The use of lumpy skin disease virus genome termini for detection and phylogenetic analysis

During 2006 and 2007 there were two outbreaks of lumpy skin disease (LSD) in Israel. An LSD virus (LSDV)-specific PCR assay was developed that can detected specifically LSDV even though the number of tested LSDV isolates were limited. Full-length sheep pox and LSDV genome sequences were aligned to find non-homologous regions, which were then used for preparing specific primers, whose specificity was tested against several LSDV DNA isolates and the system could detect all the different isolates. Specificity was tested with sheep pox, ORF and other DNA viruses such as bovine herpes 4: the primers did not support amplification of the expected-size fragments, therefore the system appears to be a valuable tool for detecting specifically LSDV. The newly developed system was activated at the first report of a possible disease outbreak. It confirmed the clinical picture, and was introduced subsequently into routine diagnosis. Phylogenetic analyses of a 466-bp fragment next to the genome ends showed that this system can distinguish between: sheep pox, goat pox and LSD, and the results revealed that the Israeli isolates from 2006 and 2007 are in the same clad and essentially identical to Ismaeliya 1989, Nigeria 1996, Senegal 1997, Cameroon 1996, the Kenya NI-2490 isolate, and the South African LD virulent isolate. In contrast the original 1958 LW Neethling vaccine appeared to be in a separate clad, suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the viruses tested suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the tested viruses or during the process of attenuating the virus by succession of egg inoculations.     

Polymorphisms in the repeat long regions of oncogenic and attenuated pathotypes of Marek’s disease virus 1

The nucleotide sequences of the terminal repeat long (TR_L) and internal repeat long regions (IR_L) in the genomes of 13 strains of Marek’s disease virus type 1 (MDV-1) were determined and represent the largest collection of sequencing data from a contiguous region (12.8 kb) in the serotype 1 genomes. The collection of strains used in this study has been well characterized with respect to their virulence and contains members of each pathotype (4 attenuated, 1 mildly virulent, 3 virulent, 2 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the RL regions are likely to encode virulence factors based on comparative genomic studies involving vaccine and virulent strains. Additional studies using knockout mutants have provided stronger evidence that indeed RLORF4 and meq or the overlapping genes 23 kD and RLORF6 are involved in virulence. In this report, we provide evidence that additional open reading frames (ORFs) in the RL regions differ significantly between the extremes of the pathotypes (attenuated vs. nonattenuated). A deletion of 10 base pairs has been identified in RLORF12 from two attenuated strains CVI988 BP-5, p48 and RM-1, p40; and the lower virulence strain JM/102W. A deletion of 40 bp was also identified in RLORF4 of the attenuated strain R2/23, passage 106. A 177 bp insertion within the meq loci has been identified in most of the attenuated strains examined. Interestingly, R2/23 did not contain this insertion but instead truncated proteins are predicted for the three overlapping ORFs (meq, 23 kD and RLORF6) due to a frameshift mutation. Single nucleotide polymorphisms (SNPs), which loosely partition between attenuated and nonattenuated strains, have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23 kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG. Although no single nucleotide polymorphism in the RL regions could predict virulence, their overall contribution to virulence can now be examined in defined mutants containing additional insertions or deletions in ORFs, suspected of encoding virulence factors, identified by this research.     

Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture

Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus. All the strains that were studied grew well in three different cell types. No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases. All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens. The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains. The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs. Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants. Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function. Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence. Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome. All three of these regions were different from those that had previously been shown to rescue virulence of the Bartha strain (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1987, J. Virol. 61, 796-801). Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.     

Comparative full genome analysis of four infectious laryngotracheitis virus (Gallid herpesvirus-1) virulent isolates from the United States

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.     

Characterization of the Structural-Protein-Coding Region of SAT 2 Type Foot-and-Mouth Disease Virus

The NSP4 protein of rotavirus is a nonstructural glycoprotein and has a crucial function in virus morphogenesis during infection of host cells. It was recently reported that NSP4 may also function as a viral enterotoxin in the induction of rotavirus diarrhea by causing Ca⁺⁺ influx in the cytoplasm of the infected cells. We sequenced and analyzed two (Wa and M strains) pairs of NSP4 genes of virulent (v) and attenuated (a) (after 30 to 40 passages in cell culture) human group A rotaviruses and a pair of NSP4 genes of virulent and attenuated porcine group C rotavirus (Cowden strain). These strains were previously identified as virulent (induce diarrhea) or attenuated (no diarrhea) in a gnotobiotic pig model of rotavirus infection [Bohl et al. (4), Saif et al. (13), Ward et al. (17)]. The NSP4 genes of the Wa, M and Cowden strains were amplified with RT-PCR using a proof reading polymerase (Tli) and the RT-PCR product was sequenced directly. Analysis of the NSP4 deduced amino acid sequences showed that only 3 (Wa) and 2 (M and Cowden) amino acids differed between the virulent and attenuated strains. For the Wa strain, the changes from the virulent to attenuated strain were in amino acids 13 (V to A), 16 (L to S) and 34 (P to L); in the M strain, the difference was in amino acids 53 (T to I) and 104 (K to E), and in the Cowden strains, amino acids 50 (L to F) and 97 (D to N) differed between virulent and attenuated strains. To our knowledge, this is the first sequence comparison between NSP4 of a virulent and attenuated pair of group C rotaviruses. The potential impact of these few amino acid changes on the pathogenesis of the NSP4 protein for piglets is unclear, relative to previous findings in mice (1), but requires further study using purified recombinant NSP4 proteins or peptides.     

Isolates of infectious bursal disease virus from India are of very virulent phenotype

Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538-1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2-0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6-11.6% and amino acid divergence of 2.8-3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528-541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.     

A new porcine reproductive and respiratory syndrome virus strain with highly conserved molecular characteristics in its parental and attenuated strains

Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial economic losses to many swine-producing regions. In this study, PRRSV strain NT0801-F80 was derived from its parental isolate NT0801 by 80 passages in Marc-145 cells. Experimental infection of piglets clearly demonstrated that strain NT0801-F80 is less virulent than NT0801. However, whole genome sequencing showed that the genomes of the parental and attenuated strains are highly conserved compared with those of four other pairs of virulent parental/attenuated vaccine strains (VR2332 and RespPRRS MLV, JA142 and Ingelvac^® ATP MLV, CH-1a and CH-1R, and JXA1 and JXAR). The attenuated strain NT0801-F80 has only 21 nucleotide changes, producing only 14 amino acid changes in NSP2, GP2, GP3, and GP5, compared with those aa sequences of the virulent parental strain. These mutated aa in the attenuated virus may be involved in virulence. These data provide valuable information on the attenuation mechanism of PRRSV that should be useful in future research.     

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains

A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4-6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.     

Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus

Summary.  To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3′ genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus. Accepted October 6, 1997 Received August 7, 1997     

Genetic characterization of L-Zagreb mumps vaccine strain

Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide sequence was previously determined (accession numbers , and ). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed. A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s-1970s). L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.     

Comparison of field and vaccine strains of Australian fowlpox viruses

Summary. The mild fowlpox vaccine, FPV M, widely used in Australia is composed of two predominant genotypes based upon differences identifiable in restriction enzyme analyses of plaque purified derivatives of this vaccine. The differences, where identifiable, were in the end fragments of the genomes. Five field isolates of FPV from chickens in New South Wales showed restriction enzyme profiles closely related to the more virulent (standard) vaccine strain, FPV S. The FPV S strain differs from FPV M in both terminal genome fragments and in the presence of a PstI fragment of approximately 10kb (this fragment was also present in PstI digests of all of the field isolates). Plaque purified derivatives of FPV M showed similar lesion development upon inoculation into the wing web of chickens. The field isolates showed significantly higher virulence in day-old and three-week-old chickens in comparison with FPV M. One field isolate was similar to the FPV S vaccine. Two isolates had slowly developing wing web lesions, caused significant secondary lesions in three-week-old chickens and generalised poxvirus infection when inoculated into day-old chickens. For two isolates, the primary wing web lesion took even longer to develop and resolve although these isolates did not cause generalised poxvirus infection. It was possible to identify four virulence/pathogenicity types amongst these vaccine and field isolates of FPV. These strains may allow the characterisation of FPV encoded virulence factors. The field strains with higher virulence may be suitable as parent strains for the construction of FPV recombinants with enhanced immune responses to co-expressed vaccine antigens when compared with current FPV M strain based recombinants. Received July 19, 1996 Accepted September 26, 1996     

Antigenic and Molecular Characterization of Recent Infectious Bursal Disease Virus Isolates in China

Eleven infectious bursal disease virus (IBDV) strains isolated recently from China were compared with the early classical virulent strain CJ801, the chicken embryo fibroblast-adapted (CEF) variant strain GZ902, and the attenuated vaccine strains BJ836, BK912, and LM to discern the evolutionary characteristics of IBDV in China at both antigenic and genetic levels. Virus neutralization (VN) assay showed that all ten very virulent (vv) IBDV strains belong to the same subtype as attenuated strains, whereas the other variant isolate strain BX could be attributed to other subtype of the variant strain GZ902. Antigen-capture ELISA (AC-ELISA) determined by a panel of monoclonal antibodies (Mabs) against classical and variant strains showed further that among these vv strains, nine strains except for strain NC had no reaction with neutralizing Mab B69. The vv strains SC and YV had no reaction with non-neutralizing Mabs 2B8 and 2C4, respectively, whose epitopes were located in classical IBDV strains. On the other hand, there is no alteration in antigenic epitopes located in the variant strain BX as that of the variant GZ902. Sequence comparison of the highly variable region (HVR) of the VP2 proteins showed that these vv strains had 98.6–100.0% identities to European and Asian vv strains at amino acid level. For the vv strains NC, SC, and YV, all had one amino acid substitution at the major hydrophilic domains, indicating that new vv strains are evolving. In addition, the vv strains DMS and NC had amino acid residue 279N as well, showing that the substitution of amino acid at this position might not be related to the virulence of IBDV. The variant strain BX had one amino acid substitution in the two major hydrophilic domains and two unique amino acids 249K and 254S as the other early variant strains, and shared 97.3% of amino acid identity to the variant strain VarE. Phylogenetic analysis suggests that the recent Chinese vvIBDVs and the previous European and Asian vv strains still belong to a genetic group and the variant strain BX to the other genetic group, which is more closely related to the European classical virulent strain F52/70 and the American classical virulent strain STC than to the early Chinese classical virulent strain CJ801, showing that the recent vv and variant strains that spread widely in the country might be derived from Europe and America than from early Chinese classical virulent strains.     

Amino acid substitutions in the structural or nonstructural proteins of a vaccine strain of equine arteritis virus are associated with its attenuation

Comparative sequence analysis of a series of strains of equine arteritis virus (EAV) of defined virulence for horses, ranging from the horse-adapted virulent Bucyrus (VB) strain to a fully attenuated vaccine strain derived from it, identified 13 amino acid substitutions associated with attenuation. These include 4 substitutions in the replicase proteins and 9 in the structural proteins. Using reverse genetic techniques, these amino acid substitutions were introduced into a virulent infectious cDNA clone pEAVrVBS derived from the VB strain of EAV. Inoculation of horses with the recombinant viruses clearly demonstrated that changes in either the replicase (nsp1, nsp2 and nsp7) or structural proteins (GP2, GP4, GP5 and M) resulted in attenuation of the virulent VB strain. The recombinant virus with substitutions in the structural proteins was more attenuated than the recombinant virus with substitutions only in the replicase proteins.     

Genomic Features of Attenuated Junín Virus Vaccine Strain Candidate

Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper, we report the nucleotide sequences of L RNA of Candid #1 and examine the relationship to its more virulent ancestors Junin virus XJ#44 and XJ 13 (prototype) and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of L and Z genes of Candid #1 and its progenitor strains revealed twelve point mutations in the L polypeptide that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype. In contrast, Z ORF was completely conserved among all strains. The nucleotide sequences data of the of the L RNA of the Junin virus strains reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: AY819707.     

Genetic variation in the dimorphic regions of rap-1 genes and rap-1 loci of Babesia bigemina

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4⁺ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4⁺ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99% nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3′ ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.     

Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates

In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10(-5) to 10(-7) to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >10(7) CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.     

Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA-14-14-2

The attenuated SA-14-14-2 strain of Japanese encephalitis (JE) virus has been used to immunize people in the People's Republic of China. Oligonucleotide fingerprints of the parent SA-14 and vaccine strain indicate that multiple genetic changes occurred during attenuation of the virus. We have cloned and sequenced the genomes of both the virulent SA-14 and attenuated SA-14-14-2 viruses to define molecular differences in the genomes. Forty-five nucleotide differences, resulting in 15 amino acid substitutions, were found by comparing sequences of the SA-14 and SA-14-14-2 genomes. Transversion of U to A occurred at position 39 in the 5'-noncoding region of SA-14-14-2 and another SA-14 vaccine derivative SA-14-5-3. A single nucleotide change in the capsid gene of SA-14-14-2 altered a single amino acid which changed its predicted secondary structure. A silent nucleotide change was found in the prM gene sequence and the M-protein was unchanged. There are seven nucleotide differences, resulting in five amino acid changes, in the E glycoprotein sequence of the two viruses. Nine amino acid differences were found in the nonstructural proteins of SA-14 and SA-14-14-2: one in NS2A, two in NS2B, three in NS3, one in ns4a, and two in NS5. A single nucleotide change at position 10,428 in the 3'-noncoding region is vaccine virus-specific. The nucleotide and deduced amino acid sequences of the vaccine strain SA-14-14-2, the parent virus SA-14, and virulent strains JaOArS982 and Beijing-1 have been compared and are highly conserved.     

Soluble antigens of virulent and attenuated biotypes of Brucella abortus.

Several methods were used to characterize three Brucella abortus biotypes (1, 5, and 7), including the attenuated vaccine strain S-19. Chemical analysis did not reveal remarkable differences among these strains, and only minor differences were noted in elution patterns of soluble extracts subjected to column chromatography. Qualitative and quantitative differences in extract components were demonstrated, however, by polyacrylamide gel isoelectric focusing. A distinctive difference was the presence of components in extracts from one or more of the virulent biotypes that were absent in similar preparations from the attenuated strain. In addition, one component common to all virulent strains was absent in strain S-19. Results of immunodiffusion experiments employing adsorbed and unadsorbed antisera also suggested that the quantity, quality, and surface distribution of various cellular antigens differed among the biotypes studied.     

Molecular epidemiology of bluetongue virus serotype 4 isolated in the Mediterranean Basin between 1979 and 2004

The nucleotide sequences of genome segments 2, 7, 8, 9 and 10, coding for viral proteins (VP) and non-structural proteins (NS)--VP2, VP7, NS2, VP6 and NS3/NS3A, respectively, were determined and compared for 10 strains of bluetongue virus (BTV) serotype 4 isolated in the Mediterranean Basin between 1979 and 2004, and the South African attenuated BTV 4 vaccine strain. The sequence data generated for the BTV 4 strains isolated in Greece in 1979, 1999 and 2000 showed that they had a common origin but were distinct from the lineage of the BTV 4 strains isolated from 2003 onward in the western Mediterranean Basin (Italy, Morocco, Spain and Corsica). The nucleotide and deduced amino acid (aa) sequences of the BTV 4 strains within each lineage were identical to each other, irrespective of the year of isolation or the geographical location. Although the sequence of VP2 from the Turkish and Greek strains were highly similar, there were sufficient differences in the VP6, VP7 and NS2 proteins to suggest that the Turkish BTV 4 belongs to a third lineage. Alignment of the NS3 sequences from the attenuated BTV 4 vaccine strain and the field strains showed 13 aa substitutions, which may, either singularly or together, be responsible for attenuation and hence determining the virulence of the virus.