Deduced Amino Acid Level Research Articles

Characterization and transcript expression analyses of four Atlantic salmon (Salmo salar) serpinh1 paralogues provide evidence of evolutionary divergence

Atlantic salmon (Salmo salar) are not only the world’s most economically important farmed fish in terms of total value, but also a salmonid, which means that they are invaluable for studies of the evolutionary fate of genes following multiple whole-genome duplication (WGD) events. In this study, four paralogues of the molecular chaperone serpinh1 were characterized in Atlantic salmon, as while this gene is considered to be a sensitive biomarker of heat stress in salmonids, mammalian studies have also identified it as being essential for collagen structural assembly and integrity. The four salmon paralogues were cloned and sequenced so that in silico analyses at the nucleotide and deduced amino acid levels could be performed. In addition, qPCR was used to measure: paralogue- and sex-specific constitutive serpinh1 expression across 17 adult tissues; and their expression in the liver and head kidney of male Atlantic salmon as affected by stress phenotype (high vs. low responder), increased temperature, and injection with a multi-valent vaccine. Compared to the other three paralogues, serpinh1a-2 had a unique constitutive expression profile across the 17 tissues. Although stress phenotype had minimal impact on the transcript expression of the four paralogues, injection with a commercial vaccine containing several formalin inactivated bacterins increased the expression of most paralogues (by 1.1 to 4.5-fold) across both tissues. At 20 °C, the expression levels of serpinh1a-1 and serpinh1a-2 were generally lower (by −1.1- to −1.6-fold), and serpinh1b-1 and serpinh1b-2 were 10.2- to 19.0-fold greater, in comparison to salmon held at 12 °C. With recent studies suggesting a putative link between serpinh1 and upper thermal tolerance in salmonids, the current research is a valuable first step in elucidating the potential mechanisms involved. This research: supports the use of serpinh1b-1 and serpinh1b-2 as a biomarkers of heat stress in salmon; and provides evidence of neo- and/or subfunctionalization between the paralogues, and important insights into how multiple genome duplication events can potentially lead to evolutionary divergence.

Basal and immune-responsive transcript expression of two Atlantic salmon interferon regulatory factor 2 (irf2) paralogues.

Atlantic salmon (Salmo salar) is one of the most economically important aquaculture species globally. However, disease has become a prevalent threat to this industry. A thorough understanding of the genes and molecular pathways involved in the immune responses of Atlantic salmon is imperative for selective breeding of disease-resistant broodstock, as well as developing new diets and vaccines to mitigate the impact of disease. Members of the interferon regulatory factor (IRF) family of transcription factors play roles in the induction of interferons and other cytokines involved in host immune responses to intracellular and parasitic pathogens. IRF family members also play diverse roles in other biological processes, such as stress response, reproduction and development. The current study focused on one member of the IRF family: interferon regulatory factor 2 (irf2). As previously shown, due to the genome duplication that occurred ∼80 million years ago in the salmonid lineage, there are two irf2 paralogues in the Atlantic salmon genome. In silico analyses at the cDNA and deduced amino acid levels were conducted followed by phylogenetic tree construction with IRF2 amino acid sequences from various ray-finned fishes, cartilaginous fish and tetrapods. qPCR was then used to analyze paralogue-specific irf2 constitutive expression across 17 adult tissues, as well as responses to the viral mimic pIC (i.e., synthetic double-stranded RNA analog) in cultured macrophage-like cells (in vitro) and to infection with the Gram-negative bacterium Moritella viscosa in skin samples (in vivo). The qPCR studies showed sex- and paralogue-specific differences in expression across tissues. For example, expression of both paralogues was higher in ovary than in testes; expression (considering both sexes together) was highest for irf2-1 in gonad and for irf2-2 in hindgut. Both irf2 paralogues were responsive to pIC stimulation, but varied in their induction level, with irf2-1 having an overall stronger response than irf2-2. Only one paralogue, irf2-2, was significantly responsive to M. viscosa infection. Differences in irf2-1 and irf2-2 transcript expression levels constitutively across tissues, and in response to pIC and M. viscosa, may suggest neo- or subfunctionalization of the duplicated genes. This novel information expands current knowledge and provides insight into how genome duplication events may impact host regulation of important immune markers.

Resilience response to yellow leaf curl disease and identification of resistance gene analogs (RGA) in pepper (Capsicum annuum)

Abstract. Ayu DK, Maharijaya A, Syukur M, Hidayat SH. 2021. Resilience response to yellow leaf curl disease and identification of resistance gene analogs (RGA) in pepper (Capsicum annuum). Biodiversitas 22: 4731-4739. Pepper yellow leaf curl disease (PYLCD) caused by infection of Begomovirus is a serious threat to pepper production worldwide. Identification of the resistance gene analogs (RGA) and resilience response of pepper against PYLCD is needed especially for selection resistance genotype. Evaluation of resistance response involving 28 pepper genotypes was carried out through transmission of Begomovirus using whitefly (Bemisia tabaci) as vector. The result showed that IPB C12 and F4-012328-6-3 were potential resistance genotypes. A total of 15 R gene analogs (CaRGA) containing NBS motif, namely CARGA1 to CARGA15, were identified by degenerated PCR amplification and database mining. The alignment of deduced amino acid sequence revealed conservation of subdomains Ploop (GKTT), kinase2 (LVVLDDV), RNBSB/kinase3 (IILTTR) and GLPL. BLASTp analysis indicated that 15 RGA showed high homology at deduced amino acid level with R gene identified such as whitefly resistance protein Mi-1.2, Pvr 9 gene for potyvirus, Begomovirus resistance protein, TRGA15 and RGA 13 for putative late blight resistance. Phylogenetic analysis exhibited that isolated sequences distinguished into CNL-NBS groups. These pepper RGA could be considered as candidate sequences of resistance genes.

First isolation and characterization of Getah virus from cattle in northeastern China

BackgroundGetah virus (GETV) is a neglected mosquito-borne Alphavirus that causes pyrexia, body rash, and leg oedema in horses and foetal death and reproductive disorders in pigs. Infected animals may play a critical role in the amplification and circulation of the virus. The present study aimed to investigate GETV infection in clinically infected cattle and vector mosquito species in northeastern China.ResultsSerum samples were collected from beef cattle that presented sudden onset of fever in forest grazing areas, and metagenomic sequencing was conducted, revealing 29 contigs from ten serum samples matching the GETV genome. Quantitative RT-PCR (RT-qPCR) was performed with GETV RNA from 48 beef cattle serum samples, showing that the overall prevalence of GETV in the beef cattle samples was 6.25% (3/48). Serological investigation indicated that GETV neutralizing antibodies were detected in 83.3% (40/48, 95% CI 67–100) of samples from the study region. The GETV JL1808 strain was isolated from clinically infected cattle showing fever. Sequence comparisons showed high identity with the HuN1 strain, a highly pathogenic swine epidemic isolate obtained in Hunan province in 2017, at the nucleotide level (99.5%) and at the deduced amino acid level (99.7–99.9%). The phylogenetic analysis of JL1808 clustered in Group III, and also revealed a close genetic relationship with the HuN1 strain. Additionally, about 12,000 mosquitoes were trapped in this region. The presence of GETV infection was detected in mosquitoes, suggesting that the minimum infection rate (MIR) was 1.50‰, with MIRs of 1.67‰ in Culex pseudovishnui, 1.60‰ in Culex tritaeniorhynchus, and 1.21‰ in Anopheles sinensis.ConclusionsTo the best of our knowledge, this is the first report of GETV infection in cattle. These results demonstrated that a highly pathogenic, mosquito-borne swine GETV can infect and circulate in cattle, implying that it is necessary to conduct surveillance of GETV infection in animals in northeastern China.

Isolation and genomic characterization of lymphocytic choriomeningitis virus in ticks from northeastern China.

Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne arenavirus that is considered a neglected cause of neurologic diseases in humans. In this study, we described genomic characterization of newly isolated LCMVs in Haemaphysalis longicornis, Dermacentor nuttalli, Dermacentor silvarum and Ixodes persulcatus in Jilin Province, northeastern China. The complete sequences of the small (S) and large (L) segments of LCMVs in ticks contained 3,375 and 7,235-7,241 nucleotides, respectively. Sequence comparison showed 82.1%-86.0% identity of S segment with other lineage I strains at the nucleotide level and 91.2%-97.5% at the deduced amino acid level, while a lower identity was observed in the L segment at both nucleotide (75.4%-82.2%) and amino acid (82.4%-93.4%) levels. Phylogenetic analysis grouped the tick LCMVs together with the lineage I strains, but in an isolated cluster with a high bootstrap value. Bayesian analysis indicated that the molecular evolutionary rate was estimated to be 3.3×10-4 substitutions/site/year for the S segment and 6.3×10-4 substitutions/site/year for the L segment, and the time to most recent common ancestor was 1980 and 1970years ago, respectively, showing that tick LCMVs were predicted to originate between 1970s and 1980s. A long evolutionary history and high prevalence of LCMV in H.longicornis were found compared to other tick species. This study represented the first report on isolation of LCMV in China, showing that LCMV is circulating among ticks in Jilin Province, but the role of ticks in the epidemiology of LCMV remains to be explored.

Molecular cloning and expression of FGF2 gene in pre-implantation developmental stages of in vitro-produced sheep embryos.

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor-2 (FGF-2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non-pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2 , 95% humidity for 22-24hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6-7days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n=100), mature oocytes (n=100) and different stages of embryos such as 2 cell (n=50), 4 cell (n=25), 8 cell (n=12), 16 cell (n=6), morula (n=5) and blastocyst (n=3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real-time polymerase chain reaction using sequence-specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2-cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p<.05) were observed between immature oocytes and all pre-implantation stages of embryo. It can be concluded that FGF-2 plays a significant role in pre-implantation and early development of embryos in sheep.

Molecular genomic characterization of tick- and human-derived severe fever with thrombocytopenia syndrome virus isolates from South Korea.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (SFTSV) from Bunyaviridae that is endemic in East Asia. However, the genetic and evolutionary characteristics shared between tick- and human-derived Korean SFTSV strains are still limited.Methodology/Principal findingsIn this study we identify, for the first time, the genome sequence of a tick (Haemaphysalis longicornis)-derived Korean SFTSV strain (designated as KAGWT) and compare this virus with recent human SFTSV isolates to identify the genetic variations and relationships among SFTSV strains. The genome of the KAGWT strain is consistent with the described genome of other members of the genus Phlebovirus with 6,368 nucleotides (nt), 3,378 nt, and 1,746 nt in the Large (L), Medium (M) and Small (S) segments, respectively. Compared with other completely sequenced human-derived Korean SFTSV strains, the KAGWT strain had highest sequence identities at the nucleotide and deduced amino acid level in each segment with the KAGWH3 strain which was isolated from SFTS patient within the same region, although there is one unique amino acid substitution in the Gn protein (A66S). Phylogenetic analyses of complete genome sequences revealed that at least four different genotypes of SFTSV are co-circulating in South Korea, and that the tick- and human-derived Korean SFTSV strains (genotype B) are closely related to one another. Although we could not detect reassortant, which are commonly observed in segmented viruses, further large-scale surveillance and detailed genomic analysis studies are needed to better understand the molecular epidemiology, genetic diversity, and evolution of SFTSV.Conclusions/SignificanceFull-length sequence analysis revealed a clear association between the genetic origins of tick- and human-derived SFTSV strains. While the most prevalent Korean SFTSV is genotype B, at least four different genotypes of SFTSV strains are co-circulating in South Korea. These findings provide information regarding the molecular epidemiology, genetic diversity, and evolution of SFTSV in East Asia.

Host Specificity of Flagellins from Segmented Filamentous Bacteria Affects Their Patterns of Interaction with Mouse Ileal Mucosal Proteins.

Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-κB signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome- and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples.IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.

Isolation, characterization and genetic diversity of NBS-LRR class disease-resistant gene analogs in multiple virus resistant line of chilli (Capsicum annuum L.)

Viruses are serious threat to chilli crop production worldwide. Resistance screening against several viruses resulted in identifying a multiple virus resistant genotype ‘IHR 2451’. Degenerate primers based on the conserved regions between P-Loop and GLPL of Resistance genes (R-genes) were used to amplify nucleotide binding sites (NBS)—encoding regions from genotype ‘IHR 2451’. Alignment of deduced amino acid sequences and phylogenetic analyses of isolated sequences distinguished into two groups representing toll interleukin-1 receptor (TIR) and non-TIR, and different families within the group confirming the hypotheses that dicots have both the types of NBS-LRR genes. The alignment of deduced amino acid sequences revealed conservation of subdomains P-loop, RNBS-A, kinase2, RNBS-B, and GLPL. The distinctive five RGAs showing specific conserved motifs were subjected to BLASTp and indicated high homology at deduced amino acid level with R genes identified such as Pvr9 gene for potyvirus resistance, putative late blight resistance protein homolog R1B-23 and other disease resistance genes suggesting high correlation with resistance to different pathogens. These pepper RGAs could be regarded as candidate sequences of resistant genes for marker development.

First identification and phylogenetic analysis of equine hepacivirus in Korea

Non-primate hepacivirus (NPHV) corresponds a group of isolates recently characterized in horses and dogs that present similar genomic organization and are closely related to hepatitis C virus. Since canine hapacivirus, NPHV identified in dogs, was first discovered in dogs in the United States, equine hepacivirus (EqHV, NPHV identified in horses) has been identified in horses in several countries. However, no epidemiological studies have investigated EqHV in horses in Korea. In this study, a total of 74 (n=74) serum samples collected from horses in four regions of Korea were tested for EqHV RNA using nested RT-PCR. Overall, 14 samples were identified as positive (18.9%) and further analyzed according to gender, age, breed, and region. There were high positive rates in males, young horses, and Thoroughbreds; however, these rates differed regionally. Sequencing of the partial NS3 region of 12 samples and the polyprotein encoding regions of two samples positive for EqHV RNA revealed that the Korean EqHV isolates shared approximately 85.3–99.6% and 97.7–100% homology at the nucleotide and deduced amino acid level, respectively. Phylogenetic analysis revealed that the partial NS3 genes clustered with sequences previously reported as NPHV. Notably, sequences of EqHV detected in horses in the same region showed sequence divergence. The sequences of the polyprotein encoding region of two representative EqHVs shared 83.9% and 95.7% homology with each other at the nucleotide and deduced amino acid level, respectively. Comparison of the sequences of polyprotein encoding regions of Korean EqHV isolates and hepaciviruses from different hosts revealed that the NS3 and NS5B regions were most conserved among hepaciviruses. The results of the present study demonstrate that there is a high positive rate of EqHV in Korea and provide significant information regarding the geographical distribution and genetic variability of Korean EqHV isolates that will help improve global epidemiology of EqHV.

Molecular characterization of two papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander, Colombia

Papaya ringspot virus is an RNA virus that belongs to the genus Potyvirus, family Potyviridae and affects both papaya and cucurbits, causing great economic losses. PRSV isolates are divided into biotypes P and W; both biotypes naturally infect plants in the family Cucurbitaceae, whereas the P type also naturally infects papaya (Carica papaya L). In the present study, we report the full-length genome sequence of two PRSV-P isolates sampled from the Campo Hermoso (PRSV-CH) and Villa del Rosario (PRSV-VR) localities in Norte de Santander, Colombia. The genomes of these PRSV isolates are 10,326 nt in length and have a predicted ORF of 3344 aa. The identity among Colombian PRSV isolates is 96.9% and 97.3% at the nucleotide and deduced amino acid levels, respectively. PRSV isolates from China had the lowest identity at 78.3% and 89.2% (nucleotide-amino acid), whereas the highest identities were detected in PRSV isolates from Mexico, Venezuela and Hawaii. At the polyprotein level, the amino acid composition surrounding the active polyprotein cleavage sites differ in the Colombian PRSV sequences. The predicted cleavage site in P1/HC-Pro is LEQY/N – LEQY/S instead of MEQY/N. Both of the Colombian PRSV isolates have a putative recombination event in the P1 coding region, which is common in all PRSV isolates from the American continent. The new full-length PRSV sequences from Colombia provide a better understanding of the dynamics of papaya ringspot virus infections in papaya in Colombia and worldwide.

Isolation and full-length genome analysis of mosquito-borne Manzanilla virus from Yunnan Province, China.

BackgroundThere have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People’s Republic of China.ResultsThe DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments.ConclusionsDHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1198-5) contains supplementary material, which is available to authorized users.

Protea hybrid ‘Carnival’ (P. compacta x P. neriifolia) expresses multiple copies of a LEAFY homologue involved in floral and vegetative development

Proteas are woody perennial plants that develop large, complex inflorescences which are popular as cut flowers. Four paralogous genes with sequence similarity to the meristem identity gene LEAFY (LFY) of Arabidopsis thaliana were identified in the Protea cultivar ‘Carnival’ (Protea compacta x Protea neriifolia). The deduced amino acid sequences of Protea LFY (ProLFY) paralogues displayed highly conserved N- and C-terminal regions comparable to that of other LFY proteins. Paralogous sequences shared 97–99% similarity at nucleotide and deduced amino acid levels. To determine if these ProLFY paralogues are involved in floral transition and development, three paralogues were heterologously expressed in A. thaliana Col-0 wild-type and lfy-2 mutant plants. These three paralogues were chosen as they had the greatest sequence dissimilarities which may suggest a different function. Expression of the ProLFY-WL4 paralogue rescued the low fertility of the lfy-2 mutant by restoring seed yield to wild-type levels, and resulted in the conversion of ‘leaf-like’ inflorescences to flowers with a wild-type appearance. ProLFY-WL4 expression levels during ‘Carnival’ meristem development confirmed a role for this paralogue in inflorescence development. The other two paralogues used in heterologous A. thaliana expression studies, ProLFY-L5 and ProLFY-D1, did not rescue fertility or the abnormal floral phenotype of lfy-2. However, transgenic plants developed either smaller leaves when expressing 35S::ProLFY-L5, or larger leaves when expressing 35S::ProLFY-D1, compared to the non-transgenic lfy-2 plants. Together these results suggest that ProLFY paralogues may be involved in both floral and vegetative development of Protea.

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1ng/ml and 10ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.

Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes

IntroductionWe cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing–thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. Material and methodsH1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen–thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. ResultsThe yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05). ConclusionsThis is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles.

Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by severe diarrhea, dehydration and high mortality in the affected pigs. Recently, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China. In this study, molecular detection of PEDV was conducted using RT-PCR (targeting the M gene) on samples collected from piglets with watery diarrhea from 15 pig farms, and phylogenetic analysis of PEDV field strains was carried out based on their M and S genes. In addition, the complete genome sequence of a PEDV field strain was determined. PEDV was detected in 92.7 % of the samples (267/288). The 15 M genes that were amplified shared 99.6-100 % nucleotide identity and 99.1-100 % amino acid similarity with each other. The 15 S genes exhibited 98.6-99.9 % homology, both at the nucleotide level and at the deduced amino acid level. Phylogenetic analysis showed that all of the amplified M genes grouped in cluster 3, together with some Chinese, Korean and Thai strains, while all of the amplified S genes were in cluster 3 and were closely related to Korean strains. Compared with previous PEDV strains, all of the S genes have common characteristics, namely, a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156, similar or identical to Korean KNU-serial strains reported in recent years. The genome of the sequenced PEDV field strain is 28,038 nucleotides in length, excluding the poly (A) tail. Our findings suggest that a novel PEDV with a characteristic variant S gene is responsible for recent outbreaks of clinical diarrhea in piglets in China.

Molecular epidemiological study of human rhinovirus species A, B and C from patients with acute respiratory illnesses in Japan

Recent studies suggest that human rhinovirus species A, B and C (HRV-ABCs) may be associated with both the common cold and severe acute respiratory illnesses (ARIs) such as bronchiolitis, wheezy bronchiolitis and pneumonia. However, the state and molecular epidemiology of these viruses in Japan is not fully understood. This study detected the genomes of HRV-ABCs from Japanese patients (92 cases, 0-36 years old, mean±sd 3.5±5.0 years) with various ARIs including upper respiratory infection, bronchiolitis, wheezy bronchiolitis, croup and pneumonia between January and December 2010. HRV-ABCs were provisionally type assigned from the pairwise distances among the strains. On phylogenetic trees based on the nucleotide sequences of the VP4/VP2 coding region, HRV-A, -B and -C were provisionally assigned to 14, 2 and 12 types, respectively. The present HRV-A and -C strains had a wide genetic diversity (>30 % divergence). The interspecies distances were 0.230±0.063 (mean±sd, HRV-A), 0.218±0.048 (HRV-B) and 0.281±0.105 (HRV-C), based on nucleotide sequences, and 0.075±0.036 (HRV-A), 0.049±0.022 (HRV-B) and 0.141±0.064 (HRV-C) at the deduced amino acid level. Furthermore, HRV-A and -C were the predominant species and were detected throughout the seasons. The results suggested that HRV-A and -C strains have a wide genetic divergence and are associated with various ARIs in Japan.

Highly divergent dimorphic alleles of the proteasome subunit beta type-8 (PSMB8) gene of the bichir Polypterus senegalus: implication for evolution of the PSMB8 gene of jawed vertebrates

The proteasome subunit beta type-8 (PSMB8) gene encodes a catalytic subunit of the immunoproteasome, which is involved in the generation of peptides presented by MHC class I molecules. To date, highly diverged dichotomous alleles of PSMB8 have been reported in Oryzias species (actinopterygian teleosts) and Xenopus species (sarcopterygian amphibians). These dimorphic alleles share a similar substitution (A/V(31)F/Y) at the 31st position of the mature protein, which is most probably involved in formation of the S1 pocket. This substitution likely confers different cleavage specificities on the dimorphic PSMB8s. In addition, two paralogous PSMB8 genes possessing the A and F residues at the 31st position have been reported in sharks. Phylogenetic analysis indicated that the two types of PSMB8 of Oryzias, Xenopus, and sharks arose by independent evolutionary events. Here, we identified another pair of dimorphic alleles of PSMB8, which have the A and F residues at the 31st position of the mature protein, from bichir, Polypterus senegalus, a basal actinopterygian. The sequences of the mature proteins-encoding region of the dimorphic alleles of bichir PSMB8, the A and F types, showed only 72.7% and 77.5% identities at the nucleotide and the deduced amino acid levels, respectively. Their intronic sequences show almost no similarity, indicating that the dimorphic alleles of bichir PSMB8 have a very ancient origin. However, phylogenetic analysis showed that the dimorphisms of PSMB8 of bichir, Xenopus, and Oryzias arose by independent evolutionary events, suggesting the presence of a strong selective pressure for possessing the dimorphism.

Genomic analysis reveals Nairobi sheep disease virus to be highly diverse and present in both Africa, and in India in the form of the Ganjam virus variant

Nairobi sheep disease (NSD) virus, the prototype tick-borne virus of the genus Nairovirus, family Bunyaviridae is associated with acute hemorrhagic gastroenteritis in sheep and goats in East and Central Africa. The closely related Ganjam virus found in India is associated with febrile illness in humans and disease in livestock. The complete S, M and L segment sequences of Ganjam and NSD virus and partial sequence analysis of Ganjam viral RNA genome S, M and L segments encoding regions (396 bp, 701 bp and 425 bp) of the viral nucleocapsid (N), glycoprotein precursor (GPC) and L polymerase (L) proteins, respectively, was carried out for multiple Ganjam virus isolates obtained from 1954 to 2002 and from various regions of India. M segments of NSD and Ganjam virus encode a large ORF for the glycoprotein precursor (GPC), (1627 and 1624 amino acids in length, respectively) and their L segments encode a very large L polymerase (3991 amino acids). The complete S, M and L segments of NSD and Ganjam viruses were more closely related to one another than to other characterized nairoviruses, and no evidence of reassortment was found. However, the NSD and Ganjam virus complete M segment differed by 22.90% and 14.70%, for nucleotide and amino acid respectively, and the complete L segment nucleotide and protein differing by 9.90% and 2.70%, respectively among themselves. Ganjam and NSD virus, complete S segment differed by 9.40–10.40% and 3.2–4.10 for nucleotide and proteins while among Ganjam viruses 0.0–6.20% and 0.0–1.4%, variation was found for nucleotide and amino acids. Ganjam virus isolates differed by up to 17% and 11% at the nucleotide level for the partial S and L gene fragments, respectively, with less variation observed at the deduced amino acid level (10.5 and 2%, S and L, respectively). However, the virus partial M gene fragment (which encodes the hypervariable mucin-like domain) of these viruses differed by as much as 56% at the nucleotide level. Phylogenetic analysis of partial sequence differences suggests considerable mixing and movement of Ganjam virus strains within India, with no clear relationship between genetic lineages and virus geographic origin or year of isolation. Surprisingly, NSD virus does not represent a distinct lineage, but appears as a variant with other Ganjam virus among NSD virus group.

Hg bioavailability and impact on bacterial communities in a long-term polluted soil

Different soil samples characterised by a long-term Hg-pollution were studied for Hg total content, fractionation, phytotoxicity and influence on the bacterial community. Hg pollution ranged from 1 to 50 mg kg(-1) and most of it was speciated in scarcely soluble forms. In agreement with this, the biochemical quality indexes were investigated (biomass, enzyme activities) and the bacterial community (viable heterotrophic (VH) bacteria, functional diversity) apparently was not influenced by the degree of Hg pollution. In particular, the investigated soils exhibited a low percentage of Hg-resistant (Hg(R)) bacteria ranging from less than 0.001% to 0.25% of the VH and the addition of available Hg in the form of HgCl(2) induced an enrichment of resistant Hg(R) populations. The general biodiversity of the bacterial community was evaluated by denaturing gradient gel electrophoresis of DNA of Hg spiked soil microcosms and of control soils. Hg(R) bacteria capable to grow in a minimal medium containing HgCl(2) were also isolated and identified. MerA and merB gene PCR fragments were obtained from different Hg(R) strains and the range of similarities at the DNA level and at the deduced amino acid level showed that they carried mercuric reductase and lyase. Differently from bacteria, some influence of soil Hg content on seeds' germination and root elongation was observed for Lepidium sativum L. and Solanum lycopersicum L. In conclusion, most of the Hg in these long-term polluted soils was scarcely mobile and available and did not significantly influence the soil bacterial community. The risk of potential Hg remobilization over time, that could be naturally favoured by the activity of plant roots or other inorganic processes occurring in soil, can be extenuated since bacterial community was resistant and resilient to subsequent Hg stress.