The clinical response of cattle experimentally infected with lumpy skin disease (Neethling) virus

Summary British cattle were inoculated with lumpy skin disease (Neethling) virus and their clinical signs observed over a three week period. Elevation of body temperature following infection was not found to be a consistent feature, and even in severe cases was limited to a peak temperature of 41 °C. Generalised lesions were seen 9–14 days post infection (p.i.), and the development of generalised infections did not appear to be dose related. Following intradermal inoculation lesions were detected from day 2 p.i. and first appearance and severity of local reaction appeared to be related to dose. Virus isolation was carried out on ocular, nasal and saliva swabs, and on buffy coat preparations. A transient viraemia was detected in two of eleven animals that did not show generalized signs; virus was not isolated from the secretions of seven animals without generalised signs. Virus was isolated from the peripheral secretions of an animal with generalised disease between 9 and 15 days p.i. and viraemia was detected in each of five animals with generalized signs. Delayed-type hypersensitivity reactions following intradermal inoculation of immune cattle with LSDV were found to be maximal at 24 h after challenge.     

Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants

Lumpy skin disease virus (LSDV) is being developed as a vector for recombinant vaccines against diseases of veterinary importance. A strategy for generating viral thymidine kinase (TK) gene-disrupted recombinants which are stable and homogeneous using the South African Neethling vaccine strain of LSDV as vector has been developed. To assist with the selection process, the Escherichia coli beta-galactosidase (lacZ) visual marker gene was incorporated into the constructs. However, the use of lacZ has certain limitations. An improved strategy was then devised substituting lacZ with the enhanced green fluorescent protein (EGFP) under control of the vaccinia virus (VV) P11K late promoter. The EGFP marker was found to enhance the selection process, and with the inclusion of additional sonication and filtration steps the number of passages required to select recombinants to homogeneity has been reduced. In support of the improved method for generation and selection of recombinants described, three different LSDV recombinants expressing the glycoprotein genes of bovine ephemeral fever virus, Rift Valley fever virus and rabies virus were prepared and characterised.     

Importance of thymidine kinase activity for normal growth of lumpy skin disease virus (SA-Neethling)

Summary.  In order to study the importance of an intact thymidine kinase (TK) gene for the vaccine strain of a southern African capripoxvirus, namely, lumpy skin disease virus (LSDV) (type SA-Neethling), a TK disruption recombinant was generated expressing the Escherichia coliβ-galactosidase (lacZ) reporter gene. A comparative growth study of the recombinant and wild-type (wt) LSDV in TK-positive primary and secondary cells and TK-negative secondary cells was performed. It was found that although recombinant and wt virus both grew in TK-positive cells without selection, the recombinant was unable to grow in TK-negative cells (with or without selection), indicating that TK activity is important, if not essential, for normal growth of LSDV. Received June 12, 2001 Accepted July 10, 2001     

The use of serological differentiation indices for the phylogenetic analysis of plant virus relationships

Summary The potential use of serological differentiation indices (SDIs) in the phylogenetic analysis of plant virus coat protein relationships is presented. Relationship dendrograms were constructed from SDI data for viruses in the tymovirus and Subgroup I geminivirus taxonomic groups, using distance or phenetic methods embodied in the computer programmesFitch andNjtree. Relationship dendrograms for geminiviruses agreed well with those constructed by others from sequence or other data; the dendrogram for tymoviruses was not in agreement with one calculated from amino acid composition data, or from other properties, but appears consistent with recently-shown sequence similarities between tymoviruses. Implications of these results for the classification and taxonomy of plant viruses are discussed.     

The termini of the Chlorella virus PBCV-1 genome are identical 2.2-kbp inverted repeats

The Chlorella virus PBCV-1 genome is a linear nonpermuted 333-kbp dsDNA molecule with covalently closed hairpin termini. The termini (minus the hairpin) are identical inverted repeats of at least 2185 bases after which the sequence diverges. The inverted repeats contain two small potential open reading frames and several direct repeats. However, neither the open reading frames nor the remainder of the inverted repeats are transcribed during PBCV-1 replication. Twenty-nine other Chlorella virus DNAs, of 36 tested, hybridized to the PBCV-1 terminal fragments.     

基孔肯雅病毒深圳分离株的全序列分析

目的了解基孔肯雅病毒SZ_20101028株的全基因组分子遗传特征。方法通过C6／36细胞从患者血清中分离得到CHIKV,针对病毒的基因组设计了6条特异性引物,对病毒的全基因组进行扩增,测序并通过生物信息学软件分析该病毒株的全基因组分子遗传特征。结果病毒SZ_20101028株的全基因组核苷酸序列长度为12377nt,含两个开放阅读框。编码3722个氨基酸,两个编码区之间含有68nt非编码连接区;发现该毒株与2010年引起东莞基孔肯雅热疫情暴发的病原体核苷酸的同源性达到了99％,进化树分析结果表明,SZ_20101028株与2010年引起东莞疫情暴发的病原体的亲缘性最高,并且和2004年引起印度洋地区疫情暴发流行的病原体同属于印度洋亚型。结论深圳市首例输人性基孔肯雅病毒属于印度洋亚型,与2010年引起东莞疫情暴发的病原体的亲缘性最高。     

Sequence and phylogenetic analysis of the large (L) segment of the Tahyna virus genome

The Tahyna virus (TAHV) is an important human pathogen in the Bunyaviridae family. To date, only the S and M segments of this virus have been sequenced, but the sequence of the L segment hasn’t been established yet. In this study, we sequenced 963 nucleotides of the L segment of TAHV, comprising pre-motif A and motif A in region 3 of the RNA polymerase gene. The nucleotide sequence data reported in this paper was submitted to the GenBank nucleotide sequence database: EU185046.     

Sequencing and phylogenetic analysis of an avian reovirus genome

Avian reovirus infection causes considerable economic loss to the commercial poultry industry. Live-attenuated vaccine strain S1133 (v-S1133, derived from parent strain S1133) is considered the safest and most effective vaccine and is currently used worldwide. To identify the genes responsible for its attenuation, DNA sequences of open reading frames (ORF) of S1133 and its parent strains S1133, 1733, 526, and C78 along with three field isolates (GuangxiR1, GuangxiR2, and GX110058) and one isolate (GX110116) from a vaccinated chicken were performed. The sequence data were compared with available sequences in nucleotide sequence databases of American (AVS-B, 138, 176) and Chinese (C-98 and T-98) origin. Sequence analysis identified that several v-S1133 specific nucleotide substitutions existed in the ORFs of λA, λB, λC, μA, μB, μNS, σA, σB, and σNS genes. The v-S1133 strain could be differentiated from the field-isolated strains based on single nucleotide polymorphisms. Phylogenetic analysis revealed that v-S1133 shared the highest sequence homologies with S1133 and reovirus isolates from China, grouped together in one cluster. Chinese isolates were clearly more distinct from the American reovirus AVS-B strain, which is associated with runting–stunting syndrome in broilers.     

Nucleotide sequence,genome organisation and phylogenetic analysis of Indian citrus ringspot virus. Brief report

The sequence of the single-stranded RNA genome of Indian citrus ringspot virus (ICRSV) consists of 7560 nucleotides. It contains six open reading frames (ORFs) which encode putative proteins of 187.3, 25, 12, 6.4, 34 and 23 kDa respectively. ORF1 encodes a polypeptide that contains all the elements of a replicase; ORFs 2, 3 and 4 compose a triple-gene block; ORF5 encodes the capsid protein; the function of ORF6 is unknown. Phylogenetic analysis of the complete genome and each ORF separately, and database searches indicate that ICRSV, though showing some similarities to potexviruses, is significantly different, as in the presence of ORF6, the genome and CP sizes, and particle morphology. These differences favour its inclusion in a new virus genus.     

The use of monoclonal antibody probes for the detection of infectious bursal disease virus antigens

Two monoclonal antibodies (MAb), 2E6 and 2G10, were used against infectious bursal disease virus (IBVD) P3009 in an immuno-dot assay to detect IBDV antigens from cell culture, and from bursa and spleen tissue samples of chickens. The limit of viral antigens detected by using both MAb probes was 48 ng. The probes were used to detect five serotype 1 IBDV isolates and one serotype 2 IBDV strain. The result indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from seven unrelated avian viruses. The probes detected IBDV antigens in bursa and spleen tissues collected from chicks as early as 2 days after inoculation. The IBDV antigens in bursa tissue samples from six poultry farms were detected by both probes. However, tissue samples from two of six farms did not react with probe 2E6. The data suggest that it is possible to use two MAb probes for diagnosis of IBDV infection in poultry farms.     

Molecular characterization and phylogenetic analysis of the complete genome of a hepatitis E virus from European swine

Hepatitis E virus (HEV) has been detected in humans and in a broad range of animals, including pigs. For the first time the full-length genomic sequence of a HEV of European porcine origin, termed swX07-E1, was determined. Comparative analysis of 76 complete or nearly complete nucleotide sequences showed that swX07-E1 shares the highest nucleotide identity with Japanese swine HEV swJ8-5 and swJ12-4. The whole-genome phylogenetic analysis showed that swX07-E1 from Europe belongs to genotype-3 HEV, clusters with variants from Japan, Mongolia and Kyrgyzstan in subgroup 3c, but it is divergent from the prototype US HEV. Our analysis indicates that swX07-E1 represents a new subgroup of genotype-3 and that analysis of full-length sequences is necessary to discover new subgroups of HEV. According to our knowledge, swX07-E1 is the first full-length genome sequence of HEV from European swine. Knowledge about the full length HEV sequence from European swine is very important for understanding the HEV evolutionary events and the molecular mechanism of infection in human and in animals.     

Sequence and phylogenetic analysis of a Chinese very virulent infectious bursal disease virus

The complete genome sequence of a Chinese very virulent infectious bursal disease virus (vvIBDV) strain, Harbin-1, was determined. Based on the sequence analysis, the molecular characteristics and potential virulence determinants and origin of vvIBDV strains were identified. Phylogenetic analysis indicated that a reassortment and/or recombination event may have occurred in the emergence of Chinese vvIBDV strains.     

Molecular differentiation and phylogenetic analysis of the Egyptian foot-and-mouth disease virus SAT2

In February 2012, a massive new foot-and-mouth disease (FMD) outbreak struck Egypt. In this work, one-step RT-PCR assays were used for in-house detection and differentiation of foot-and-mouth disease virus (FMDV) in Egypt in this year using pan-serotypic and serotype-targeting sequence primers. FMDV SAT2 was the dominant virus in the examined isolates from the epidemic. The complete VP1 coding regions of two isolates were sequenced. The two isolates had 99.2 % sequence identity to most contemporary Egyptian SAT2 reference viruses, whereas they had 89.7-90.1 % identity to the SAT2/EGY/2/2012 isolate, which was collected from Alexandria, Egypt, and previously sequenced by WRLFMD. Phylogenetic analysis showed that Egypt had one topotype and two lineage of FMDV SAT2 in 2012. The Egyptian and the Palestinian 2012 strains were associated mainly with topotype VII, lineage SAT2/VII/Ghb-12, while the virus isolated from Alexandria Governorate belonged to the SAT2/VII/Alx-12 lineage. Topotype VII also comprised lineages that included strains isolated from Libya in 2012 and 2003. Furthermore, within the same topotype, the Egyptian SAT2/2012 isolates were related to strains from Saudi Arabia, Sudan, Eritrea, Cameroon and Nigeria. Nevertheless, more epidemiological work with neighboring countries is needed to prevent cross-border spread of disease and to reach a precise conclusion about the origin of the 2012 FMDV SAT2 emergency in the Middle East.     

Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from Turkish patients with chronic HBV infection

Hepatitis viruses are the leading causes of chronic liver disease resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and also in Turkey. Although Turkey has an intermediate rate of hepatitis B virus (HBV) infection with a prevalence reported as 5%, a complete HBV genome sequence has not been published. In this study, the molecular characterization and phylogenetic analysis are described of 11 complete HBV genomes isolated from 11 naïve patients (5 male, 6 female; ages: 18–54 years old, median 35 years old) with chronic HBV infection. Of 11 patients, 7 and 4 were HBeAg positive/anti‐HBe negative and HBeAg negative/anti‐HBe positive, respectively. All patients had no co‐infection with HCV, HDV, or HIV. HBV DNA was extracted from the sera of the patients. The complete genome was amplified by PCR and cloned into a TA vector. The PCR products were sequenced directly and the complete HBV genome sequences were determined. Ten HBV genomes were 3182 base pairs in length. There was a 183 bp deletion (between nucleotides 2987–3169) in pre‐S region in one HBeAg positive patient. There were two pre‐core stop codons (G1896A) in two HBeAg negative and three core promoter dual mutations (T1762/A1764) in one HBeAg positive and two HBeAg negative patients' HBV genomes. Phylogenetic analysis of all complete genomes yielded that all Turkish sequences were clustered in genotype D branch (ten in subgenotype D1 and one in subgenotype D2). The analysis of S gene amino acid sequences revealed that surface gene subtypes of one and ten HBV strains were subtype ayw3 and ayw2, respectively. This study indicates that Turkish patients with chronic hepatitis B infection show very little genotypic heterogeneity. Genotype D of HBV DNA and subtype ayw2 of surface gene represent almost the whole Turkish patient population infected with HBV. J. Med. Virol. 76:476–481, 2005. © 2005 Wiley‐Liss, Inc.