Small ruminant lentivirus genetic subgroups associate with sheep TMEM154 genotypes

Small ruminant lentiviruses (SRLVs) are prevalent in North American sheep and a major cause of production losses for the U.S. sheep industry. Sheep susceptibility to SRLV infection is influenced by genetic variation within the ovine transmembrane 154 gene (TMEM154). Animals with either of two distinct TMEM154 haplotypes that both encode glutamate at position 35 of the protein (E35) are at greater risk of SRLV infection than those homozygous with a lysine (K35) haplotype. Prior to this study, it was unknown if TMEM154 associations with infection are influenced by SRLV genetic subgroups. Accordingly, our goals were to characterize SRLVs naturally infecting sheep from a diverse U.S. Midwestern flock and test them for associations with TMEM154 E35K genotypes. Two regions of the SRLV genome were targeted for proviral amplification, cloning, sequence analysis, and association testing with TMEM154 E35K genotypes: gag and the transmembrane region of env. Independent analyses of gag and env sequences showed that they clustered in two subgroups (1 and 2), they were distinct from SRLV subtypes originating from Europe, and that subgroup 1 associated with hemizygous and homozygous TMEM154 K35 genotypes and subgroup 2 with hemi- and homozygous E35 genotypes (gag p < 0.001, env p = 0.01). These results indicate that SRLVs in the U.S. have adapted to infect sheep with specific TMEM154 E35K genotypes. Consequently, both host and SRLV genotypes affect the relative risk of SRLV infection in sheep.     

Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.     

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.     

Recombinant small ruminant lentivirus subtype B1 in goats and sheep of imported breeds in Mexico

Nucleotide sequences of small ruminant lentiviruses (SRLVs) were determined in sheep and goats, including progeny of imported animals, on a farm in Mexico. On the basis of gag-pol, pol, env and LTR sequences, SRLVs were assigned to the B1 subgroup, which comprises caprine arthritis-encephalitis virus (CAEV)-like prototype sequences mainly from goats. In comparison with CAEV-like env sequences of American and French origin, two putative recombination events were identified within the V3-V4 and V4-V5 regions of the env gene of a full length SRLV sequence (FESC-752) derived from a goat on the farm.     

Phylogenetic analysis of small ruminant lentiviruses detected in Slovenia

Small ruminant lentiviruses (SRLV), which belong to the Retroviridae family, infect goats and sheep worldwide. The aim of this study was to characterize the SRLV strains circulating in Slovenia, by phylogenetic analysis of two genomic regions, 1.8 kb gag-pol fragment and 1.2 kb pol fragment. The results of our study revealed that Slovenian SRLV strains are highly heterogeneous, with ovine strains belonging to genotype A and caprine strains to genotypes A and B. The closest relatives of sheep virus sequences from two flocks that clustered together (SLO 35, 36) were found to be in subtype A5. A cluster composed of four sheep virus sequences (SLO 31) was clearly divergent from all other subtypes in group A and could not be assigned to any of them. The virus sequences from one goat flock belonged solely to subtype B1, whereas virus sequences from more than one genotype were found to circulate within the other two goat flocks, belonging to subtype B1 (SLO 1 and SLO 37) and to genotype A (SLO 2 and 78–88 g). Two goat virus sequences (SLO 2) were found to belong to genotype A and could not be assigned to existing subtypes. One goat virus sequence (37–88 g) from flock 37 was clearly different from other sequences of this flock and was more closely related to genotype A sequences. We propose two new subtypes within genotype A, subtype A14 (SLO 2) and A15 (SLO 31).     

Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA

The leukotoxin of Mannheimia haemolytica has a very high degree of amino acid diversity because the lktA gene has a complex mosaic structure that has been derived by horizontal DNA transfer and intragenic recombination. The objective of the present study was to determine the effect of this amino acid diversity on leukotoxin cytotoxicity against bovine and ovine cell types. This was done by comparing the luminol-dependent chemiluminescence response of bovine and ovine neutrophils after pre-incubation with the various leukotoxin types. The LktA1.1-type leukotoxin associated with bovine serotype A1 and A6 strains differs from the LktA1.2-type leukotoxin produced by ovine serotype A1 and A6 strains at a single amino acid position and has enhanced activity against bovine but reduced activity against ovine neutrophils. These findings, together with the exclusive association of the LktA1.1-type leukotoxin with bovine strains, suggest that this leukotoxin type has an adaptive advantage in the bovine host. Leukotoxins LktA6-LktA10 are associated with ovine strains and have complex mosaic structures and diverse amino acid sequences but similar levels of cytotoxic activity against bovine and ovine neutrophils, respectively. However, ovine neutrophils were more sensitive to the cytotoxic activities of these leukotoxins than were bovine neutrophils. LktA8- and LktA10-type leukotoxins are associated with serotype A2 and A7 strains that are responsible for the majority of ovine disease cases, but LktA6-, LktA7- and LktA9-type leukotoxins are associated with less common serotypes. These findings contribute to the growing body of evidence suggesting that factors other than leukotoxin cytotoxicity are responsible for the full expression of virulence in M. haemolytica. Overall, the extensive recombinational exchanges within the lktA gene of M. haemolytica have had little effect on leukotoxin function which is highly conserved.     

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.     

禽呼肠孤病毒μNS非结构蛋白基因的克隆及序列分析

根据GenBank中禽呼肠孤病毒(ARV)M3基因序列,设计并合成了一对跨越μNS非结构蛋白基因完整开放阅读框(ORF)的特异性引物,对ARV的10个毒株进行RT-PCR扩增、克隆及序列测定。结果表明,10个毒株μNS蛋白基因ORF的核苷酸序列全长均为1908 bp,编码635个氨基酸。这10个毒株之间的核苷酸及推导的氨基酸同源性分别都在98%以上。将它们与哺乳动物呼肠孤病毒(MRV)、番鸭呼肠孤病毒(DRV)等进行核苷酸及推导的氨基酸序列比较,并进行遗传系统树分析,结果表明ARV与MRV有较大的差异,与DRV差异较小。     

Genetic characterization of maedi-visna virus (MVV) detected in Finland

The aim of the study was to characterize the small-ruminant lentiviruses (SRLVs) detected in Finland by defining their phylogenetic relationships and by studying the evolution of the virus based on a well-known epidemiology. The study material comprised lung tissue samples of 20 sheep from 5 different farms, a cell-cultured virus from one of the original sheep lung samples, and a blood sample of a goat. The sheep were identified as positive during seroepidemiologic screenings in 1994-1996 and the goat in 2001. Initial classification of a 251 nucleotide sequence within gag gene amplified from the uncultured samples as well as from the cell-cultured virus showed that the SRLVs were genetically close and that they were more closely related to the prototype ovine maedi-visna viruses (MVVs) than to the caprine arthritis-encephalitis virus (CAEV). The lentivirus detected from the goat aligned within the cluster of the Finnish ovine viruses, demonstrating a natural sheep-to-goat transmission. Further phylogenetic analysis of the proviral gag, pol and env sequences confirmed the initial classification and showed that they constituted a new subtype within the diverse MVV group. The sequence analyses also showed that the virus had remained genetically relatively stable, in spite of the time given for virus evolution, an estimated 20 years, and in spite of the virus crossing the host species barrier.     

Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.     

禽呼肠病毒P10、P17非结构蛋白基因的克隆及序列分析

根据GenBank上的禽呼肠病毒(ARV)S1基因序列,设计并合成了一对跨越P10和P17非结构蛋白基因的特异性引物,对13个ARV毒株进行RT-PCR扩增、克隆及序列测定。结果显示,13个ARV毒株的P10蛋白基因ORF全长均为297bp,编码98个氨基酸;P17蛋白基因ORF全长为441bp,编码146个氨基酸。这13个ARV毒株P10、P17蛋白基因核苷酸同源性分别在96.6%~100%和95.2%~99.3%之间,推导的氨基酸同源性分别在98.2%~100%和91.9%~99.0%之间。将这13个ARV毒株与GenBank上其他正呼肠病毒毒株,包括番鸭株(DRV)和飞狐上分离到的内尔森海湾病毒(NelsonBayvirus,NBV)及两个澳洲分离株(ARM-1和SOM-4)进行同源性比较和遗传进化树分析,结果表明,呼肠病毒有地域和种类的差别。     

云南蓝舌病病毒1型毒株M6基因序列分析

为了解云南蓝舌病病毒(Bluetongue virus,BTV) 1型M6基因流行株的遗传变异及其与国内外流行病毒的遗传进化关系,试验从细胞培养物中分别提取4株云南分离株BTV-1 (Y863、SZ120169、6-12和7-12) RNA,用M6基因特异引物进行RT-PCR扩增和测序,采用生物信息学软件对获得的M6基因编码区序列进行核苷酸、氨基酸同源性比对及遗传进化分析.结果表明,分别获得4株云南分离株BTV-1 M6基因1 763 bp序列;4株云南分离株BTV-1核苷酸同源性在95.2%~99.9%之间,氨基酸同源性在97.6%~99.8%之间,1979年师宗分离的Y863病毒毒株与2012年师宗(SZ120169)、2013年江城(6-12、7-12)分离的3株病毒毒株核苷酸同源性分别为95.5%、95.2%和95.2%,氨基酸同源性分别为97.6%、98.4%和98.2%,而近两年(2012、2013)分离病毒核苷酸和氨基酸同源性较高,分别在96.9%~99.9%和99.1%~99.8%之间;遗传进化分析发现,4株云南分离株BTV-1为Eastern基因群病毒,它们之间核苷酸和氨基酸同源性分别为95.2%~99.9%和97.6%~99.8%;进一步分析发现4株云南分离株BTV-1与希腊及澳大利亚 BTV-1型毒株亲缘关系较近,核苷酸和氨基酸同源性分别为90.4%~95.6%和95.1%~99.1%,而与地中海国家(意大利、法国、阿尔及利亚、摩洛哥和突尼斯)和南非毒株关系较远,核苷酸和氨基酸同源性分别在83.8%和95.7%以下.4株云南分离株BTV-1属于Eastern基因群病毒,云南分离株BTV-1 M6基因在自然进化中发生遗传变异缓慢,该基因可以用来进行BTV-1基因群分布及毒株的地理区域来源相关的研究.     

2015－2020年天津地区猪伪狂犬病病毒gB、gC、gE基因遗传变异分析

为掌握天津地区猪伪狂犬病病毒（Pseudorabies virus,PRV）的流行及遗传变异情况,本研究对2015－2020年天津地区分离的20个分离株的gB、gC和gE基因进行扩增、测序,并与参考毒株序列进行比对分析。相似性分析结果显示,分离株与2012年后中国变异株相比,3种基因核苷酸及编码氨基酸序列相似性分别为：gB基因均为99.9%~100%;gC基因为99.7%~100%和99.4%~100%;gE基因为99.7%~100%和99.6%~100%。遗传进化和序列比对分析结果显示,依据gB、gC、gE基因绘制遗传进化树均可将PRV毒株分为GⅠ型和GⅡ型,天津分离株属于GⅡ型;其中19个分离株与PRV变异株遗传关系较近,属于同一亚分支,并存在相同的氨基酸变异位点;另外1个分离株（TJBD6株）gB和gE基因与PRV变异株遗传关系较近,氨基酸变异位置与PRV变异株一致,但其gC基因与经典株Ea株遗传关系较近,且核苷酸和氨基酸序列相似性为100%。上述结果表明,2015年以来天津地区流行的PRV毒株存在经典株和变异株2种类型,其中变异株为主要流行株。本次研究初步调查了天津地区PRV分子流行特征,可为猪伪狂犬病防控提供依据。     

Molecular characterization of lentiviruses from goats from Poland based on gag gene sequence analysis

Caprine arthritis-encephalitis virus (CAEV) infection in goats is worldwide but with higher prevalence in industrialized countries. While positive serology of CAEV in Polish goats was reported there was no genetic study of this virus. In this study, we described the molecular characterization of lentiviruses isolated from seropositive goats from Poland. We cloned and sequenced a fragment from the gag gene covering part of the coding sequences for the matrix (MA) p17 and for the capsid (CA) p25 proteins. Resulting nucleotide sequences were aligned with those from other ovine/caprine lentivirus isolates. We present data showing that the sequences of most goat lentivirus isolates are closer to the prototypic CAEV-Co isolate, nevertheless from one goat we isolated a virus that is closer to the sheep Maedi Visna virus (MVV) isolate. This might indicate a recent cross-species infection from sheep to goat.     

Sequence Analysis of Bluetongue Virus 1 M6 Gene in Yunnan Province

The object of this study was to investigate the evolution and variation of NS1 encoding gene (M6) of Bluetongue virus 1 (BTV-1) from Yunnan province and the evolutionary relationship of strains which from Yunnan province and other countries.RNA were extracted from four strains (Y863,SZ120169,6-12 and 7-12),and M6 gene were amplified by using specific primer and sequenced,which were analyzed by using bioinformatics software for nucleotides homology and phylogenetic relationships.The results showed that four strains M6 gene were 1 763 bp;The homology of nucleotides among four strains M6 gene were 95.2% to 99.9%,the amino acids among four strains M6 gene were 97.6% to 99.8%,the homology of nucleotides between Y863 (1979) and 3 strains (SZ120169,6-12 and 7-12) were 95.5%,95.2% and 95.2%,the amino acids between Y863 (1979) and 3 strains were 97.6%,98.4% and 98.2%,the homology of nucleotides and amino acids were high (96.9% to 99.9% and 99.1% to 99.8%,respectively) among four Yunnan strains.BTV was divided into two clusters (Western and Eastern) and four strains (BTV-1) from Yunan province belong to Eastern cluster.The homology of nucleotides and amino acids among four Yunnan strains was 95.2% to 99.9% and 97.6% to 99.8% respectively;The genetic distance were close among four Yunnan strains and strains from Greece and Australia,the homology of nucleotides and amino acids between them were 90.4% to 95.6% and 95.1% to 99.1%;The genetic distance were distinct among four Yunnan strains and strains from Mediterranean countries (Italy,Fance,Algeria,Morocco and Tunisia) and South Africa;The homology of nucleotides and amino acids between them were below 83.8% and 95.7%,so we found that gene clusters distribution was related to the geographical distribution for BTV.In a conclusion,four Yunnan strains belong to Eastern cluster and the speed of genetic variation of M6 from Yunnan province was slow,so M6 gene could be used in study of gene group of distribution and origin of geographical area.     

山西猪瘟野毒株E_2基因序列测定与遗传变异多样性分析

为了解山西地区猪瘟病毒（CSFV）流行毒株的遗传变异情况,采用RT-PCR方法,2013年从山西部分地区分离出5株CSFV流行毒株,并进行了E2全基因扩增、克隆与序列测定,应用DNAStar分析软件对所测定的5株毒株与国内外参考毒株的相应序列进行了同源性分析,绘制系统发育进化树。结果表明：5株CSFV流行毒株之间E2基因核苷酸序列与所推导氨基酸序列的同源性分别为81.4%~100%和87.9%~100%,与CSFV石门毒株（Shimen株）的核苷酸与氨基酸的同源性分别为82.9%~94.8%和89.0%~94.9%,与CSFV兔化弱毒株（HCLV株）的核苷酸与氨基酸的同源性分别为81.6%~99.6%和87.9%~99.5%,与17株来自各国不同地区的CSFV参考毒株的核苷酸与氨基酸的同源性分别为81.5%~99.6%和86.3%~99.7%。经系统发育关系分析,4株属于基因2群,且705、713、725、729、734和738位氨基酸发生置换,另外1株属于基因1群。本研究揭示了山西猪瘟流行毒株的遗传变异多样性现状。     

Nucleotide sequence of UK and Australian isolates of feline calicivirus (FCV) and phylogenetic analysis of FCVs.

We have determined the first complete genome sequence and capsid gene sequences of feline calicivirus (FCV) isolates from the UK and Australia. These were compared with other previously published sequences. The viruses used in the comparisons were isolated between 1957 and 1995 from various geographical locations and obtained from cats showing a range of clinical signs. Despite these diverse origins, comparisons between all strains showed a similar degree of sequence variation within both ORF1 (non-structural polyprotein) and ORF2 (major capsid protein) (amino acid distances of 7.7-13.0% and 8.8-18.6%, respectively). In contrast, ORF3 (putative minor structural protein) sequences indicated a more heterogenous distribution of FCV relatedness (amino acid distances of 1.9-17.9%). Phylogenetic analysis suggested that, unlike some other caliciviruses, FCV isolates within the current data set fall into one diverse genogroup. Within this group, there was an overall lack of geographic or temporal clustering which may be related to the epidemiology of FCV infection in cats. Analysis of regions of variability in the genome has shown that, as well as the previously identified variable regions in ORF2, similar domains exist within ORFs 1 and 3 also, although to a lesser extent. In ORF1, these variable domains largely fall between the putative non-structural protein functional domains.     

2005年-2010年我国部分地区PRRSV流行毒株的遗传变异分析

为了掌握高致病性猪繁殖与呼吸综合征病毒(PRRSV)的变异情况,揭示该病的发生规律,根据GenBank登录的PRRSV基因序列设计引物,采用RT-PCR法对2005年-2010年间送检的282份病料进行了PRRSV核酸检测,对其中9份阳性样品进行了ORF5～7基因片段扩增和测序,所得序列与GenBank下栽的PRRSV...