Complete sequence and variability of a new subgroup B nepovirus infecting potato in central Peru
The complete bipartite genome (RNA1 and RNA2) of a new nepovirus infecting potato was obtained using small RNA sequencing and assembly complemented by Sanger sequencing. Each RNA encodes a single polyprotein, flanked by 5’ and 3’ untranslate regions (UTR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs which are characteristic of viruses in the genus Nepovirus. Sequence comparisons using the Pro-Pol region and the coat protein, including phylogenetic analysis of these regions, showed closest relationships with nepoviruses. The data obtained support the taxonomical status of this new virus (putative named Potato virus B, PVB) as a member of the genus Nepovirus, subgroup B.
The genus Nepovirus (order Picornavirales, family Secoviridae, sub-family Comovirinae) of plant viruses has been divided into three subgroups (A, B, C) based on the length and packaging of RNA2, serological properties, cleavage site specificity of the proteinase, and phylogenetic analysis of the coat protein sequence . There are few nepoviruses infecting potato. In the subgroup A, there are reports of a calico strain of Tobacco ring spot virus (TRSV)  and Potato black ring spot virus (PBRSV) ; however, a recent report  confirmed that the calico strain of TRSV, the only strain of TRSV that was reported to infect potato , is actually a strain of PBRSV. Accordingly, no reports exist of TRSV infecting potato. In the subgroup C, the only virus reported infecting potato is Potato virus U (PVU) , but there is no sequence information available yet that can confirm its classification in subgroup C. In subgroup B, Tomato black ring virus (TBRV) has been reported to infect potato, but only in Europe. Infection of TBRV in potatoes is normally only sporadic, and the virus is considered of only minor importance in this crop . In this study, the complete genome of a new nepovirus from subgroup B infecting potato in Peru was determined from native potato plants showing calico-like symptoms and subsequently found to be relatively common in such plants in the central Peruvian highlands. This virus was tentatively named Potato virus B (PVB). Whereas the name PVB has been used before for a potato virus, the virus concerned was soon shown to be a strain of PVX [7, 8].
Primers and linkers used in this study and the corresponding virus region amplified
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A5’pp CTG TAG GCA CCA TCA AT/ddC/
ATC GTrA rGrGrC rArCrC rUrGrA rArA
Primers, that amplify the polymerase region and were used to confirm the genome obtained, were also used to detect this virus in leaves of additional potato plants with calico symptoms collected from Pasco and Junín (departments in Peru) (Figure S2). From 151 samples from Pasco, 28 were positive (19%); however, out of 67 samples evaluated from Junín, only two were positive (3%) (Table S2). The amplified 1,977 bp RdRp fragment of some positive samples were also sequenced to evaluate the PVB variability. In the phylogenetic tree produced with these sequences, two groups could be distinguished (86.8% average nt identity between the two groups and >98.7% or 95.7% identity within the two groups respectively), but there seemed to be little geographic relationship (Fig. S3)
The sequence similarity found, the organization genome and the phylogenetic analysis indicated that PVB corresponds to a new virus species belonging to the genus Nepovirus within subgroup B, according to . Many nepoviruses are transmitted by soil-inhabiting nematodes belonging to three closely related genera Xiphinema, Longidorus, or Paralongidorus in the order Dorylaimida, family Londigoridae . Longidorus is so far the only nematode genus reported to transmit subgroup B nepoviruses, whereas the genus Xiphinema and Paralongidorus were reported to transmit subgroup A and C nepoviruses. Genus Longidorus is considered a quarantine species by the National Agriculture Health Service (SENASA, its acronym Spanish) . However Jones et al.  reported preliminary experiments with Longidorus spp., which had been collected in Peru (Cesar Fribourg, personal communication) and Ciancio et al [15, 16] also reported the presence of longidorid nematodes in Peru. It would be a priority to confirm what the vector of PVB is and confirm the presence of Longidorus spp. in Peru. The phylogenetic tree using the symptomatic plants sampled from Pasco and Huancayo show that variability is present in this virus, similar to GFLV as determined by Oliver et al.  where RdRp also was used as a source to analyze virus variability. On the other hand, whereas PVB was always found associated with calico like symptoms (all positive samples had calico symptoms), not all sampled plants with these symptoms tested positive for the virus. Thus it remains unclear to what extent PVB contributes to the observed symptoms. Future studies into this aspect, including tentative yield impacts should be an additional priority.
We gratefully acknowledge PhD Dina Gutierrez, Biol. Rocío Silvestre, Carlos Chuquillanqui and Maria Scurrah for critical review of the manuscript and Eng. Ricardo Orrego for sample collection.
Compliance with ethical standards
This study was funded by the CGIAR research program on Roots, Tubers and Bananas (RTB). All authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.
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