Matrix Protein Gene Sequence Analysis of Avian Paramyxovirus 1 Isolates Obtained from Pigeons

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.     

Pathogenicity and phylogenetic evaluation of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses” based on the nucleotide sequence of the fusion protein gene

Summary The nucleotide sequences of the entire F genes of two isolates of the pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) were determined using RTPCR. The deduced amino acid sequences of the F0 protein showed four differences between isolate 760/83 which had been passaged 4 times in chickens and gave an intravenous pathogenicity index in chickens (IVPI) of 2.01 and isolate 1168/84 which had received six passages in chickens and had an IVPI of 0.00. The F genes of virus from two passage levels of isolate 1447/84, 0 with IVPI value 0.00 and six with IVPI value 0.58, were partially sequenced to cover the areas of variation between 760/83 and 1168/84. The two passage levels of 1447/84 showed identical sequences in these areas which in turn were identical to those of 760/83. It was concluded that the recorded differences in intravenous pathogenicity were unlikely to be associated with differences in the primary structure of the F0 protein. Phylogenetic comparisons of the F gene sequences of the two PPMV-1 viruses with those published for other NDV strains and isolates showed that the PPMV-1 viruses formed a new fourth lineage but were closely related to strain Warwick with which they presumably shared a common origin.     

A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene

A sequence 375 nucleotides in length, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 174 isolates of Newcastle disease virus (avianparamyxovirus type 1). These were compared with the sequences of 164 isolates published on GenBank, and the resulting alignment was analysed phylogenetically using maximum likelihood. The results are presented asunrooted phylogenetic trees. Briefly, the isolates divided into six broadly distinct groups (lineages 1 to 6).Lineages 3 and 4 were further subdivided into four sublineages (a to d) and lineage 5 into five lineages (a to e).Considerable genetic heterogeneity was detected within avian paramyxoviruses type 1, which appears to beinfluenced by host, time and geographical origin. It is concluded that by using this dataset it will be possible totype future virus isolates rapidly on the basis of their nucleotide sequence and make inferences about theirorigins.     

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.     

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses”

Summary The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif¹¹²G-R-Q-K-R-F¹¹⁷. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence¹¹²R-R-Q-K-R-F¹¹⁷ at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN₅₇₁. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.     

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1

Summary.  Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia 1 virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I–IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 × 10⁻² synonymous substitutions per nucleotide per year. Received May 7, 2001 Accepted August 1, 2001     

Sequence and phylogenetic analysis of the L and VP1 genes of foot-and-mouth disease virus serotype Asia1

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, we determine the nucleotide (nt) sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype Asia 1 isolates recovered from different outbreaks in India, and compared with each other and the vaccine strain, IND 63/72, used in the country. Independent phylogenetic analyses on both the aligned gene sequences identified two major lineages (designated A & B) in the Asia 1 isolates. Both L- and VP1-based trees were congruent with respect to the major branching pattern of the isolates. The lineage A is represented by the isolates of 1986-2000 including the vaccine strain IND 63/72, whereas, lineage B appeared to be dominant and responsible for most of the recent outbreaks. A correlation was observed between the clustering of the isolates in the phylogenetic tree and the amino acid changes at many of the positions in VP1 as well as in L protein. The annual rate of evolution in L and VP1 genes was found similar and estimated to be 4.0 x 10(-3) and 3.8 x 10(-3) substitutions per nucleotide, respectively. Our result, largely from the congruence in phylogenetic trees and the rate of evolution in both the genes, suggests the possibility for the use of L gene sequence in phylogenetic comparison of FMDV.     

Molecular epidemiology of rabies in Indonesia

In order to clarify the genetic relationships and dynamics of rabies viruses that are epidemic in Indonesia, we determined and analyzed 1307 nucleotides of nucleoprotein genes of 34 rabies field isolates collected from Sumatra, Java, Kalimantan, Sulawesi and Flores islands. Results of phylogenetic analysis indicated that rabies isolates in Indonesia formed one cluster, were of Asian lineage, and were closely related to a rabies isolate in China rather than to rabies isolates in Thailand, India or Sri Lanka. Rabies isolates in Indonesia were divided into three phylogroups (ID1, ID2 and ID3) that included seven lineages. There was a correlation between phylogroup and geographical distribution of the isolates. Isolates in four lineages (SC1, SC2, SC3 and ST) of the ID1 phylogroup were mainly present in Sumatra. Isolates in the ST lineage were distributed widely in Sumatra, while isolates in the SC1, SC2 and SC3 lineages were limited to central Sumatra. ID2 and ID3 phylogroups included one lineage (JA) and two lineages (KS and SF), respectively. Results of phylogenetic analysis and historical background suggest that rabies viruses in China might have been transferred to Indonesia and spread to each island due to human activities.     

Phylogeny and evolution of the NS1 and VP1/VP2 gene sequences from porcine parvovirus

The objective of the present study was to gain new insights on the evolution and phylogeny of the PPV genome, specifically on the NS1 and VP1/VP2 genes. Moreover, two new complete sequences from PPV isolates from China (BQ and ZJ strains) were generated and included in the study. The data set studied contained available NS1 and VP1/VP2 sequences at the GenBank database, plus those corresponding to the mentioned Chinese isolates. PPV sequences were divided into two major groups, with one group separated into two branches. Both phylogenetic groups were homogeneous and several marker aminoacidic changes and synapomorphic positions were identified along both genes. Despite the two genes were satisfactory molecular markers, the absence of selection pressure on the VP1/VP2 fragment makes it a preferential option compared to the NS1 one. Furthermore, NS1 gene showed a biased mutation pattern compared with VP1/VP2 genes, which is compatible with the existence of selection in the first but not in the second gene (as indicated by the negative difference between non-synonymous and synonymous values). No correlation between NS1 and VP1/VP2 phylogenetic groups and/or branches and health status was observed. However, a relationship among virulence and the absence of the 127-bp repeat located downstream the part of ORF2 encoding the structural proteins VP1 and VP2 cannot be excluded.     

Phylogenetic analysis of alphaviruses in the Venezuelan equine encephalitis complex and identification of the source of epizootic viruses.

We studied the evolution of alphaviruses in the Venezuelan equine encephalitis (VEE) complex using phylogenetic analysis of RNA nucleotide sequences from limited portions of the nsP4, E1, and 3' untranslated genome regions of representative strains. The VEE complex constituted a monophyletic group of viruses (descended from a common ancestor); some serologic VEE varieties such as subtype III formed monophyletic groups while subtype I did not. Subtype II Everglades and variety ID enzootic viruses formed a monophyletic group which also included all epizootic variety IAB and IC VEE isolates. Everglades virus diverged from this ID lineage (colonized North America) ca. 100-150 years ago, followed by divergence of variety IAB and IC epizootic viruses. Variety IAB viruses probably emerged from the variety ID lineage once during the early part of this century, while variety IC viruses evolved at least two times. These results identify the source of epizootic VEE viruses as the variety ID enzootic virus lineage which occurs in northern South America and Panama. Even if variety IAB and IC viruses are extinct, recent, multiple emergences of epizootic viruses from an enzootic lineage suggests that other epizootic VEE viruses may evolve again in the future. The close genetic relationship of subtype II Everglades virus to the variety ID lineage also implies the potential for emergence of equine-virulent VEE viruses in Florida.     

Evolution of pigeon Newcastle disease virus strains

Twenty-seven Newcastle disease virus isolates obtained during the years 1998 and 1999 from racing pigeons were shown to be antigenically indistinguishable from the pigeon paramyxovirus type 1 (PPMV-1) viruses isolated in the years 1983 and 1984. Partial sequencing of 240 base pairs of the F gene demonstrated at least 94.7% identity at the nucleotide level between isolates from 1983 and 1984, and more recent viruses isolated in 1998 and 1999. Most of the nucleotide changes observed were silent mutations as only six amino acid changes were observed. Three amino acid substitutions were observed in the F2/F1 cleavage site. The sequence of the F2/F1 cleavage site of all isolates was typical for pathogenic paramyxovirus 1 viruses. Amino acids at the F2/F1 cleavage site changed from 112 GRQKRF 117 to 112 RRQKRF 117 , 112 RRKKRF 117 or 112 RRRKRF 117 . The motif 112 RRQKRF 117 was present in the majority of the isolates but the intracerebral pathogenicity indexes of PPMV-1 isolates having this motif was highly variable but largely lower (mean, 0.69) than that reported for PPMV-1 viruses isolated in the years 1983 and 1984 (mean, 1.44).     

Clinical and genetic characterization of severe influenza B-associated diseases during an outbreak in Taiwan.

BACKGROUND: Mismatches between circulating and vaccine strains of influenza virus had been observed in Taiwan. A comprehensive clinical and genetic analysis of influenza B viruses-associated important diseases was lacking. OBJECTIVES: Clinical and phylogenetic analysis of influenza B viruses during an outbreak in Taiwan. STUDY DESIGNS: Clinical manifestations of hospitalized, culture-confirmed patients were analyzed from July 2004 to June 2005. Partial genome sequence analysis of hemagglutinin (HA), neuraminidase (NA), and nonstructural (NS) genes were performed in 54 influenza B isolates during the study period, and nine srandomly chosen isolates during 2000 and 2003. RESULTS: Three specific diseases were found in these patients, including 13 of encephalitis/encephalopathy, 28 of influenza-associated myositis (IAM), and one of acute respiratory distress syndrome (ARDS). Three phylogenetic groups were identified, including reassortant strains-group 1 (Victoria lineage of HA, Yamagata lineage of NA, clade A of NS), group 2 (Yamagata lineage of HA, Yamagata lineage of NA, clade A of NS), and group 3 (Yamagata lineage of HA, Yamagata lineage of NA, clade B of NS). CONCLUSIONS: Severe influenza B-associated disease in children was not rare and might be fatal. We offered the evidence of co-circulation of the two HA lineages in the same outbreak.     

Phylogenetic analysis of avian infectious bronchitis virus strains isolated in Japan

Summary To define the origin and evolution of recent avian infectious bronchitis virus (IBV) in Japan, a genetic analysis was performed. By phylogenetic analysis based on the S1 gene including the sequence of the hypervariable regions, IBV isolates in Japan were classified into five genetic groups, which included two already-known groups (Mass and Gray). Among them, three major genetic groups were associated with the recent outbreaks of IB in Japan. One group is indigenous to Japan and could not be placed within the known existing groups in other countries. The remaining two groups, which have emerged recently, are related to isolates in China and Taiwan.     

Genetic diversity in the coat protein coding region of eighty-six sugarcane mosaic virus isolates from eight countries,particularly from Cameroon and Congo

Summary.  Fifty-eight sugarcane virus isolates were obtained from leaves showing mosaic symptoms, and collected in Cameroon (26 isolates), Congo (20 isolates), Egypt (1 isolate), South Africa (3 isolates) and the U.S.A. (8 isolates). All these isolates belonged to Sugarcane mosaic virus (SCMV) based on the amplification product obtained by RT-PCR with SCMV-specific primers. The amplicons (0.9 kb) from the coat protein (CP) coding region were cloned, sequenced and compared to each other as well as to the sequences (GenBank accessions) of 16 SCMV isolates from sugarcane (Australia, South Africa and U.S.A.) and 12 SCMV isolates from maize (Australia, Germany and China). Maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the SCE or sugarcane group that included all isolates from sugarcane and the MZ or maize group that contained all isolates from maize. The 86 virus isolates were distributed in 13 minor phylogenetic groups, four (I–IV) restricted to maize and nine (V–XIII) to sugarcane. A strong correlation was observed between the sugarcane groups and the geographical origin of the SCMV isolates. Each SCMV type strain from sugarcane (A, B, D, E and SC) was distributed in a different phylogenetic group or subgroup. The 26 isolates from Cameroon constituted a relatively homogenous group (group V) whereas the 20 isolates from Congo belonged to two other groups (VI and VII). All the isolates from Cameroon and Congo were different from the SCMV type strains and other strains or isolates studied so far. It appears, therefore, that the population of SCMV from sugarcane in Africa contains virus genotypes that have not yet been described. Received December 10, 2001; accepted July 24, 2002     

Genetic diversity of the coat protein of olive latent virus 1 isolates

The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very few nucleotide sequence differences were observed among the isolates, olive isolates exhibited lower diversity (0.012). In addition, at position 158 (157 in C7 and G1S and 159 in G6) of the deduced aa sequences, an alanine residue was found to be conserved among the olive isolates. In citrus and tulip isolates, a threonine residue was present at position 158, whereas a valine was present at this same position in tomato isolates. Phylogenetic analysis indicated that OLV-1 isolates clustered in five groups according to original host. However, G6, originally recovered from olive but repeatedly inoculated and maintained in N. benthamiana plants for 8 years in our laboratory, was separated from other isolates. This may be attributable to adaptation to the experimental host over time. There was no correlation of phylogenetic grouping of isolates based on geographical location or year of collection. Strong negative selection may have contributed to the low diversity among the OLV-1 CP isolates.     

Clonal associations among Staphylococcus aureus isolates from various sites of infection

A molecular epidemiological analysis was undertaken to identify lineages of Staphylococcus aureus that may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405 S. aureus clinical isolates collected from various infection types and geographic locations was performed. Five distinct S. aureus lineages (SALs 1, 2, 4, 5, and 6) were identified, which accounted for 19.01, 9.14, 22.72, 10.12, and 4.69% of isolates, respectively. In addition, 85 lineages which occurred with frequencies of <2.5% were identified and were termed "sporadic." The most prevalent lineage was methicillin-resistant S. aureus (SAL 4). The second most prevalent lineage, SAL 1, was also isolated at a high frequency from the anterior nares of healthy volunteers, suggesting that its prevalence among clinical isolates may be a consequence of high carriage rates in humans. Gene-specific PCR was carried out to detect genes for a number of staphylococcal virulence traits. tst and cna were found to be significantly associated with prevalent lineages compared to sporadic lineages. When specific infection sites were examined, SAL 4 was significantly associated with respiratory tract infection, while SAL 2 was enriched among blood isolates. SAL 1 and SAL 5 were clonally related to SALs shown by others to be widespread in the clinical isolate population. We conclude from this study that at least five phylogenetic lineages of S. aureus are highly prevalent and widely distributed among clinical isolates. The traits that confer on these lineages a propensity to infect may suggest novel approaches to antistaphylococcal therapy.     

A new genotype of the Sacbrood virus in honeybee Apis mellifera

The sacbrood virus (SBV) causes the death of honeybee larvae. Until recently, three SBV genotypes were known: European, Asian, and South African [7, 9]. Serological assay, sequencing, and phylogenetic analysis of the SBV RNA polymerase gene fragment have revealed two genotypes of SBV circulating among honeybee Apis mellifera in the European region of the Russian Federation (RF); one of them forms an independent, not previously described genetic lineage (genotype 4), which occupies an intermediate position on the phylogenetic tree between the Asian and the South African virus genotypes. The strain 202/8 isolated in 2006 in Moscow oblast from honeybees previously imported from Uzbekistan and two variants (Tr5 and 6/A3) of the laboratory strain isolated in Kaluga oblast in 1986 belong to this group of viruses. The homology between them is 94.2%, while that with representatives of other groups does not exceed 80.6?C83.5%. Another group of SBVs comprise European genotype viruses circulating in Stavropol krai, Adygea, and Moscow oblast (isolates 207/4 and 212/3). The homology between them is 99%. The two SBV genotypes found in the RF were differentiated by PCR and RID (radial immunodiffusion test). As for RID, both genotypes react with antiserum no. 6(1), but only the fourth genotype reacts with antiserum no. 58.