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Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1?C22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1?C22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.

Necrotic disorder of freesia (Freesia refracta hyb., family Iridaceae) was first described in The Netherlands before 1970. In following years, the disorder was widely reported in other European countries including Northern Italy (Vaira & Milne 2008). Very recently, the same necrotic disease was also detected in Virginia, United States (Vaira et al. 2009) and New Zealand (Pearson et al. 2009). Presence of the Ophiovirus Freesia sneak virus (FreSV) has been widely associated with the necrotic disease in Europe, the United States and New Zealand but some uncertainty remains (Brandvagt et al. 2008). The freesia leaf necrosis complex has been shown to be soilborne, transmitted by Olpidium brassicae, but other infectious agents (e.g. the Varicosavirus tentatively named Freesia leaf necrosis virus) could be naturally transmitted by the same vector, thickening the plot about the disease causal agent. In 2002 Dr. Morikawa was able to differentiate a Mild mottle mosaic- and Streaking disease in tulips: the first was linked to the Tulip mild mottle mosaic virus (TMMMV, genus Ophiovirus) infection and the second to an unknown Tulip streaking associated agent, with unstable infectivity (Morikawa 2002). He also showed that development of both diseases was suppressed by fungicidal treatments of the soil, suggesting soil-borne transmission for both agents. In 2005 the same team preliminarily described Tulip streak virus, a novel virus strictly associated with tulip streak disease. This new viral agent, morphologically resembling Tenuiviruses or supercoiled Ophioviruses, has a coat protein of c. 30kDa sharing some homology with Phlebovirus (Bunyaviridae) (Morikawa et al. 2005). Freesia plants showing necrotic disease were collected in Liguria (Northern Italy) during the 2011-2012 growing season. About 40-50% of the plants were affected by typical leaf necrosis and were assayed for FreSV infection by electron microscopy (TEM) and RT-PCR tests. FreSV was detected using FreSV-specific primers in all six samples obtained by pooling several Freesia plants from different areas of the parcel, and elongated supercoiled virus particles were detected in freesia crude sap by TEM. Freesia tissue was purified using Ophiovirus procedure and the product obtained was used for total RNA extraction and for TEM visualization. Several differently shaped virus particles were visualized in the mixture and total RNA has been used for sequence-independent amplification (SIA) for the identification of RNA virus, following a published procedure (Agindotan 2010). Results of virus detection in infected freesia tissue are reported.

Human papillomavirus 16 (HPV 16) L1 self-assembles into pentamers or virus-like particles (VLPs) that are highly immunogenic. We investigated the potential for using HPV-16 L1 as a carrier of heterologous epitopes of influenza A virus: (i) M2e, ectodomain of the M2 protein, highly conserved among all influenza A isolates and (ii) M2e8, a shorter version including the N-terminal highly conserved SLLTEVET epitope, common for both M1 and M2 proteins. Structural models available for HPV-16 L1 showing that helix 4 (h4) and the coil region linking h4 and ?-sheet J are projected outwards in each of the five L1 monomers led us to choose these two positions for inserting the epitopes. A synthetic HPV-16 L1 gene optimized with human codon usage and fused to a calreticulin-endoplasmic reticulum targeting signal was used as a master gene to create four chimeric sequences: ChiM2e_h4, ChiM2e_c (M2e epitope into h4 and coil, respectively), ChiM2e8_h4, and ChiM2e8_c (M2e8 epitope into h4 and coil, respectively). All chimeric constructs transiently expressed in plants using the Cowpea mosaic virus-derived vector pEAQ-HT (Sainsbury et al., 2009) were recognized by a panel of linear and conformational-specific anti HPV-16 L1 MAbs. ChiM2e_h4 appeared to be the most highly expressed construct. This chimeric protein also strongly reacted with an anti-influenza A M2 MAb. Electron microscopy showed that plant-made chimeric proteins assembled into small VLPs of approximately 30 nm in diameter. This study further confirms the use of papillomavirus particles as carriers of heterologous epitopes and their potential as a plant-made vaccine platform. Work supported by EU FP7 Grant Agreement No. KBBE-227056 (PLAPROVA).

Human papillomavirus 16 (HPV 16) L1 self-assembles into pentamers or virus-like particles (VLPs) that are highly immunogenic. We investigated the potential for using HPV-16 L1 as a carrier of heterologous epitopes of influenza A virus: (i) M2e, ectodomain of the M2 protein, highly conserved among all influenza A isolates and (ii) M2e8, a shorter version including the N-terminal highly conserved SLLTEVET epitope, common for both M1 and M2 proteins. Structural models available for HPV-16 L1 showing that helix 4 (h4) and the coil region linking h4 and ?-sheet J are projected outwards in each of the five L1 monomers led us to choose these two positions for inserting the epitopes. A synthetic HPV-16 L1 gene optimized with human codon usage and fused to a calreticulin-endoplasmic reticulum targeting signal was used as a master gene to create four chimeric sequences: ChiM2e_h4, ChiM2e_c (M2e epitope into h4 and coil, respectively), ChiM2e8_h4, and ChiM2e8_c (M2e8 epitope into h4 and coil, respectively). All chimeric constructs transiently expressed in plants using the Cowpea mosaic virus-derived vector pEAQ-HT (Sainsbury et al., 2009) were recognized by a panel of linear and conformational-specific anti HPV-16 L1 MAbs. ChiM2e_h4 appeared to be the most highly expressed construct. This chimeric protein also strongly reacted with an anti-influenza A M2 MAb. Electron microscopy showed that plant-made chimeric proteins assembled into small VLPs of approximately 30 nm in diameter. This study further confirms the use of papillomavirus particles as carriers of heterologous epitopes and their potential as a plant-made vaccine platform. Work supported by EU FP7 Grant Agreement No. KBBE-227056 (PLAPROVA).

The capsid protein (CP) of the begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is indispensable for systemic plant infection and vector transmission and a region between aa 129-152 is critical for virion assembly and insect transmissibility. Three non-transmissible (NT) mutants, one with the double Q129P and Q134H mutation (PNHD), a second with a further D152E change (PNHE) and another with a single N130D change (QDQD) were analyzed in their relationships with the whitefly vector Bemisia tabaci and the nonvector Trialeurodes vaporariorum, using the wild-type (wt) virus as reference. Retention kinetics experiments performed on whiteflies fed on plants infected by the mutants revealed that the QDQD mutant was hardly detectable in either whitefly species at any sampling time. It produced geminate virions that appeared nongeminate following purification. While PNHD was acquired and circulated in both whitefly species as the wt virus, PNHE circulated in B. tabaci only. Immunogold labelling experiments on whiteflies that acquired the different mutants showed the presence of both PNHD and PNHE CPs in salivary glands (SGs), as for the wt virus, while no labelling was found in tissues from whiteflies that acquired QDQD. Besides, the transmission of the wt virus was significantly inhibited after prior feeding on plants infected by the PNHE mutant. A model describing the circulation of TYLCSV virions in Bemisia will be presented where both virion stability and ability to cross the SG barrier are fundamental, but interactions with molecular components inside the SGs are also critical for transmission.

The capsid protein (CP) of the geminivirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is indispensable for plant infection and vector transmission. A region between amino acids 129 and 152 is critical for virion assembly and insect transmissibility. Three nontransmissible (NT) mutants, one with a double Q129P Q134H mutation (PNHD), the second with a further D152E change (PNHE) and the third with a single N130D change (QDQD) were compared to the wild-type (wt, QNQD) virus in their relationships with the whitefly vector Bemisia tabaci and the nonvector Trialeurodes vaporariorum. Retention kinetics of NT mutants in whiteflies fed on infected plants were analyzed by quantitative dot blot hybridization. Virions of the QDQD mutant appeared nongeminate following purification and its DNA was hardly detectable in either whitefly species at any sampling time. The PNHD mutant was acquired and circulated for up to 10 days in both whitefly species, like the wt virus, while PNHE circulated in B. tabaci only. Both PNHD and PNHE CPs were immunogold labelled in B. tabaci salivary glands (SGs) like the wt virus, while no labelling was found in whitefly tissue with the QDQD mutant. Furthermore, a significant inhibition of transmission of the wt virus was observed after prior feeding of the insects on plants infected with the PNHE mutant, but not on plants infected with the other mutants. Virion stability and ability to cross the SG barrier are necessary for TYLCSV transmission, but interactions with molecular components inside SGs are critical for transmissibility.

Two viruses, Tobacco rattle virus (TRV), genus Tobravirus and Raspberry ringspot virus (RRSV), genus Nepovirus, have been isolated from plants of Japanese anemone (Anemone x hybrida = Anemone hupehensis var. japonica x A. vitifolia) growing in nurseries in southern Germany and in north western Italy. TRV was detected, in the samples checked in Italy, by one-step RT-PCR using TRV-specific primers and total RNA as template. On indicator plants, the four isolates studied induced disease characteristic of TRV infection. Two of the isolates formed typical tobravirus particles. The other two caused milder symptoms on indicators and no virus particles were seen in these plants; these isolates were assumed to be of the MN-type, lacking the gene for the coat protein. RRSV was detected in one sample tested in Germany and in one of the samples collected in Italy. This last was freed from mixed infection with TRV by using Chenopodium quinoa as differential host. The experimental host range of both German and Italian isolates was in agreement with what is known for RRSV. Both isolates were identified by electron microscopy decoration, using antisera to five RRSV isolates. In comparative tests, titres of four of these antisera were almost identical for both isolates, while the fifth antiserum, to the Italian isolate, differentiated between the two isolates. No reaction occurred with antisera to 22 other nepoviruses, four fabaviruses and five comoviruses. A major coat protein of Mr slightly over 50 kDa and two RNA species of about 8 kb (RNA1) and 4 kb (RNA2) were found for the Italian isolate.

Ranunculus white mottle virus (RWMV), genus Ophiovirus, a possibly tripartite RNA virus, has been reported in commercial Ranunculus hyb. crops since 1990, in North-West Italy, with uncertain pathogenic impact as it was always found in mixed infection with the most common viruses infecting ranunculus and with a low incidence. Cloning of RWMV isolate Rn3 produced several clones targeting RNAs 1 and 2. Two clones were used to obtain non-radioactive probes. In Northern blot experiments with total RNAs from infected plants, probe RW35 recognized RNA 2 while probe RW380 recognized RNA 1. Probe RW35 was chosen as a diagnostic tool. Tissue-print experiments were performed with indicator plants mechanically inoculated with RWMV and showed sensitive detection of the virus. Ranunculus plants, mechanically inoculated with various isolates of RWMV, were tested for infection by stem-squash, and the results were equally reliable. Recently, RWMV was also detected in field plants of anemone hybrids by Northern blotting of total RNA.

Isolates of Tomato spotted wilt virus (TSWV) severely and systemically infecting commercial pepper cultivars with resistance introgressed from Capsicum chinense PI15225 were found in Albenga (NW Italy) in July 2000. Experimentally, these resistance-breaking (RB) isolates overcame the resistance in C. chinense PI152225, but they produced infection in other hosts similarly to non-RB isolates from the same area. The RB isolates were indistinguishable from TSWV by serology and electron microscopy and they were efficiently transmitted by Frankliniella occidentalis. Such isolates were recovered on the same farm in tomato, pepper and artichoke 2 and 12 months later, suggesting natural spread from the resistant plants and survival. The RB isolates survived in experimental mixed infections with a non-RB isolate in susceptible pepper and C. chinense, but cross protection in pepper acted against them. Commercial TSWV-resistant pepper but not resistant tomato cvs from different companies were susceptible to these RB isolates after mechanical inoculation. Similar isolates were not detected among TSWV samples collected from 1993 to 2000 in the area. The management of TSWV and thrips using resistant pepper cvs is discussed.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sie, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sie, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.