The Complete CDS of the Prion Protein (<Emphasis Type="Italic">PRNP</Emphasis>) Gene of African Lion (<Emphasis Type="Italic">Panthera leo</Emphasis>)

We provide the complete PRNP CDS sequence for the African lion, which is different from the previously published sequence and more similar to other carnivore sequences. The newly obtained prion protein sequence differs from the domestic cat sequence at three amino acid positions and contains only four octapeptide repeats. We recommend that this sequence be used as the reference sequence for future studies of the PRNP gene for this species.     

Amino acid sequence of the Amur tiger prion protein

Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank.     

Sequence analysis of the <Emphasis Type="Italic">Meq</Emphasis> gene in the predominant Marek’s disease virus strains isolated in China during 2006–2008

The main aim of the present study were to investigate sequence diversity in the Meq gene of Marek’s disease viruses (MDV) isolated in China and to determine the most prevalent MDV strains. The 19 MDV strains were isolated from dead or diseased chickens from different chicken farms in China during 2006–2008, and the Meq gene was sequenced from each of these strains. Sequence analysis showed that all of the isolates contained an open reading frame of 1020 nucleotides, which encoded a 339 amino acid peptide. Compared with reference MDV strains, 12 of the 19 MDV isolates possessed two amino acid substitutions, (T → A) at position 139 and (P → R) at position 176, one isolate shared sequence similarity with the attenuated strain CVI988, and five of the other six isolates exhibited one amino acid change (P → T) at position 177 or 176. The 19 MDV isolates shared between 99.0 and 100% nucleotide sequence homology, and between 97.7 and 100% amino acid sequence homology. The nucleotide and amino acid sequence identity between the 19 MDV isolates and the 25 reference MDV strains varied from 97.6 to 100% and 94.4 to 100%, respectively. Based on the phylogenetic relationships between Meq gene sequences, Chinese MDV isolates constituted a separate clade to MDV reference strains, demonstrating that a different genotype of MDV was prevalent in China between 2006 and 2008.     

Single nucleotide polymorphisms in the cadherin 23 (CDH23) gene in Polish workers exposed to industrial noise

Single nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome and may underlie differential susceptibility to common genetic diseases. A candidate gene for susceptibility to noise‐induced hearing loss (NIHL) is Cadherin 23 (CDH23). This study aimed to analyze genetic variation in the CDH23 gene in a group of 10 individuals derived from a cohort of 949 workers exposed to noise, and consisted of five persons from each of the resistant and susceptible extremes. DNA samples were collected and the coding exons of CDH23 were sequenced. We identified a total of 35 SNPs: 11 amino acid substitutions, 8 silent nucleotide changes, and 16 substitutions in intervening sequences. Ten of the 11 amino acid substitutions were previously shown also to segregate in a Cuban population. The nonsynonymous SNPs localized to the part of the gene encoding the extracellular domain of Cadherin 23, in particular ectodomains 5, 13, 14, 15, 16, 17, 19, and 22. One amino acid change occurred at a conserved position in ectodomain 5. Our results provide a framework for future study of polymorphisms in CDH23 as risk factor for NIHL. Am. J. Hum. Biol., 2008. Published 2008 Wiley‐Liss, Inc.     

Sequence analysis of the prion protein gene in Mongolian gazelles (<Emphasis Type="Italic">Procapra gutturosa</Emphasis>)

Prion diseases are a group of human and animal neurodegenerative conditions, which are caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (Prnp). In sheep, genetic variations of Prnp were found to be associated with the incubation period, susceptibility, and species barrier to the scrapie disease. We investigated the sequence and polymorphisms of the prion protein gene of Mongolian gazelles (gPrnp). gPrnp gene sequence analysis of blood samples from 26 Mongolian gazelles showed high identity within species. The gPrnp gene was closely related to the Prnp genes of Thomson’s gazelle, blackbuck, and cattle with 100, 100, and 98.5% identity, respectively, whereas the gPrnp gene with a deletion was closely related to the Prnp genes of wildebeest, Western roe deer, and sheep with 99.3, 99.3, and 98.9% identity, respectively. Polymorphisms of the open reading frame of Prnp as amino acid substitutions were detected at codons 119(N → S), 143(S → G) or 160(Y → H), 172(V → A), 182(N → S) and 221(V → A). There was also deletion of one octapeptide repeat at the N-terminal octapeptide repeat region. The polymorphisms of gPrnp will assist the study of prion disease pathogenesis, resistance, and cross species transmission.     

Molecular and clinical characteristics of hepatitis B virus in Korea

Korea is an endemic area of hepatitis B virus (HBV) infection but very little is known about the molecular characteristics of HBV isolates from Korean patients or the association with disease progression. The complete HBV genome sequences from 53 Korean patients with chronic hepatitis B, advanced cirrhosis, or hepatocellular carcinoma (HCC) were analyzed to identify (i) subgenotype distribution and genetic diversity and (ii) signature mutations associated with liver disease progression. With the exception of 1 patient infected with HBV/B, all 52 patients (98.1%) were infected with HBV/C, subgenotype C2. These strains were 98.4% identical and the frequency of amino acid substitutions occurring within key immunological epitopes increased with disease severity. A number of amino acid/nucleotide substitutions were associated with HCC, namely sR24K (HBsAg), SI126T (HBsAg), and pcA1846T (precore gene) mutations (P = 0.029, 0.001, and 0.008, respectively). HBV harboring deletions in the pre‐S region were also associated with increased liver disease severity (chronic hepatitis B vs. cirrhosis, P = 0.040; chronic hepatitis B vs. HCC, P = 0.040). Despite the high degree of sequence conservation, several key HBV mutations were associated with disease progression. Prospective studies with larger cohorts of patients are required to evaluate further the clinical manifestation of HBV/C2 in Korea. J. Med. Virol. 82: 1126–1134, 2010. © 2010 Wiley‐Liss, Inc.     

Variation in the coat protein sequence of British isolates of <Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> and comparison with previously published isolates

Summary. Isolates of Turnip yellow mosaic virus (TYMV) were collected from wild cabbage (Brassica oleracea) on a 400 m stretch of Dorset coastline. The coat protein genes of four isolates showed high homology in nucleotide sequence (0.970–1.000, mean 0.987). Lower levels of homology where found to previously published sequences of Australian isolates [10] (0.725–0.775, mean 0.741). The amino acid composition of the Dorset isolates showed high levels of homology (0.964–1.000, mean 0.986). Numerous amino acid substitutions occurred between the Dorset and Australian isolates (0.705–0.819, mean 0.742). Comparison with other isolates showed large genetic distances between the Dorset isolates and both European and Australian isolates.     

The<Emphasis Type="Italic"> Aa-Pri4</Emphasis> gene,specifically expressed during fruiting initiation in the<Emphasis Type="Italic"> Agrocybe aegerita</Emphasis> complex,contains an unusual CT-rich leader intron within the 5′ uncoding region.

The Aa1-Pri4 gene was cloned from the edible mushroom Agrocybe aegerita. The gene, specifically expressed during fruiting initiation, encodes a glycine-rich protein of 116 amino acids, with no homology to already known proteins. Homologous genes were amplified from two other strains belonging to the Agr. aegerita complex and originating from South-East Asia; and a comparison of the three genes revealed a high conservation of the coding sequences (72.8–97.8%). The PRI4 putative protein sequences were highly similar (87.5–100.0%); and all of them contained two protein kinase C sites, suggesting a potential supplementary regulation by phosphorylation at the protein level. The 5 uncoding regions all presented a leader intron, very variable in sequence (45.7% identity), but with a high C+T content (74.5–79.0%). The presence of such CT-rich sequences previously described in the promoter of highly expressed fungal genes suggests that the leader intron of the Aa1-Pri4 gene could be involved in the high-level, stage-specific expression.Communicated by U. KückP. Sirand-Pugnet and C. Santos contributed equally to this work     

Prolonged Infection in Rhesus Macaques with Simian Immunodeficiency Virus (SIVmac239) Results in Animal-Specific and Rarely Tissue-Specific Selection ofnefVariants

We analyzed the sequence ofnefgenes from different tissues of three rhesus macaques that had been infected with molecularly cloned SIV_mac239 for 88 to 92 weeks. Comparison of the predicted amino acid sequences revealed that each macaque had selected out specific amino acid substitutions and that most of this variation (70%) was confined to four regions, amino acids 39 to 75, 90 to 105, 153 to 167, and 191 to 217, comprising 36% of the protein. Thenefgenes in these animals underwent extensive genetic variation with average nucleotide and amino acid substitution rates varying from 0.86 to 2.84% and 2.47 to 6.27%, respectively, although tissue-specific selection ofnefvariants occurred in only 1 of 14 tissues examined in this study. Comparison of the rate of nucleotide and amino acid substitutions in thenefgenes to those previously reported in theenvin the central nervous system (CNS) and lymph node (LN) revealed that the predicted amino acid substitution rates for Nef were much higher than for the gp120 region ofenvin the CNS and LN tissues for one macaque. In the two other macaques, the predicted amino acid substitution rates were similar between these two proteins in LN tissues, but the amino acid substitution rates in Nef were significantly higher than in the gp120 from the CNS. Comparison of the nucleotide substitutions in the region of overlap between theenvand thenefrevealed that approximately 83% of the nucleotide substitutions in this area resulted in a Nef amino acid sequence change, 26% of the nucleotide substitutions resulted in a gp41 amino acid change, and 9.5% of nucleotide substitutions resulted in amino acid sequence changes in both proteins, suggesting a preference for the selection of amino acid substitutions in the Nef in these animals. Our results indicate that in animals infected with SIV_mac239 for prolonged periods, variation in thenefoccurs at rates similar to or exceeding that observed for theenvgene.     

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Total RNA from infectedPhysalis floridana was isolated to generate complementary DNA corresponding to the coat protein (CP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1–3) for fusion protein expression inE. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97–99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRV_cub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

Extensive sequence variation of feline immunodeficiency virusenv genes in isolates from naturally infected cats

Summary In an investigation of the evolution of feline immunodeficiency virus (FIV) in vivo, sequential isolates from a persistently infected cat were examined by direct sequencing following amplification of selected subgenomic regions by polymerase chain reaction (PCR). Three isolates, T 90, T 91, and T 92, obtained over a three-year period revealed no changes to regions known to be conserved withingag andpol genes. Additionally, no change occurred withingag andpol in an isolate recovered from a second cat which was experimentally infected with T 90. Changes were detected within an N-terminal region of the envelope glycoprotein gp 120 (env). These consisted of point mutations, some of which would result in amino acid substitutions and the predicted amino acid changes tended to cluster within variable domains. Inoculation of T 90 into a second cat resulted in a different pattern of mutations than that observed for the three isolates from the first cat. In all cases, virus isolates derived from the same cat were much more highly related to each other (extent ofenv variation was 0.5–1.5%) than to isolates from other cats (10–12%env variation). The rate of change of FIV was estimated to be 3.4×10^–3 nucleotide substitutions per site per year for theenv gene and less than 10^–4 nucleotide substitutions per site per year for thegag andpol genes, values concordant with that found for human immunodeficiency virus 1. Both nucleotide and amino acid changes in the gp 120 region were found to be directional, suggesting that selective pressures influence FIV envelope gene sequences.     

Polymorphism of the PRNP gene in the main breeds of indigenous Chinese goats

The polymorphism of the PRNP gene plays a key role in susceptibility to prion disease. Scrapie is a neurodegenerative disease affecting sheep and goats and belongs to the group of prion diseases. We isolated DNA from 333 goat samples representing the main local goat breeds in six provinces in China to identify PRNP polymorphisms and to determine whether these breeds were at risk for developing scrapie. Two novel amino acid polymorphisms (R211G and T219I) and a novel silent mutation at codon 125 as well as nine previously reported polymorphisms were observed. Twenty-eight alleles and forty-nine different genotypes were obtained. The codon 142M associated with resistance of goat scrapie was not found in this study. The codon 143R was relatively rare. The codon 222K, a potentially useful candidate site for selecting for scrapie resistance, was also rare in indigenous Chinese goats. These results could provide some useful data for assessing the risk of scrapie in Chinese indigenous goats.     

Genetic diversity of human papillomavirus type 16 E6, E7, and L1 genes in Italian women with different grades of cervical lesions

High‐risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high‐grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI‐loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high‐grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian‐American lineage. The European‐350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients. J. Med. Virol. 81:1627–1634, 2009. © 2009 Wiley‐Liss, Inc.     

Phylogeny of Thogoto Virus

Thogoto virus is a tick-borne member of the family Orthomyxoviridae. Previously, based on the similarity in antigenic relationship by cross-neutralization test, all virus strains were concluded to have derived from the same origin. In this study, we obtained partial gene sequences of 4 genes (PB1-like protein, PA-like protein, glycoprotein, and nucleoprotein) of 8 Thogoto virus strains isolated in Africa, Asia, and Europe and studied the genetic variation and phylogeny. Unrooted phylogenetic trees created by both neighbor-joining and maximum likelihood methods based on nucleotide and amino acid sequences for 4 genes were mostly similar and revealed two lineages, Euro-Asian and African. Intra-lineage nucleotide sequence variation was greater in the Euro-Asian lineage than in the African lineage for all 4 genes. Furthermore, for the strains of Euro-Asian lineage, variations for two genes associated with RNA-dependent RNA polymerase activities were greater than those for glycoprotein or nucleoprotein gene, based on both nucleotide and amino acid sequence differences as well as on synonymous and nonsynonymous differences, indicating greater mutation rates for the polymerase activity genes in these strains.     

Sequence analysis of selected regions of theenv (V 3 loop and gp 41) andgag (p 7) reading frames of Ethiopian human immunodeficiency virus type 1 strains

Summary Amplified polymerase chain reaction (PCR) products, corresponding to the V3 loop and gp 41 of theenv, and p 7 of thegag region, from proviral DNA of several Ethiopian and Swedish HIV-1 strains were sequenced. Of the six amino acids (GPGRAF) that constitute the principal neutralizing determinant (PND) within the V 3 loop, the Ethiopian isolates all showed two amino acid changes (GPGQTF). Four to five other substitutions were found in the amino acids flanking the PND. Substitution of alanine (A) for threonine (T) should result in a change in the predicted secondary structure, i.e., disappearance of a coil structure. Percentage similarity data on a stretch of 22 amino acids within the V 3 loop showed a concordance of the Ethiopian HIV-1 isolates with the sequences of published macrophage-T-cell tropic HIV isolates. Additionally derived protein sequences in two other regions showed two common substitutions in p 7 and one to two substitutions in gp 41 compared to a recent consensus sequence. These changes are hitherto unique for the Ethiopian strains, and suggest the presence of a clustering of a divergent HIV-1 strain in Addis Ababa, Ethiopia.     

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belém VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.     

Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses

Summary.   Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR-1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al. [7]. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group “b” while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90. Received November 6, 2000 Accepted January 23, 2001     

The basic DNA-binding protein of Bombyx mori nuclear polyhedrosis virus: The existence of an additional arginine repeat

The basic DNA-binding protein of the Bombyx mori nuclear polyhedrosis virus (BmNPV) was purified by HPLC and a sequence of 45 amino acids from the N-terminus was determined. There were no detectable modifications such as N-terminal blockage, glycosylation, or phosphorylation. The amino acid sequence showed high homology to the predicted amino acid sequences of the basic proteins of Autographa californica NPV (AcNPV) and Orgyia pseudotsugata NPV (OpNPV) (90 and 76%, respectively), however, the BmNPV basic protein possessed an additional sequence of 10 amino acids. A DNA fragment encoding the basic protein was identified in a BmNPV DNA library by screening for possible DNA sequences coding for the basic protein's amino acid sequence. The nucleotide sequence of the basic protein of BmNPV was more similar to that of AcNPV (97%) than to that of OpNPV (62%). Homology plot analysis of the nucleotide sequence indicates that the BmNPV basic protein internal repeat evolved very recently.     

Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas.

The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan.