Identification and molecular characterization of Taro bacilliform virus and Taro bacilliform CH virus from East Africa.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) are important root crops cultivated mainly by small-scale farmers in sub-Saharan Africa and the South Pacific. Viruses are known to be one of the most important constraints to production, with infections resulting in severe yield reduction. In 2014 and 2015, surveys were conducted in Ethiopia, Kenya, Tanzania and Uganda to determine the identity of viruses infecting taro in East Africa. Screening of 392 samples collected from the region using degenerate badnavirus primers revealed an incidence of 58-74% among the four countries surveyed, with sequence analysis identifying both Taro bacilliform virus (TaBV) and Taro bacilliform CH virus (TaBCHV). TaBCHV was identified from all four countries while TaBV was identified in all except Ethiopia. Full-length sequences from representative TaBV and TaBCHV isolates showed that the genome organization of TaBV isolates from East Africa was consistent with previous reports while TaBCHV isolates from East Africa were found to encode only four ORFs, distinct from a previous report from China. Phylogenetic analysis showed that all East African TaBV isolates form a single subgroup within known TaBV isolates, while TaBCHV isolates form at least two distinct subgroups. To the authors' knowledge, this is the first report describing the occurrence and genome organization of TaBV and TaBCHV isolates from East Africa and the first full-length sequence of the two viruses from tannia.

Development of a Novel Rolling-Circle Amplification Technique to Detect Banana streak virus that also Discriminates Between Integrated and Episomal Virus Sequences.

Banana plants are hosts to a large number of Banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV, and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.

Identification and sequence analysis of potyviruses infecting crops in Vietnam.

Fifty-two virus isolates from 13 distinct potyvirus species infecting crops in Vietnam were identified and the 3' region of each genome was sequenced. The viruses were: bean common mosaic virus (BCMV), potato virus Y (PVY), sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV), chilli veinal mottle virus (ChiVMV), zucchini yellow mosaic virus (ZYMV), leek yellow stripe virus (LYMV), shallot yellow stripe virus (SYSV), onion yellow dwarf virus (OYDV), turnip mosaic virus (TuMV), dasheen mosaic virus (DsMV), sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, tentatively named chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses of the entire CP-coding region revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV.

Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses.

Two pairs of degenerate primers were designed from sequences within the potyviral CI (CIFor/CIRev) and HC-Pro-coding regions (HPFo/HPRev), and these were shown to be highly specific to members of the genus Potyvirus. Using the CIFor and CIRev primers, three novel potyviruses infecting crop and weed species from Vietnam were detected, namely telosma mosaic virus (TelMV) infecting telosma (Telosma cordata, Asclepiadaceae), peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii, Araceae) and wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum, Solanaceae). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these viruses and a banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to chilli veinal mottle virus (ChiVMV) and pepper veinal mottle virus (PVMV), while PeLMV, TelMV and BBrMV were related to different extents to members of the bean common mosaic virus (BCMV) subgroup.

Mapping the 5' ends of banana bunchy top virus gene transcripts.

Banana bunchy top virus (BBTV), a multi-component circular ssDNA virus, replicates via a dsDNA intermediate that also serves as a template for virion sense transcription. Seven virus-derived transcripts have been previously identified and analysed in BBTV-infected bananas by northern analysis and 3' rapid amplification of cDNA ends (3' RACE). In this study, we have used RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-RACE) to complete the mapping of the BBTV gene transcripts and have now fully mapped the transcribed regions of each BBTV component and effectively defined the upstream regulatory region.

Predicted declines in sickle allele frequency in Jamaica using empirical data.

The high frequency of the sickle allele in some parts of Africa is understood to be a consequence of high malarial endemicity. One corollary of this is that the sickle allele frequency should be declining in populations of African ancestry that are no longer exposed to malaria. We have previously shown that there has been no change in sickle allele frequency in malaria-free Jamaica between two large-scale neonatal screening exercises conducted in 1973-1981 and 1995-2003. To evaluate the determinants of, and derive expected values for, sickle allele frequency in Jamaica, local empirical data were used to estimate the parameters of deterministic models of allele frequency decline. We found that although model predictions were broadly consistent with observed values in the 1973-1981 cohort, the predicted change in allele frequency between the two cohorts was larger than the observed, nonsignificant, reduction. Close agreement between predicted and observed values was only achieved by simulating a recent, marked increase in HbSS fitness. Thus, the "unexpected" persistence of the sickle allele in Jamaica may reflect the fact that the actual fitness among SS individuals is higher than that previously realized. If true, our models suggest that without substantial changes in current screening and counseling practice, there will be little "natural" reduction in sickle allele frequency for several hundred years. Better estimates of relative fitness will be helpful in refining these predictions and may aid in assigning health care priorities in Jamaica and the African Diaspora.

The frequency of the sickle allele in Jamaica has not declined over the last 22 years.

The 'malaria hypothesis' predicts that the frequency of the sickle allele, which is high in malaria-endemic African populations, should decline with each generation in populations of African descent living in areas where malaria is no longer endemic. In order to determine whether this has been the case in Jamaica, we compared haemoglobin electrophoresis results from two hospital-based screening programmes separated by more than 20 years (i.e. approximately one generation). The first comprised 100,000 neonates screened between 1973 and 1981, the second, 104,183 neonates screened between 1995 and 2003. The difference in frequency of the sickle allele was small (5.47% in the first cohort and 5.38% in the second screening cohort) and not significant (Z = 1.23, P = 0.22). The same was true of the sickle trait frequency (10.05% in the first cohort and 9.85% in the second, Z = 1.45, P = 0.15). These differences were smaller than predicted under simple deterministic models based on the malaria hypothesis, and suggest that these models may not capture important determinants of allele and trait frequency decline (or persistence) in contemporary populations. Refining the expectations for allele and trait frequency change for Jamaica and other similar populations is an area for future study.

HLA class-I and class-II allele frequencies and two-locus haplotypes in Melanesians of Vanuatu and New Caledonia.

HLA class-I and class-II allele frequencies and two-locus haplotypes were examined in 367 unrelated Melanesians living on the islands of Vanuatu and New Caledonia. Diversity at all HLA class-I and class-II loci was relatively limited. In class-I loci, three HLA-A allelic groups (HLA-A*24, HLA-A*34 and HLA-A*11), seven HLA-B alleles or allelic groups (HLA-B*1506, HLA-B*5602, HLA-B*13, HLA-B*5601, HLA-B*4001, HLA-B*4002 and HLA-B*2704) and four HLA-C alleles or allelic groups (HLA-Cw*04, HLA-Cw*01, HLA-Cw*0702 and HLA-Cw*15) constituted more than 90% of the alleles observed. In the class-II loci, four HLA-DRB1 alleles (HLA-DRB1*15, HLA-DRB1*11, HLA-DRB1*04 and HLA-DRB1*16), three HLA-DRB3-5 alleles (HLA-DRB3*02, HLA-DRB4*01 and HLA-DRB5*01/02) and five HLA-DQB1 alleles (HLA-DQB1*0301, HLA-DQB1*04, HLA-DQB1*05, HLA-DQB1*0601 and HLA-DQB1*0602) constituted over 93, 97 and 98% of the alleles observed, respectively. Homozygosity showed significant departures from expected levels for neutrality based on allele frequency (i.e. excess diversity) at the HLA-B, HLA-Cw, HLA-DQB1 and HLA-DRB3/5 loci on some islands. The locus with the strongest departure from neutrality was HLA-DQB1, homozygosity being significantly lower than expected on all islands except New Caledonia. No consistent pattern was demonstrated for any HLA locus in relation to malaria endemicity.

Molecular analysis of Fiji disease virus genome segments 5, 6, 8 and 10.

The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150 nt, 2831 nt, 1959 nt and 1819 nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115 kDa, 97 kDa, 69 kDa and 63.0 kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.

Sugarcane bacilliform virus encapsidates genome concatamers and does not appear to integrate into the Saccharum officinarum genome.

Sugarcane bacilliform virus (SCBV) DNA molecules larger than the complete genome length of 7.6 kbp were detected in infected plants and in virions. We have confirmed that these high molecular weight nucleic acids were open circular DNA and viral in origin. Due to their open circular conformation, accurate size determination of the DNA molecules was not possible using conventional electrophoresis. Using field inversion gel electrophoresis (FIGE), however, the DNA appeared to increase in genome size increments, with sizes ranging from 1 to 4 genomes (31 kbp) detected. The DNA was packaged into virions, which may explain the observation of purified virions with lengths corresponding to one, two or three times the modal length of 130 nm. The DNA products were possibly concatamers formed during replication as a result of a terminal overlap on the sense strand, and were shown to be overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands. Southern analysis indicated that SCBV sequences are not integrated into the sugarcane genome and that the high molecular weight DNA observed in the sugarcane accessions analysed represents SCBV concatamers.

Genetic diversity of Campylobacter jejuni isolates from farm animals and the farm environment.

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.

Sequence diversity of South Pacific isolates of Taro bacilliform virus and the development of a PCR-based diagnostic test.

We have analysed the sequence variability in the putative reverse transcriptase (RT)/ribonuclease H (RNaseH) and the C-terminal coat protein (CP)-coding regions from Taro bacilliform virus (TaBV) isolates collected throughout the Pacific Islands. When the RT/RNaseH-coding region of 22 TaBV isolates from Fiji, French Polynesia, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands and Vanuatu was examined, maximum variability at the nucleotide and amino acid level was 22.9% and 13.6%, respectively. Within the CP-coding region of 13 TaBV isolates from Fiji, New Caledonia, PNG, Samoa and the Solomon Islands, maximum variability at the nucleotide and amino acid level was 30.7% and 19.5%, respectively. Phylogenetic analysis showed that TaBV isolates from the Solomon Islands showed greatest variability while those from New Caledonia and PNG showed least variability. Based on the sequences of the TaBV RT/RNaseH-coding region, we have developed a PCR-based diagnostic test that specifically detects all known TaBV isolates. Preliminary indexing has revealed that TaBV is widespread throughout Pacific Island countries. A sequence showing approximately 50% nucleotide identity to TaBV in the RT/RNaseH-coding region was also detected in all taro samples tested. The possibility that this may represent either an integrated sequence or the genome of an additional badnavirus infecting taro is discussed.

A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants.

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

Genomic characterisation of taro bacilliform virus.

Taro bacilliform virus (TaBV) has been classified as a putative badnavirus based on its non-enveloped, bacilliform virion morphology and transmission by mealybugs. The complete nucleotide sequence of a Papua New Guinea isolate of TaBV has now been determined and comprises 7458 bp. The genome contains four open reading frames (ORFs) on the plus-strand that potentially encode proteins of 17, 16, 214 and 13 kDa. The size and organisation of TaBV ORFs 1-3 is similar to that of most other badnaviruses, while the location of ORF 4 is similar to that of ORF 4 and ORF X of the atypical badnaviruses Citrus yellow mosaic virus and Cacao swollen shoot virus, respectively. The putative amino acid sequence of TaBV ORF 3 contained motifs that are conserved amongst badnavirus proteins including aspartic protease, reverse transcriptase (RT) and ribonuclease H (RNase H). The highly conserved putative plant tRNA(met)-binding site was also present in the 935 bp intergenic region of TaBV. Phylogenetic analysis using the amino acid sequence of ORF 3 showed that TaBV branched most closely to Dioscorea bacilliform virus. These results confirm that TaBV is a pararetrovirus of the genus Badnavirus, family Caulimoviridae.

Sequence analysis of an Australian isolate of sugarcane bacilliform badnavirus.

The genome of an Australian isolate of Sugarcane bacilliform virus (SCBV-IM) was cloned, sequenced and analysed. The genome consisted of 7687 nucleotides and contained three open reading frames which were similar in size and organisation to those of other badnaviruses. SCBV-IM was found to be most similar to the SCBV-Morocco isolate with amino acid sequence similarity of 91.4 %, 83.8 % and 85.3 % in the ORF I, II and III coding regions, respectively. Phylogenetic analysis of the SCBV-IM ORF III deduced amino acid sequence showed that SCBV isolates were more closely related to each other than to other badnaviruses. Amplification of SCBV sequences from three different sugarcane varieties revealed considerable variability in the viral populations, both within single infected plants as well as between infected plants, suggesting that the SCBV isolates sequenced to date may not be representative of the range of virus variability.

Additional rep-encoding DNAs associated with banana bunchy top virus.

Banana bunchy top nanovirus (BBTV) has a multicomponent circular single-stranded DNA (cssDNA) genome consisting of at least six components. We have cloned, sequenced and analysed two additional cssDNA components, designated BBTV-S1 and S2, associated with a Taiwanese BBTV isolate. The sequences of BBTV-S1 and S2 comprised 1109 and 1095 nucleotides (nt), respectively, and like BBTV DNA-1, potentially encoded replication initiation proteins (Reps). However, the genome organisation of BBTV-S1 and S2 differed from that of BBTV DNA-1 in that (i) the stem sequence of the CR-SL was not conserved, (ii) the internal gene was absent and (iii) the probable TATA boxes were located 5' of the stem-loop. Further, sequence and phylogenetic analysis of the Rep genes indicated that BBTV DNA-S1 and S2 were distinct from BBTV DNA-1. When different geographical isolates of BBTV were tested for the presence of BBTV-S1/S2, these components were detected in various isolates from Vietnam, Taiwan, the Philippines, Tonga and Samoa but were not detected in isolates from Australia, Egypt, Fiji, and India. Based on these results, BBTV-S1 and S2 do not appear to be integral components of the BBTV genome and represent additional Rep-encoding DNAs associated with BBTV.

Sequence variability in the coat protein gene of two groups of banana bunchy top isolates.

Complete nucleotide sequences of the coat protein gene (DNA-3) of banana bunchy top virus (BBTV) were obtained from five geographical isolates by PCR. Analysis of these sequences revealed two distinct groups of BBTV isolates with those from the Philippines, Taiwan and Vietnam forming the Asian group while the South Pacific/African group consisted of isolates from Australia, Burundi and Fiji. At the nucleotide level, the sequences of DNA-3 were more similar between isolates from the same group (maximum 5.86%) than between members of the two different groups (maximum 13.05%). At the amino acid level, the BBTV coat protein remained highly conserved, with a maximum of < 3% sequence variation between all isolates in this study. There was a significantly higher degree of divergence between the Asian isolates, which may indicate that BBTV has been present in this region for an extended period of time or that there have been multiple introductions of BBTV into bananas. The high level of conservation in the BBTV coat protein suggests that any of the DNA-3 sequences presented in this study would probably be equally effective as transgene in attempts to generate transgenic banana plants with resistance to both groups of BBTV isolates.