonstrated that BAY41-4109 generally remained fully active across GT A-H clinical isolates. In addition, core aa substitutions within the CAM-binding pocket replicated in vitro and variants at positions 29, 33, and 118 were identified to reduce antiviral activity.

Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B.

BackgroundThe fusion (F) protein of RSV is the major vaccine target. This protein undergoes a conformational change from pre-fusion to post-fusion. Both conformations share antigenic sites II and IV. Pre-fusion F has unique antigenic sites p27, ø, α2α3β3β4, and MPE8; whereas, post-fusion F has unique antigenic site I. Our objective was to determine the antigenic variability for RSV/A and RSV/B isolates from contemporary and historical genotypes compared to a historical RSV/A strain.MethodsThe F sequences of isolates from GenBank, Houston, and Chile (N = 1,090) were used for this analysis. Sequences were compared pair-wise to a reference sequence, a historical RSV/A Long strain. Variability (calculated as %) was defined as changes at each amino acid (aa) position when compared to the reference sequence. Only aa at antigenic sites with variability ≥5% were reported.ResultsA total of 1,090 sequences (822 RSV/A and 268 RSV/B) were analyzed. When compared to the reference F, those domains with the greatest number of non-synonymous changes included the signal peptide, p27, heptad repeat domain 2, antigenic site ø, and the transmembrane domain. RSV/A subgroup had 7 aa changes in the antigenic sites: site I (N = 1), II (N = 1), p27 (N = 4), α2α3β3β4(AM14) (N = 1), ranging in frequency from 7–91%. In comparison, RSV/B had 19 aa changes in antigenic sites: I (N = 3), II (N = 1), p27 (N = 9), ø (N = 4), α2α3β3β4(AM14) (N = 1), and MPE8 (N = 1), ranging in frequency from 79–100%.DiscussionAlthough antigenic sites of RSV F are generally well conserved, differences are observed when comparing the two subgroups to the reference RSV/A Long strain. Further, these discrepancies are accented in the antigenic sites in pre-fusion F of RSV/B isolates, often occurring with a frequency of 100%. This could be of importance if a monovalent F protein from the historical GA1 genotype of RSV/A is used for vaccine development.

Allele-Specific Effects on Extracellular Superoxide Dismutase Synthesis and Secretion

Plasma extracellular superoxide dismutase (ecSOD) concentrations are significantly (2 - 3-fold) higher in 129 mice than in the C57 strain. We reported earlier that the 129 strain carries a different allele of this enzyme, characterized by two amino acid substitutions and a 10 bp deletion in the 3’UTR of the mRNA. One of the substitutions is at the signal peptide cleavage site (position 21) while the other is within the catalytic site. Since the differences in plasma enzyme concentrations persist in congenic mice expressing the different alleles, the differences in plasma concentrations appear to be largely allele-driven and independent of the remainder of the genome. As expected, in vitro translation using rabbit reticulocyte lysate showed no differences in translational efficiency of transcripts and processing of the newly synthesized enzymes. Determinations of the rates of synthesis and secretion of the two isoforms of this enzyme in stably transfected CHO cells clearly demonstrate that the mature 129 variant is synthesized at rates nearly twice those of the wild-type isoform and has an intracellular pool size twice as large. However, both isoforms are secreted at the same fractional rate and have identical intracellular T1/2 as well as similar extent of degradation. These data explain, at least partially, the higher levels of ecSOD in congenic mice expressing the 129 variant, as well as enzyme levels in the 129 strain of mice. In silico calculations support this conclusion and indicate that the aa change at the signal peptide cleavage site (N21D) results in a substantial increase in cleavage probability at this site for the 129 variant. Our results also highlight the importance of signal peptide cleavage in determining steady-state levels of secretory proteins.

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.

Comparative Genomic Analysis of Classical and Variant Virulent Parental/Attenuated Strains of Porcine Epidemic Diarrhea Virus

Since 2010, the variant porcine epidemic diarrhea virus (PEDV) has been the etiological agent responsible for the outbreak of porcine epidemic diarrhea (PED) worldwide. In this study, a variant PEDV strain YN1 was isolated, serially propagated on the Vero cells and was characterized for 200 passages. To better elucidate the molecular basis of Vero cell adaptation of variant PEDV strains, we sequenced, compared, and analyzed the full-genome sequences of parental YN1 and passages 15, 30, 60, 90, 144, and 200. The results showed that the variations increased with the viral passage. The nucleotides sequences of non-structural protein (NSP)2, NSP4-7, NSP10, NSP12 and NSP13 genes did not change during the Vero cell adaptation process. After comparison of the variation characteristic of classical, variant virulent/attenuated strains, it was found that attenuation of PEDV virus was associated with 9−26 amino acid (aa) changes in open reading frames (ORF) 1a/b and S protein, early termination in ORF3, 1–3 aa changes in E, M and N protein and some nucleotide sequences’ synonymous mutations. The aa deletion at about 144 aa of S protein could be the attenuation marker for the PEDV. The pig study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation.

Sequence analysis of Epstein-Barr virus (EBV) early genes BARF1 and BHRF1 in NK/T cell lymphoma from Northern China.

BackgroundNK/T cell lymphoma is an aggressive lymphoma almost always associated with EBV. BamHI-A rightward open reading frame 1 (BARF1) and BamHI-H rightward open reading frame 1 (BHRF1) are two EBV early genes, which may be involved in the oncogenicity of EBV. It has been found that V29A strains, a BARF1 mutant subtype, showed higher prevalence in NPC, which may suggest the association between this variation and nasopharyngeal carcinoma (NPC). To characterize the sequence variation patterns of the Epstein-Barr virus (EBV) early genes and to elucidate their association with NK/T cell lymphoma, we analyzed the sequences of BARF1 and BHRF1 in EBV-positive NK/T cell lymphoma samples from Northern China.MethodsIn situ hybridization (ISH) performed for EBV-encoded small RNA1 (EBER1) with specific digoxigenin-labeled probes was used to select the EBV positive lymphoma samples. Nested-polymerase chain reaction (nested-PCR) and DNA sequence analysis technique were used to obtain the sequences of BARF1 and BHRF1. The polymorphisms of these two genes were classified according to the signature changes and compared with the known corresponding EBV gene variation data.ResultsTwo major subtypes of BARF1 gene, designated as B95-8 and V29A subtype, were identified. B95-8 subtype was the dominant subtype. The V29A subtype had one consistent amino acid change at amino acid residue 29 (V → A). Compared with B95-8, AA change at 88 (L → V) of BHRF1 was found in the majority of the isolates, and AA79 (V → L) mutation in a few isolates. Functional domains of BARF1 and BHRF1 were highly conserved. The distributions of BARF1 and BHRF1 subtypes had no significant differences among different EBV-associated malignancies and healthy donors.ConclusionThe sequences of BARF1 and BHRF1 are highly conserved which may contribute to maintain the biological function of these two genes. There is no evidence that particular EBV substrains of BARF1 or BHRF1 is region-restricted or disease-specific.

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

SLC47A1 gene rs2289669 G > A variants enhance the glucose-lowering effect of metformin via delaying its excretion in Chinese type 2 diabetes patients

AimsThe SLC47A1 gene encodes the multi-drug and toxic excretion-1(MATE1) protein, which plays a key role in the transport and excretion of metformin. This study is to clarify the influence of variants in SLC47A1 (rs2289669 G→A) on metformin pharmacokinetics and the long-term glucose-lowering effect of metformin. MethodsA total of 220 newly diagnosed type 2 diabetes patients were recruited, genotyped and divided into three groups by SLC47A1 genotypes (G/G, G/A, A/A). Ten patients in each group were randomly selected for metformin pharmacokinetics. All the participants received metformin oral treatment and were followed for one year. ResultsAfter one-year follow-up, the decline of HbA1c level was significantly greater in subjects with variant genotype (AA) than other two groups (−2.32% [−25.4mmol/mol] in AA vs. −1.16% [−12.7mmol/mol] in GA, −1.07% [−11.7mmol/mol] in GG, P<0.05). Then taking GG genotype as the referent, the association between AA genotype and change of HbA1c still existed after adjusted for age, sex, BMI, baseline HbA1c and diabetes duration (P<0.05). Pharmacokinetic parameters of metformin indicated that patients carrying MATE1 homozygous A had higher area under the plasma concentration versus time curve (AUC12h), but lower renal clearance (CLR) and renal clearance by secretion (CLSR) than other patients (all P<0.01). Multivariate lineal stepwise analysis further revealed that SLC47A1 genotype was an independent impact factor for urine excretion of metformin (P<0.01). ConclusionsSLC47A1 rs2289669 G>A variants improve the glucose-lowering effect of metformin through slowing its excretion in type 2 diabetes populations.

Effects of Adaptation of Infectious Bronchitis Virus Arkansas Attenuated Vaccine to Embryonic Kidney Cells.

The population structure of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas (Ark) Delmarva Poultry Industry (DPI)-derived vaccine was characterized during serial passages in chicken embryo kidney (CEK) cells and after back-passage in embryonated chicken eggs (ECE) and in chickens. Both conventional and deep-sequencing results consistently showed population changes occurred during adaptation to CEK cells. Specifically, 13 amino acid (aa) positions seemed to be targets of selection when comparing the vaccine genome prior to and after seven passages in CEK (CEKp7). Amino acid changes occurred at four positions in the spike (S) gene and, at two positions in the S gene, large shifts in frequencies of aa encoding were observed. CEK adaptation shifted the virus population towards homogeneity in S. The changes achieved in the S1 gene in CEKp7 were maintained after a back-passage in ECE. Outside the S gene, aa changes at three positions and large shifts in frequencies at four positions were observed. Synonymous nucleotide changes and changes in noncoding regions of the genome were observed at eight genome positions. Inoculation of early CEK passages into chickens induced higher antibody levels and CEKp4 induced increased respiratory signs compared to CEKp7. From an applied perspective, the fact that CEK adaptation of embryo-attenuated Ark vaccines reduces population heterogeneity, and that changes do not revert after one replication cycle in ECE or in chickens, provides an opportunity to improve commercial ArkDPI-derived vaccines.

Comparative analysis of ABCB1 reveals novel structural and functional conservation between monocots and dicots.

Phytohormone auxin plays a critical role in modulating plant architecture by creating a gradient regulated via its transporters such as ATP-binding cassette (ABC) B1. Except for Arabidopsis and maize, where it was shown to interrupt auxin transport, ABCB1's presence, structure and function in crop species is not known. Here we describe the structural and putative functional organization of ABCB1 among monocots relative to that of dicots. Identified from various plant species following specific and stringent criteria, ZmABCB1's “true” orthologs sequence identity ranged from 56–90% at the DNA and 75–91% at the predicted amino acid (aa) level. Relative to ZmABCB1, the size of genomic copies ranged from −27 to +1.5% and aa from −7.7 to +0.6%. With the average gene size being similar (5.8 kb in monocots and 5.7 kb in dicots), dicots have about triple the number of introns with an average size of 194 bp (total 1743 bp) compared to 556 bp (total 1667 bp) in monocots. The intron-exon junctions across species were however conserved. N-termini of the predicted proteins were highly variable: in monocots due to mismatches and small deletions of 1–13 aa compared to large, species-specific deletions of up to 77 aa in dicots. The species-, family- and group- specific conserved motifs were identified in the N-terminus and linker region of protein, possibly responsible for the specific functions. The near-identical conserved motifs of Nucleotide Binding Domains (NBDs) in two halves of the protein showed subtle aa changes possibly favoring ATP binding to the N-terminus. Predicted 3-D protein structures showed remarkable similarity with each other and for the residues involved in auxin binding.

Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012.

BackgroundData about molecular diversity of commonly circulating type A influenza viruses in Ontario swine are scarce. Yet, this information is essential for surveillance of animal and public health, vaccine updates, and for understanding virus evolution and its large-scale spread.MethodsThe study population consisted of 21 swine herds with clinical problems due to respiratory disease. Nasal swabs from individual pigs were collected and tested by virus isolation in MDCK cells and by rtRT-PCR. All eight segments of 10 H3N2 viruses were sequenced using high-throughput sequencing and molecularly characterized.ResultsWithin-herd prevalence ranged between 2 and 100%. Structurally, Ontario H3N2 viruses could be classified into three different groups. Group 1 was the most similar to the original trH3N2 virus from 2005. Group 2 was the most similar to the Ontario turkey H3N2 isolates with PB1 and NS genes originating from trH3N2 virus and M, PB2, PA and NP genes originating from the A(H1N1)pdm09 virus. All Group 3 internal genes were genetically related to A(H1N1)pdm09. Analysis of antigenic sites of HA1 showed that Group 1 had 8 aa changes within 4 antigenic sites, A(1), B(3), C(2) and E(2). The Group 2 viruses had 8 aa changes within 3 antigenic sites A(3), B(3) and C(2), while Group 3 viruses had 4 aa changes within 3 antigenic sites, B(1), D(1) and E(2), when compared to the cluster IV H3N2 virus [A/swine/Ontario/33853/2005/(H3N2)].ConclusionsThe characterization of the Ontario H3N2 viruses clearly indicates reassortment of gene segments between the North American swine trH3N2 from cluster IV and the A(H1N1)pdm09 virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0194-z) contains supplementary material, which is available to authorized users.

Sequencing and molecular characterization of CTNCEC25, a China fixed rabies virus vaccine strain CTN-1 adapted to primary chicken embryo cells.

BackgroundRabies virus is the main etiologic agent of the widespread neurological disease rabies. Recently, the China rabies virus vaccine strain CTN-1 adapted to chicken embryo cells, which has been designated as CTNCEC25, was obtained and demonstrated to have high immunogenicity. However, the full genome sequence of CTNCEC25 and its phylogenetic relationship with other rabies virus street and vaccine strains have not been characterized.ResultsThe complete genome of CTNCEC25 was sequenced and analyzed. The length of CTNCEC25 genome is 11,924 nucleotides (nt), comprising a 3′ leader sequence of 59 nt, nucleoprotein (N) gene of 1,425 nt, phosphoprotein (P) gene of 989 nt, matrix protein (M) gene of 803 nt, glycoprotein (G) gene of 2,067 nt, RNA-dependent RNA polymerase gene (L) of 6,474 nt and a 5′ trailer region of 71 nt. A comparison of the entire genomes of CTN-1 and CTNCEC25 identified 16 nt substitutions and 1 deletion, resulting in 8 amino acid (aa) changes in the five structural proteins with one in L (aa 1602), two in M (aa 99 and 191) and six in mature G (aa 147, 333, 389, 421 and 485). The percentage homology of the CTNCEC25 genomic sequence with other fully sequenced rabies virus strains ranged from 81.4% to 99.9%. Phylogenetic analysis indicated that CTNCEC25 was more closely related with those recently isolated China street strains than other vaccine strains. Virus growth analysis showed that CTNCEC25 achieved high rate of propagation in cultured cells.ConclusionsIn this study, the complete genome of CTNCEC25 was sequenced and characterized. Our results showed that CTNCEC25 was more closely related to wild street strains circulating in China than other vaccine strains. Sequence analysis showed that the G protein ectodomain amino acid sequence identity between CTNCEC25 and other rabies virus strains was at least 90% identical. Furthermore, CTNCEC25 achieved high virus titers in cultured cells. Given that CTNCEC25 has high immunogenicity and induced strong protective immune response in animals, these results collectively demonstrated that CTNCEC25 is an ideal vaccine strain candidate for producing human vaccine with high quality and safety in China.

Two missense mutations in melanocortin 1 receptor (MC1R) are strongly associated with dark ventral coat color in reindeer (Rangifer tarandus).

The protein-coding region of melanocortin 1 receptor (MC1R) was sequenced to identify potential variation affecting coat color in reindeer (Rangifer tarandus). A T→C sequence variation at nucleotide position 218 (c.218T>C) causing an amino acid (aa) change from methionine to threonine at aa position 73 (p.Met73Thr) was identified. In addition, a T→G sequence variation was found at nucleotide position 839 (c.839T>G), causing phenylalanine to be exchanged by cysteine at aa position 280 (p.Phe280Cys). The two sequence variants (c.218C and c.839G) were found to be closely associated with a darker belly coat compared with animals not having any of these two variants. The aa acid change p.Met73Thr affects the same position as p.Met73Lys previously reported to give constitutive activation of MC1R in black sheep (Ovis aries), whereas p.Phe280Cys is identical to one of two variants previously reported to be associated with dark coat color in Arctic fox (Alopex lagopus), supporting that the two variants found in reindeer are functional. The complete absence of Thr73 and Cys280 among the 51 wild reindeer analyzed provides some evidence that these variants are more common in the domestic herds.

P1241 A FULLY-AUTOMATED PIPELINE FOR SEQUENCING WHOLE VIRAL GENOMES WITHOUT VIRUS-SPECIFIC PCR AMPLIFICATION TO DETERMINE HCV RESISTANCE TO ANTIVIRAL THERAPY

of HCV-PI resistance in liver but not in plasma compartment of 3 pts: 2 of these 3 pts harbored D168E amino acid (aa) substitution associated with macrocyclic PI resistance and one harbored V55A aa change associated with boceprevir resistance. The remaining 2 pts exhibited a wild-type sequence in both compartments. Conclusions: The detection of discordant G1 subtypes in liver and plasma may be consequent to a mixed infection and selection of a specific subtype with greater adaptability to different environmental condition. The presence of natural mutations associated with HCV-PI resistance in liver tissue may explain the early emergence of these strains during triple therapy including HCV-PI.