High Degree Of Genetic Stability Research Articles

Comprehensive characterization of the differences in metabolites, lipids, and volatile flavor compounds between Ningxiang and Berkshire pigs using multi-omics techniques

This study was conducted to comprehensively characterize, metabolites, lipids, and volatile flavor compounds of NingXiang (NX) pigs, Berkshire (BKS) pigs, and their crossbred (Berkshire × Ningxiang, BN) pigs using multi-omics technique. The results showed that NX had high intramuscular fat (IMF) content and meat redness. The metabolite and lipid compositions were varied greatly among three pig breeds. The NX pigs exhibited distinctive sweet, fruity, and floral aroma while BN pigs have inherited this flavor profile. 2-pentylfuran, pentanal, 2-(E)-octenal, and acetic acid were the key volatile flavor compounds (VOC) of NX and BKS pork. The VOCs were influenced by the composition and content of metabolites and lipids. The NX pigs have excellent meat quality traits, unique flavor profiles, and high degree of genetic stability regarding flavor. The study deepens our understanding of the flavor of Chinese indigenous pigs, providing theoretical basis to understand the meat flavor regulation under different feeding conditions.

Evaluation of lettuce germplasm for resistance to impatiens necrotic spot virus

Recently, impatiens necrotic spot virus (INSV) has emerged as a major pathogen of lettuce in the Salinas Valley of California. The virus is a member of the genus Orthotospovirus, and under natural conditions, is transmitted by the tiny insect, western flower thrips. Due to the high efficiency of virus transmission, chemical management of thrips vectors is often ineffective in preventing the spread of the disease. Utilization of genetic resistance will be a critical component for managing the disease; however, little is known about genetic sources of resistance in lettuce. Here, we evaluated 189 lettuce lines under natural disease pressure in four field experiments to identify INSV resistant germplasm. The stability of resistance was also evaluated using different inoculation methods under controlled conditions. Our results showed a significant variation (p < 0.0001) among lettuce cultivars for both INSV severity and incidence. We identified INSV resistant germplasm that belongs to different horticultural types and leaf color, including a red-leaf cultivar ‘Eruption’ with a high degree of genetic stability. Moreover, we characterized several factors influencing thrips and host plant relationships, including horticultural type, leaf color, and polyphenol concentration. Overall, INSV resistant germplasm identified in this study provide valuable resources for breeding of INSV resistant lettuce cultivars.

Regeneration competence of an endangered orchid, Vanda cristata Wall. ex Lindl. using leaf explants: A study in vitro

In the present paper, a protocol for plant regeneration was developed for micropropagation of an epiphytic orchid, Vanda cristata Wall. ex Lindl., by using whole leaf explants procured from five months old in vitro grown cultures. The regeneration competence of leaf explants was tested on two different nutrient media (M and KC) supplemented with auxins (IAA, IBA, NAA) and cytokinins (BAP, KN, TDZ) at different concentrations. The regeneration response was invariably initiated mostly at the bases of the leaf explants via shoot bud, protocorm like bodies (PLBs) and callus formation whereas leaf tips (apical side) showed very less response. KC medium supplemented with NAA(10.6 µM)+BAP(8.8 µM), acted synergistically; cent per cent explants initiated regeneration response via formation of callus, PLBs and shoot buds at the basal part of leaf explant and complete plantlets were obtained. Highest number of regenerants (6) was observed in this combination as compared to the control. The genetic stability analysis of in vitro regenerated plantlets of V. cristata was performed by using inter-simple sequence repeats (ISSR) primers; 6.67% polymorphism and 93.33% of monomorphism was calculated using five ISSR primers, that showed a high degree of genetic stability to obtain true-to-type plantlets. The protocol developed in this study may be useful for conserving V. cristata including other orchid species using different biotechnological approaches.

Intraspecific DNA Barcoding and Variation Analysis for Citri Reticulatae Pericarpium of Citrus reticulata "Chachi".

Citri Reticulatae Pericarpium, the desiccative mature peel of Citrus reticulata Blanco or its cultivated varieties, is a national geographical indicated product that has the concomitant function of both medicine and foodstuff. The primary source of Citri Reticulatae Pericarpium is Citrus reticulata “Chachi,” called “Guang chenpi,” while it differs in variety, propagation, grafting rootstock, and tree age, and the hereditary stability of its biological information between intraspecific plants is worthy of our attention. Homologous analysis result of 4 DNA barcodings in the ribosome or the chloroplast showed that the homology of them (ITS2, rbcl, matK, and psbA-trnH) of 22 samples was 100.00%, 99.97%, 99.99%, and 99.81%, respectively, which indicated that 4 DNA barcodes maintained a high degree of genetic stability in Citrus reticulata “Chachi.” Also, ITS2 was considered to identify Citrus reticulata “Chachi” from other varieties because it presented not only low variability within a certain taxon but also a high level of interspecies variability. Simultaneously, variant site detection of Citrus reticulata “Chachi” was analyzed by comparing with the reference Citrus reticulata genome, and 2652697 SNP sites and 533906 InDel sites were detected from whole-genome resequencing data of 22 samples, providing the data resources and theoretical foundation for the future study about the relevant molecular makers of “Guang chenpi.”

Retrospective surveillance of porcine circovirus 4 in pigs in Inner Mongolia, China, from 2016 to 2018.

A novel circovirus designated "porcine circovirus type 4" (PCV4) was recently reported in pigs with severe clinical disease in Hunan Province, China. Relatively little is known about the molecular epidemiology of this recently discovered virus. In order to assess the prevalence of PCV4 infection in pigs and to analyze its genomic characteristics, 1683 clinical samples were collected in Inner Mongolia, China, from 2016 to 2018. The overall infection rate of PCV4 was 1.6% (27/1683) at the sample level and 21.6% (11/51) at the farm level, with rates ranging from 3.2% (1/31) to 20.0% (6/30) on different PCV4-positive pig farms. In addition, the PCV4 infection rates at both the sample and farm level increased from 2016 to 2018. This also showed that PCV4 was present in pigs in 2016 in China and therefore did not arrive later than this date. Additionally, our findings showed that PCV4 infections had no association with PCV2 or PCV3 infections. We sequenced the complete genomes of three PCV4 strains and found that the PCV4 strains had a high degree of genetic stability but shared less than 80% sequence identity with other circoviruses. We identified six amino acid mutations in the Rep protein and seven in the Cap protein. Phylogenetic analysis based on Cap and Rep sequences confirmed that the PCV4 strains grouped in an independent branch. Our findings provide important information about the prevalence and genetic characteristics of PCV4 strains.

Comparative Nucleotide Sequence Analysis of Glycoprotein B, C, and G of Infectious Laryngotracheitis Virus Isolated in Egypt During 2016-2018

Infectious Laryngotracheitis (ILT) is an economically important disease of poultry, causing a high mortality rate and /or reduced egg production. Tracheas from sever morbid and recently died unvaccinated household chicken were subjected to PCR assay to detect the presence of the virus through amplification of the glycoprotein B gene. All tested samples gave positive ct ranging from 13.32 to 25.53. Virus isolation was performed by inoculation—the processed tracheal swaps onto the chorioallantoic membrane. Positive pock lesion has been developed within 7 days post-inoculation. The pock lesions were subjected to PCR assay for amplification of both the full-length glycoprotein B, C, and G for sequencing analysis. Positive amplicons migrating about 2600, 1250, and 890 bp were amplified corresponding to the orf of gpB, gpC and gpG genes. Sequence alignment and phylogenetic tree revealed that all sequenced isolates gave a high degree with wild type isolate of ILT and a high degree of genetic stability was clearly evident among strains isolated in different periods (2016-2018), indicating that these glycoproteins could be used as a vaccine candidate.

A Life Course Study of Genetic and Environmental Influences on Work Incapacity.

Work incapacity is a major public health challenge and an economic burden to both society and individuals. Understanding the underlying causes is becoming ever more relevant as many countries face an aging workforce. We examined stability and change in genetic and environmental factors influencing work incapacity from age 18 until retirement, and sex differences in these effects. The large population-based sample comprised information from 28,759 twins followed for up to 23 years combined with high-quality national registry data. We measured work incapacity as the total proportion of potential workdays lost due to sickness absence, rehabilitation and disability benefits. Structural equation modeling with twin data indicated moderate genetic influences on work incapacity throughout life in both men and women, with a high degree of genetic stability from young to old adulthood. Environmental influences were mainly age-specific. Our results indicate that largely the same genetic factors influence individual differences in work incapacity throughout young, middle and older adulthood, despite major differences in degree of work incapacity and probable underlying medical causes.

Construction and immunological evaluation of recombinant Newcastle disease virus vaccines expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP3/GP5 proteins in pigs

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) poses a significant threat to the pig industry, for which vaccination is considered to be an effective means of prevention and control. Here, we developed two recombinant Newcastle disease virus (NDV) LaSota-vectored PRRS candidate vaccines, rLaSota-GP5 and rLaSota-GP3-GP5, using reverse genetic techniques. The two recombinant viruses exhibited a high degree of genetic stability after 10 successive generations in chicken embryos. There was no significant difference in pathogenicity compared with the rLaSota parent strain in poultry, mice and pigs. The recombinant viruses could not be detected in the feeding environment of immunized pigs, but could be detected in the organs and tissues of pigs for no more than 10 days after immunization. Importantly, in contrast to rLaSota-GP5, rLaSota-GP3-GP5 elicited both significant humoral and cellular immune responses in pigs. In particular, the neutralizing antibody titer in the rLaSota-GP3-GP5 group was 1.51 times significantly higher than that of the commercial vaccine group at 42 days post-immunization. At the same time, there was significant difference in the level of IFN-γ between the rLaSota-GP3-GP5 group and the commercial vaccine group. Furthermore, the viral load in the organs and tissues of rLaSota-GP3-GP5-immunized pigs was substantially lower than that of unimmunized pigs after being challenged with HP-PRRS virus GD strain. These results suggest that rLaSota-GP3-GP5 is a safe and promising candidate vaccine, and there is potential for further development of a recombinant virus vaccine for PRRS using NDV.

Education, lifestyle and life course differences in level of work incapacity – genes and environment

Abstract Background As many countries face an aging workforce, it is becoming ever more relevant to understand the underlying causes of work incapacity across the entire working age. Previous twin studies have demonstrated that work incapacity is influenced by genetic as well as environmental factors. Furthermore, educational attainment and lifestyle behaviors are relatively stable from early adulthood and are associated with work incapacity. This association may be confounded by unobserved factors - both genetic and environmental. In two separate twin studies, we examine 1) the stability and change in genetic and environmental factors influencing work incapacity from age 18 until retirement, and sex differences in these effects, and 2) the associations and likelihood of causality between educational attainment, lifestyle and sickness absence. Methods study 1: A population-based sample of 28,759 twins were linked to high quality national registry data and followed for up to 23 years. We measured work incapacity as the total proportion of potential workdays lost due to sickness absence, rehabilitation, and disability benefits. study 2: Data from a subsample of 8,806 twins who completed health questionnaires were linked to registry data on sickness absence and educational attainment. Self-reported lifestyle behaviors were smoking, physical activity and BMI (height and weight). Results study 1: Structural equation modeling indicated moderate genetic influences on work incapacity throughout life in both men and women, with a high degree of genetic stability from young to old adulthood. study 2: Preliminary regression analyses indicated that genetic factors largely confound the associations between educational attainment, lifestyle and sickness absence. Conclusions Genetic factors seem to be a major issue in understanding causes of work incapacity. Largely the same genetic factors influence individual differences in work incapacity throughout working age.

Establishment of a novel testicular cell line from sterlet Acipenser ruthenus and evaluation of its applications.

In this study, a cell line, designated as Acipenser ruthenus testis (ART), was successfully established from testis tissues of the sterlet Acipenser ruthenus and characterized by studying and comparing the expression of specific genes between the cell line and the parent gonad tissues. The results suggested that the developed ART cell line was composed of a mixture of germ cells and somatic cells. Ploidy analysis indicated that the cell line exhibited a high degree of genetic stability and that the cells remained in a good proliferating state after being subcultured to passage 80.

Stability of the HTLV-1 glycoprotein 46 (gp46) gene in an endemic region of the Brazilian Amazon and the presence of a significant mutation (N93D) in symptomatic patients

BackgroundThe human T-lymphotropic virus type 1 (HTLV-1) affects 2–5 million people worldwide, and is associated with a number of degenerative and infectious diseases. The Envelope glycoproteins (gp) are highly conserved among the different HTLV-1 isolates, although nucleotide substitutions in the region that codifies these proteins may influence both the infectivity and the replication of the virus. The gp46 gene has functional domains which have been associated with the inhibition of the formation of the syncytium, cell-cell transmission, and the production of antibodies. The present study investigated the genetic stability of the gp46 gene of HTLV-1 in an endemic region of Brazilian Amazonia.MethodsIndex case (IC - a sample of a given family group) carriers of HTLV-1 were investigated in the metropolitan region of Belém (Pará, Brazil) between January 2010 (registered retrospectively) and December 2015. The sequences that codify the gp46 were amplified by PCR, purified and sequenced (MF084788–MF084825). The gene was characterized using bioinformatics and Bayesian Inference.ResultsThe 40 patients analyzed had a mean age of 45.2 years and 70% presented some type of symptom, with a predominance of pain and sensitivity, dysautonomia, and motor disorders. All patients presented the aA (Transcontinental Cosmopolitan) genotype, with an extremely low mutation rate, which is characteristic of the codifying region (aA – 1.83 × 10–4 mutations per site per year). The gp46 gene had a nucleotide diversity of between 0.00% and 2.0%. Amino acid mutations were present in 66.6% of the samples of individuals with signs/symptoms or diseases associated with HTLV-1 (p = 0.0091). Of the three most frequent mutations, the previously undescribed N93D mutant was invariably associated with symptomatic cases.ConclusionsThe aA HTLV-1 subtype is predominant in the metropolitan region of Belém and presented a high degree of genetic stability in the codifying region. The rare N93D amino acid mutation may be associated with the clinical manifestations of this viral infection.ImportanceLittle is known of the phylogeny of HTLV-1 in the endemic region of Brazilian Amazonia, and few complete gene sequences are available for the gp46 glycoprotein from the local population. The nucleotide sequences of the viral gp46 gene recorded in the present study confirmed the genetic stability of the region, and pointed to a homogeneous viral group, with local geographic characteristics. Further research will be necessary to more fully understand the molecular diversity of this protein, given the potential of this codifying region as a model for an effective HTLV-1 vaccine. The identification of a rare mutation (N93D), present only in symptomatic patients, should also be investigated further as a potential clinical marker.Trial registrationISRCTN 12345678, registered 28 September 2014.

Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid.

Malaxis wallichii (Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000m asl. The pseudobulbs are important ingredients of century old drug 'Ashtavarga' and a polyherbal energetic tonic 'Chyavanprash'. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for M. wallichii. In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5±0.6) was observed in MS medium supplemented with 1.0mg/l meta-topolin and 0.5mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66±0.3) was achieved in half-strength MS medium fortified with 1.0mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of M. wallichii (2n) was found to be 2C=2.760±0.02pg and therefore, 1349.64Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased H2O2 content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

High frequency regeneration protocol for Dendrobium nobile: A model tissue culture approach for propagation of medicinally important orchid species

Low rates of shoot multiplication, clonal instability, poor rooting frequency and high cost of production are major impediments challenging the micropropagation of orchids and other ornamental plants. These problems can be solved with the proper usage of plant growth regulators (PGRs) in the medium. The highest shoot proliferation rate of 21 shoots/explant was recorded in Murashige and Skoog (MS) medium containing 1mg/l meta-topolin and 0.8mg/l Putrescine. To date, this is the best frequency of shoot induction for Dendrobium nobile and one of the highest among other orchids. Higher rooting frequency was also recorded with the highest rooting of 10.1 roots/shoot achieved in plants grown in half strength MS media supplemented with 2mg/l of indole butyric acid and 0.5mg/l of phloroglucinol. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 82.3% exhibiting normal developmental patterns. The regenerated plantlets were assessed for genetic stability using the molecular markers namely SCoT and IRAP revealing high degree of genetic stability within the micropropagated plants of D. nobile. Being a highly medicinal orchid, the antioxidant potentials of the mother and the micropropagated plants were assessed using DPPH, FRAP and metal chelating assays which revealed a significantly higher antioxidant activity in the micropropagated plants. Rapid multiplication rate, higher genetic stability and significantly higher antioxidant power reported in the present study on D. nobile ensure the use of this micropropagation protocol for ex-situ conservation and commercial exploitation which could also be extended to other important orchid species.

Spontaneous modulation of a dynamic balance between bacterial genomic stability and mutability: roles and molecular mechanisms of the genetic switch

Bacteria need a high degree of genetic stability to maintain their species identities over long evolutionary times while retaining some mutability to adapt to the changing environment. It is a long unanswered question that how bacteria reconcile these seemingly contradictory biological properties. We hypothesized that certain mechanisms must maintain a dynamic balance between genetic stability and mutability for the survival and evolution of bacterial species. To identify such mechanisms, we analyzed bacterial genomes, focusing on the Salmonella mismatch repair (MMR) system. We found that the MMR gene mutL functions as a genetic switch through a slipped-strand mispairing mechanism, modulating and maintaining a dynamic balance between genetic stability and mutability during bacterial evolution. This mechanism allows bacteria to maintain their phylogenetic status, while also adapting to changing environments by acquiring novel traits. In this review, we outline the history of research into this genetic switch, from its discovery to the latest findings, and discuss its potential roles in the genomic evolution of bacteria.

Molecular Characterization of Patient-Derived Human Pancreatic Tumor Xenograft Models for Preclinical and Translational Development of Cancer Therapeutics

Preclinical evaluation of novel cancer agents requires models that accurately reflect the biology and molecular characteristics of human tumors. Molecular profiles of eight pancreatic ductal adenocarcinoma patient tumors were compared to corresponding passages of xenografts obtained by grafting tumor fragments into immunocompromised mice. Molecular characterization was performed by copy number analysis, gene expression and microRNA microarrays, mutation analysis, short tandem repeat (STR) profiling, and immunohistochemistry. Xenografts were found to be highly representative of their respective tumors, with a high degree of genetic stability observed by STR profiling and mutation analysis. Copy number variation (CNV) profiles of early and late xenograft passages were similar, with recurrent losses on chromosomes 1p, 3p, 4q, 6, 8p, 9, 10, 11q, 12p, 15q, 17, 18, 20p, and 21 and gains on 1q, 5p, 8q, 11q, 12q, 13q, 19q, and 20q. Pearson correlations of gene expression profiles of tumors and xenograft passages were above 0.88 for all models. Gene expression patterns between early and late passage xenografts were highly stable for each individual model. Changes observed in xenograft passages largely corresponded to human stromal compartment genes and inflammatory processes. While some differences exist between the primary tumors and corresponding xenografts, the molecular profiles remain stable after extensive passaging. Evidence for stability in molecular characteristics after several rounds of passaging lends confidence to clinical relevance and allows for expansion of models to generate the requisite number of animals required for cohorts used in drug screening and development studies.

Influence of Agrobacterium rhizogenes on induction of hairy roots for enhanced production of artemisinin in Artemisia annua L. plants

Artemisinin production from plant tissue cultures and induction of hairy roots in vitro have been considered to be a promising alternative, which offer a high degree of genetic stability, grow rapidly and produce the higher spectrum of secondary metabolites than wild type plants. Hairy root cultures developed from infection of different explants of in vitro germinated Artemisia annua L. plant with Agrobacterium rhizogenes LBA 9402 strain were selected on the basis of high artemisinin content and growth. Integration of the TL-DNA ( rol gene) region of the pRi plasmid was confirmed by polymerase chain reaction (PCR) analysis of the gene located in this region. The effect of different environmental factors like temperature, pH, cultivation media and carbon source on growth and artemisinin production were studied in shake flask cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 390 μg L -1 day -1 was obtained in hairy root cultures. Keywords: Agrobacterium , Artemisia annua L., artemisinin, hairy root cultures

Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq®

RotaTeq is a pentavalent rotavirus vaccine that contains five human-bovine reassortant strains (designated G1, G2, G3, G4, and P1) on the backbone of the naturally attenuated tissue culture-adapted parental bovine rotavirus (BRV) strain WC3. The viral genomes of each of the reassortant strains were completely sequenced and compared pairwise and phylogenetically among each other and to human rotavirus (HRV) and BRV reference strains. Reassortants G1, G2, G3, and G4 contained the VP7 gene from their corresponding HRV parent strains, while reassortants G1 and G2 also contained the VP3 gene (genotype M1) from the HRV parent strain. The P1 reassortant contained the VP4 gene from the HRV parent strain and all the other gene segments from the BRV WC3 strain. The human VP7s had a high level of overall amino acid identity (G1: 95-99%, G2: 94-99% G3: 96-100%, G4: 93-99%) when compared to those of representative rotavirus strains of their corresponding G serotypes. The VP4 of the P1 reassortant had a high identity (92-97%) with those of serotype P1A[8] HRV reference strains, while the BRV VP7 showed identities ranging from 91% to 94% to those of serotype G6 HRV strains. Sequence analyses of the BRV or HRV genes confirmed that the fundamental structure of the proteins in the vaccine was similar to those of the HRV and BRV references strains. Sequences analyses showed that RotaTeq exhibited a high degree of genetic stability as no mutations were identified in the material of each reassortant, which undergoes two rounds of replication cycles in cell culture during the manufacturing process, when compared to the final material used to fill the dosing tubes. The infectivity of each of the reassortant strains of RotaTeq, like HRV strains, did not require the presence of sialic acid residues on the cell surface. The molecular and biologic characterization of RotaTeq adds to the significant body of clinical data supporting the consistent efficacy, immunogenicity, and safety of RotaTeq.

Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1 NA) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1 EU). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1 EU genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1 NA and ENTV-1 EU reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K a/ K s ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.

Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain (“MAV”, Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain “Lausanne” and its attenuated field derivative strain “6918”. The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to “Lausanne” and “6918”. Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

Genetic Variability of Human Respiratory Coronavirus OC43

A recent paper by J. R. St-Jean and colleagues reported the complete genome sequences of the human coronavirus OC43 (HCoV-OC43) laboratory strain from the American Type Culture Collection (ATCC) and an HCoV-OC43 clinical isolate, designated Paris (7). The ATCC HCoV-OC43 (VR759) strain originated in 1967 and was passaged several times in suckling mouse brain and in cell culture. The contemporary HCoV-OC43 Paris strain was isolated in 2001 and was subsequently cultured in an HRT-18 cell line by the authors. A high degree of genetic stability was stated for HCoV-OC43 since only six nucleotide variations in the whole genome could be observed between both HCoV-OC43 strains, with isolation dates 34 years apart. There are, however, some arguments to suggest that the HCoV-OC43 genome is not as stable as suggested by the authors and that the HCoV-OC43 Paris strain might not be a contemporary strain but might be a result of cross-contamination with the ATCC HCoV-OC43 strain.