Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.Kassava on trooppisten alueiden merkittävimpiä ruokaturvakasveja. Kassavan ruskoviirutauti on levinnyt 2000-luvulla Intian valtameren rannikkoalueilta sisämaan ja ylänköjen kassavaviljelmille Itä-Afrikassa. Tauti pilaa ravinnoksi käytettävät varastojuuret aiheuttaen niissä kuivamätää. Tässä tutkimuksessa havaittiin, että tauti liittyy kahden toisilleen lähisukuisen viruksen tartuntaan. Taudin aiheuttajina toimivien ipomovirusten genomien havaittiin olevan rakenteeltaan poikkeuksellisia muihin Potyviridae-ryhmään kuuluviin viruksiin verrattuna. Lisäksi virukset sisältävät todennäköisesti kasvisolusta kaapatun geenin. Sen toiminta saattaa liittyä viruksen tarpeeseen suojata genominsa hajotukselta kasveissa, jotka kärsivät esimerkiksi kuumuuden aiheuttamasta stressistä. Toinen viruksista havaittiin kassavalle lähisukuisessa, villinä kasvavassa lajissa, joka sekin on tuotu Afrikkaan Etelä-Amerikasta. Ruskoviirutautia aiheuttavien virusten diagnostiikkaa varten kehitettiin PCR-testi, joka tunnistaa molemmat virukset samanaikaisesti, mutta pystyy myös erottelemaan ne. Testimenetelmän avulla virusten levinneisyyttä pystyttiin kartoittamaan aiempaa laajemmin. Molemmat virukset ja niiden sekainfektiot kassavassa havaittiin yleisiksi niin Ugandan kuin Tansaniankin viljelmillä. Tulokset tuottivat uutta tietoa virusten evoluutiosta. Tulokset edistävät myös merkittävästi kassavan ruskoviirutaudin leviämisen estämiseen tarvittavaa kasvintarkastustoimintaa, sillä toinen viruksista oli aiemmin tuntematon eikä kummallekaan virukselle ole ollut helppokäyttöistä testausmenetelmää

Endemism and reemergence potential of the ipomovirus Sweet potato mild mottle virus (family Potyviridae) in Eastern Africa: half a century of mystery

Viruses have the ability to frequently colonize new hosts and ecological niches because of their inherently high genetic and evolutionary plasticity. However, a virus may emerge and remain of no or less economic importance until changes in viral and/or environmental factors dictate its epidemiological status. An example is sweet potato mild mottle virus (SPMMV), which was first reported in the 1970s on sweetpotatoes in eastern Africa, has remained endemic in the region and poorly understood, yet accounting for 60-95% losses especially in mixed infections. Unlike other sweetpotato viruses which have a global incidence, SPMMV has never been confirmed outside eastern Africa. This implicates the region as its center of origin, but does not fully account for SPMMV’s exclusive geographic delimitation to eastern Africa. Despite its importance, several mysteries and research gaps surround SPMMV, which decelerate efforts for effective virus disease management in sweetpotato. The aim of this review is to articulate research gaps, propose pivotal scientific directions and stimulate knowledge generation for better management of virus diseases in sweetpotato. Vector-mediated transmission of SPMMV remains enigmatic. Here we postulate testable hypotheses to explain SPMMV transmission. Comparisons between SPMMV and cassava brown streak ipomoviruses demonstrate epidemiological “hallmarks” for monitoring SPMMV. Evolutionary forces on SPMMV coupled with the virus’ broad host range imply a ‘silent build up’ of better fit variants in a changing climate, and this could explode into a worse disease conundrum. These information gaps need urgent filling to ease future management of virus disease emergences in sweetpotato

Pathogenic seedborne viruses are rare but Phaseolus vulgaris endornaviruses are common in bean varieties grown in Nicaragua and Tanzania

Common bean (Phaseolus vulgaris) is an annual grain legume that was domesticated in Mesoamerica (Central America) and the Andes. It is currently grown widely also on other continents including Africa. We surveyed seedborne viruses in new common bean varieties introduced to Nicaragua (Central America) and in landraces and improved varieties grown in Tanzania (eastern Africa). Bean seeds, harvested from Nicaragua and Tanzania, were grown in insect-controlled greenhouse or screenhouse, respectively, to obtain leaf material for virus testing. Equal amounts of total RNA from different samples were pooled (30-36 samples per pool), and small RNAs were deep-sequenced (Illumina). Assembly of the reads (21-24 nt) to contiguous sequences and searches for homologous viral sequences in data-bases revealed Phaseolus vulgaris endornavirus 1 (PvEV-1) and PvEV-2 in the bean varieties in Nicaragua and Tanzania. These viruses are not known to cause symptoms in common bean and are considered non-pathogenic. The small-RNA reads from each pool of samples were mapped to the previously characterized complete PvEV-1 and PvEV-2 sequences (genome lengths ca. 14 kb and 15 kb, respectively). Coverage of the viral genomes was 87.9-99.9%, depending on the pool. Coverage per nucleotide ranged from 5 to 471, confirming virus identification. PvEV-1 and PvEV-2 are known to occur in Phaseolus spp. in Central America, but there is little previous information about their occurrence in Nicaragua, and no information about occurrence in Africa. Aside from Cowpea mild mosaic virus detected in bean plants grown from been seeds harvested from one region in Tanzania, no other pathogenic seedborne viruses were detected. The low incidence of infections caused by pathogenic viruses transmitted via bean seeds may be attributable to new, virus-resistant CB varieties released by breeding programs in Nicaragua and Tanzania.Peer reviewe

Endemism and Reemergence Potential of the Ipomovirus Sweet Potato Mild Mottle Virus (Family Potyviridae) in Eastern Africa: Half a Century of Mystery

Viruses have the ability to frequently colonize new hosts and ecological niches because of their inherently high genetic and evolutionary plasticity. However, a virus may emerge and remain of no or less economic importance until changes in viral or environmental factors dictate its epidemiological status. An example is sweet potato mild mottle virus (SPMMV), which was first reported in the 1970s on sweetpotato in eastern Africa. SPMMV has remained endemic in the region and poorly understood, yet accounting for 60 to 95% of losses, especially in mixed infections. Unlike other sweetpotato viruses which have global incidences, SPMMV has never been confirmed outside eastern Africa. This implicates the region as its center of origin but does not fully account for SPMMV's exclusive geographic delimitation to eastern Africa. Despite its importance, several mysteries and research gaps surround SPMMV, which decelerate efforts for effective virus disease management in sweetpotato. The aim of this review is to articulate research gaps, propose pivotal scientific directions, and stimulate knowledge generation for better management of virus diseases in sweetpotato. Vector-mediated transmission of SPMMV remains enigmatic. Here, we postulate testable hypotheses to explain SPMMV transmission. Comparisons between SPMMV and cassava brown streak ipomoviruses demonstrate epidemiological “hallmarks” for monitoring SPMMV. Evolutionary forces on SPMMV coupled with the virus' broad host range imply a “silent build up” of more fit variants in a changing climate, and this could explode into a worse disease conundrum. These information gaps need urgent filling to ease future management of virus disease emergences in sweetpotato. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Next-generation sequencing-based detection of common bean viruses in wild plants from tanzania and their mechanical transmission to common bean plants

Publisher Copyright: Copyright © 2021 The Author(s).Viral diseases are a major threat for common bean production. According to recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Virus management requires an understanding of how viruses survive from one season to the next. During this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs together with conventional reverse-transcription PCRs (RT-PCRs) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. The RT-PCR analysis of all collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated during this study are crucial for the development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.Peer reviewe

Comprehensive survey of common bean viruses in Tanzania using next generation and Sanger sequencing techniques

<p>Common bean (<em>Phaseolus vulgaris</em> L.) is an important legume crop in Tanzania and elsewhere in the tropics and subtropics. We employed a next-generation sequencing technique to detect viruses in common bean plant samples collected from five agricultural research zones in the country. The aim was to target and sequence virus-derived small RNAs. To achieve this, total RNA was isolated from dry leaf samples using the CTAB method. The CTAB buffer contained 2% CTAB, 100 mM Tris–HCl, 20 mM EDTA, 2.5 M NaCl, freshly prepared 1% sodium sulfite, 2% PVP and 2.5% 2-mercaptoethanol in nuclease-free water. Total RNA was sent to Fasteris in Switzerland where the small RNA was purified by electrophoresis in an acrylamide gel. The small RNA library was prepared using the Illumina TrueSeq small RNA  sample preparation kit (Illumina Inc., San Diego, CA, USA). Viruses were detected using VirusDetect software (v.1.6 and v.1.7) (available at http://bioinfo.bti.cornell.edu/cgi-bin/virusdetect/index.cgi) and supercomputer at CSC.fi. Viruses detected belonged to at least 11 genera.</p

Symptoms observed in common bean plants in La Compañia, Nicaragua.

<p>(a), Stunting of the plant, malformation and blistering of leaves. (b), Mild epinasty and vein reversion. (c), Green-yellow chlorosis. (d), Green-yellow mosaic.</p

Conserved domains in the polyprotein encoded by PvEV-1 and PvEV-2 from Nicaragua.

<p>Numbers indicate the residues defining the conserved domains. Hel-1, helicase; CPS, putative capsular polysaccharide synthase; UGT, UDP-glycosyltransferase; RdRp, RNA-dependant RNA polymerase; and MTR, methyltransferase.</p

Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.

<p>Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.</p

Identification of PvEV-2 in the sample pool HXH8 from the Southern Highland zone of Tanzania based on small-RNA deep sequencing.

<p>(a), Viral contigs (red bars) mapped to the sequence of PvEV-2-Okada (AB719398) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref025" target="_blank">25</a>] using VirusDetect [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref051" target="_blank">51</a>]. Each nucleotide in the contigs was covered by siRNA reads at least 5 times. (b) The 21- to 24-nt reads mapped to the sequence of PvEV-2. The <i>x</i> axis and the scale below the figure depict the viral genome and nucleotide positions, respectively. The <i>y</i> axis indicates the number of siRNA reads derived from the coding strand (blue bars above the <i>x</i> axis) and complementary strand (red bars below the <i>x</i> axis).</p