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Tobacco mosaic virus in cigarettes and saliva of smokers
Journal of Clinical Virology 55 (2012) 374–376
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Tobacco mosaic virus in cigarettes and saliva of smokers Fanny Balique a,c , Philippe Colson a,b , Didier Raoult a,b,∗ a Aix-Marseille University, URMITE UM63 CNRS 7278 IRD 198 INSERM U1095, IHU Méditerranée Infection, Facultés de Médecine et de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France b Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance PubliqueHôpitaux de Marseille, 264 rue Saint-Pierre 13385, Marseille Cedex 05, France c INRA, UR407, Pathologie Végétale, 84140 Montfavet, France
a r t i c l e
i n f o
Article history: Received 3 August 2012 Accepted 6 August 2012 Keywords: Tobacco mosaic virus Tobacco Smoking Cigarettes Plant virus
a b s t r a c t Background: Tobacco mosaic virus (TMV) has been described as viable in cigarettes or cigar tobacco. It has been cultured about 50 years ago from sputa and thoracentesis fluids of cigarette smokers with a history of pulmonary disease and from lung cancerous matter. In addition, TMV RNA has been recovered recently from human stools while tobacco DNA was recovered from smokers’ bronchoalveolar lavages. Objectives: We assessed the prevalence, titers, and infectivity of TMV in tobacco cigarettes and in the saliva of smokers and non smokers. Study design: Tobacco cigarettes from 10 packs of different brands were purchased. Saliva was collected from 12 smokers and 15 non-smokers. Cigarettes and saliva samples were tested for the presence of TMV RNA using a home-made quantitative real-time RT-PCR assay. TMV RNA quantification was enabled by using dilutions of purified TMV. TMV viability was tested by inoculating leaves of Nicotiana tabacum Xanthi (NtX). Results: All 47 smoking cigarettes of six brands were TMV RNA-positive (mean titer, 9.5 log10 copies/cigarette); TMV was found viable in 53% of them. In addition, 20/44 (45%) saliva from 12 smokers compared to 0/16 saliva from 15 non-smokers tested positive for TMV RNA (mean titer, 3.8 log10 copies/ml) (p = 0.001). Conclusions: Our results indicate that the TMV genome may get access to the human body by direct exposure through smoking. Although plant viruses are considered not pathogenic for animals, these data prompt to study if TMV RNA is present and induces a modification of the transcriptional program in lung cells of cigarette smokers. © 2012 Elsevier B.V. All rights reserved.
1. Background Tobacco mosaic virus (TMV) is the first discovered virus.1 It belongs to the Tobamovirus genus and has a rod shape and a positive-sense single stranded RNA genome that is about 6400 nucleotides in length. TMV is a plant pathogen that infects plants of the Solanacae family, in particular tobacco (Nicotiana tabacum).2,3 This virus is known to be extraordinarily stable and heat resistant, and was described as viable in cigarettes or cigar tobacco.2–4 Recently, our team has reported the recovery of tobacco chloroplast DNA from the bronchoalveolar lavage of three patients with ventilator-associated pneumonia and who were smokers.5 In addition, TMV RNA has been recently recovered from the feces of
∗ Corresponding author at: Aix-Marseille Université, Unité des Rickettsies, URMITE UM63 CNRS 7278 IRD 198 INSERM U1095, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Tel.: +33 491 324 375; fax: +33 491 387 772. E-mail address: [email protected] (D. Raoult). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.08.012
non-diarrheic and diarrheic persons in two independent metagenomic studies.6,7 Notably, TMV RNA was found to be amongst the most abundant viral RNA recovered from three feces of two healthy patients.6 Interestingly, it had been also reported about 50 years ago that TMV was cultured from sputa and thoracentesis fluids of cigarette smokers with a history of pulmonary disease and from lung cancerous matter.8,9 Taken together, previous data suggest that tobacco and TMV can reach the lungs in humans. 2. Objectives We aimed to assess the prevalence, titers, and infectivity of TMV in tobacco cigarettes and in the saliva of smokers. 3. Study design We purchased 10 packs of tobacco cigarettes of different brands in tobacconists in the city of Marseille, France. Saliva was collected in sterile containers by self-sampling from 27 persons
F. Balique et al. / Journal of Clinical Virology 55 (2012) 374–376
375
Fig. 3. Tobacco mosaic virus (TMV) RNA detection by real-time reverse transcriptase-PCR in human saliva.
Fig. 1. Electron microscopy image of TMV-like viral particles in cigarette tobacco.
working at Marseille University hospitals. A total of 44 saliva samples were collected from 12 smokers before or just after smoking a cigarette and 16 saliva samples were collected from 15 non-smokers. This study has been approved by the institutional ethics committee of IFR48 (Aix-Marseille University; review no. 11-016). Cigarette tobacco was mixed with 15 ml of buffer 0.01 M Na2 HPO4 –KH2 PO4 pH 7.2. RNA extraction was performed from 200 l of the solution, or directly from saliva samples, using the EZ1 virus Mini kit v2.0 on the BioRobot EZ1 workstation (Qiagen, Courtaboeuf, France). TMV RNA testing was performed by a home-made quantitative real-time reverse transcriptase-PCR assay targeting the replicase gene of the TMV genome with SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen Life Technologies, Carlsbad, CA, USA) with primers TMV Rep Fwd: 5 -CCGTGATCAATCCGATCTCA (nucleotide (nt) 3079–3098 in reference to GenBank accession no. AB369276) and TMV Rep Rev: 5 -ATGAAGTGCAAGGCGAGACA (nt 3187–3206) and probe TMV Rep Pr: 5 -FAM-AAACCCTTGCATGGCAAGATCCTGATAMRA (nt 3099–3123). TMV RNA quantification was enabled by using dilutions of purified TMV produced by inoculating mechanically plants of Nicotiana tabacum Samsun with TMV strain tmj. Identification of TMV RNA in cigarette tobacco was performed by population sequencing of a fragment of the capsid gene using SuperScript One-Step RT-PCR System (Invitrogen
Life Technologies) then Big Dye Terminator cycle sequencing kit version 1.1 on the ABI Prism 3130 genetic analyzer (Applied Biosystems, Branchburg, NJ, USA) with primers TMV-Spe-Fwd: 5 -GGGCCGACCCAATAGAGTTA (nt 5764–5783) and TMV-SpeRev: 5 -AGTAGACGACGCRACGGTG (nt 6053–6071). Electron microscopy study of TMV in cigarette tobacco was performed by mixing tobacco in 1 ml of PBS 1× then observing the supernatant as described previously.10 TMV viability in cigarette tobacco was tested by mixing 200 l of the tobacco solution in buffer with carborundum then inoculating mechanically leaves of Nicotiana tabacum Xanthi (NtX), an hypersensitive host plant for TMV, and local necrotic lesions were searched for after one week. 4. Results All 47 purchased smoking cigarettes of six brands were TMV RNA-positive. The mean TMV RNA titer was 9.5 log10 copies/cigarette (range, 7.2–10.8). RNA sequences recovered from these tobacco cigarettes by population sequencing were identified as fragment of the TMV coat protein encoding gene with 95–100% nucleotide identity. TMV-like viral particles were observed by electron microscopy in a cigarette tobacco sample (Fig. 1). The viability of TMV in cigarettes, as indicated by the development of local lesions on leaves of Nicotiana tabacum Xanthi after inoculation, was shown for 18 (53%) of 34 processed cigarette tobacco samples (Fig. 2). In addition, 20 of 44 saliva samples (45%) from 12 smokers, compared to none of 16 saliva from 15 non-smokers tested positive for TMV RNA (p = 0.001) (Fig. 3). Overall, eight of the 12 (67%) smokers had positive saliva
Fig. 2. Leaves of Nicotiana tabacum Xanthi non-inoculated (left) or inoculated with processed TMV RNA-positive cigarette tobacco (middle and right (detail of lesions)).
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F. Balique et al. / Journal of Clinical Virology 55 (2012) 374–376
and the mean TMV RNA titer was 3.8 log10 copies/ml (range, 2.3–4.9). Although differences were not statistically significant, the proportion of positive samples was higher (59% versus 32%) and mean TMV RNA titer was greater (3.9 log10 versus 2.8 log10 ) in saliva samples collected after compared to before smoking.
Competing interest
5. Discussion
This study has been approved by the institutional ethics committee of IFR48 (Aix-Marseille University; review no. 11-016).
Our study indicates that TMV is abundant and often infectious in currently commercialized tobacco cigarettes. As over 15 billion cigarettes are smoked worldwide every day (http://www.who.int/tobacco/en/atlas8.pdf), this means that humans are massively exposed to TMV RNA. Furthermore, the present work shows that TMV RNA can be recovered in saliva from cigarette smokers, albeit at low titers. Therefore, the viral genome can get access to the human body by direct exposure through smoking. Based on the current dogma, plant viruses are not pathogenic for animals.10 Nevertheless, several findings indicate that some plant pathogenic viruses can replicate in their insect vectors.11,12 Moreover, tomato spotted wilt virus, a plant bunyavirus that can infect arthropods has been shown to directly alter their feeding behavior.12 Besides, cowpea mosaic virus, a plant comovirus in the picornavirus superfamily, has been described to bind to and enter mammalian cells and persist for several days in a wide panel of body tissues in mice, including the lung and the liver, after oral or intravenous inoculation.13 In addition, it has been described that human cell lines (HeLa and diploid fibroblasts) could be turned permissive to Tomato spotted wilt virus, and that TMV RNA can be transfected and translated into various vertebrate animal cells, including in human epithelial carcinoma (HeLa) cells.14 Furthermore, the presence in human stools of pepper mild mottle virus, another tobamovirus, has been associated with specific immune responses and clinical symptoms including fever, abdominal pains and pruritus.10 Taken together, aforementioned data and our results incite in studying if TMV RNA is present and induces a modification of the transcriptional program in lung cells of cigarette smokers. Funding None for all authors.
None for all authors. Ethical approval
References 1. Ivanovski D. Über die Mosaikkrankheit der Tabakspflanze. St Petersb Acad Imp Sci Bull 1892;35:67–70. 2. Wahyuni WS, Hanapi M, Hartana I. The presence of tobacco mosaic virus in the compost extract of cigar tobacco debris. Hayati J Biosci 2008;15: 118–22. 3. Wetter C. Tobacco mosaic virus and para-tobacco mosaic virus in cigarettes. Naturwissenschaften 1975;62:533. 4. Bothwell PW. Lung cancer tobacco mosaic virus. Lancet 1960;275: 657–8. 5. Bousbia S, Papazian L, La Scola B, Raoult D. Detection of plant DNA in the bronchoalveolar lavage of patients with ventilator-associated pneumonia. PLoS ONE 2010;5:e11298. 6. Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, et al. RNA viral community in human faeces: prevalence of plant pathogenic viruses. PLoS Biol 2006; 4:e3. 7. Nakamura S, Yang CS, Sakon N, Ueda M, Tougan T, Yamashita A, et al. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach. PLoS ONE 2009;4: e4219. 8. Le Clair RA. Recovery of culturable tobacco mosaic virus from sputum and thoracentesis fluids obtained from cigarette smokers with a history of pulmonary disease. Am Rev Respir Dis 1967;95:510–1. 9. Katsilambros L. Tobacco mosaic virus lung cancer. Lancet 1960;2:934. 10. Colson P, Richet H, Desnues C, Balique F, Moal V, Grob JJ, et al. Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. PLoS ONE 2010;5:e10041. 11. Medeiros RB, Resende Rde O, de Avila AC. The plant virus Tomato Spotted Wilt Tospovirus activates the immune system of its main insect vector, Frankliniella occidentalis. J Virol 2004;78(10):4976–82. 12. Stafford CA, Walker GP, Ullman DE. Infection with a plant virus modifies vector feeding behavior. Proc Natl Acad Sci U S A 2011;108:9350–5. 13. Koudelka KJ, Destito G, Plummer EM, Trauger SA, Siuzdak G, Manchester M, et al. Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin. PLoS Pathog 2009; 5:e1000417. 14. Li L, Wang L, Xiao R, Zhu G, Li Y, Liu C, et al. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells. Biosci Rep 2012;32:171–86.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Tobacco mosaic virus in cigarettes and saliva of smokers Fanny Balique a,c , Philippe Colson a,b , Didier Raoult a,b,∗ a Aix-Marseille University, URMITE UM63 CNRS 7278 IRD 198 INSERM U1095, IHU Méditerranée Infection, Facultés de Médecine et de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France b Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance PubliqueHôpitaux de Marseille, 264 rue Saint-Pierre 13385, Marseille Cedex 05, France c INRA, UR407, Pathologie Végétale, 84140 Montfavet, France
a r t i c l e
i n f o
Article history: Received 3 August 2012 Accepted 6 August 2012 Keywords: Tobacco mosaic virus Tobacco Smoking Cigarettes Plant virus
a b s t r a c t Background: Tobacco mosaic virus (TMV) has been described as viable in cigarettes or cigar tobacco. It has been cultured about 50 years ago from sputa and thoracentesis fluids of cigarette smokers with a history of pulmonary disease and from lung cancerous matter. In addition, TMV RNA has been recovered recently from human stools while tobacco DNA was recovered from smokers’ bronchoalveolar lavages. Objectives: We assessed the prevalence, titers, and infectivity of TMV in tobacco cigarettes and in the saliva of smokers and non smokers. Study design: Tobacco cigarettes from 10 packs of different brands were purchased. Saliva was collected from 12 smokers and 15 non-smokers. Cigarettes and saliva samples were tested for the presence of TMV RNA using a home-made quantitative real-time RT-PCR assay. TMV RNA quantification was enabled by using dilutions of purified TMV. TMV viability was tested by inoculating leaves of Nicotiana tabacum Xanthi (NtX). Results: All 47 smoking cigarettes of six brands were TMV RNA-positive (mean titer, 9.5 log10 copies/cigarette); TMV was found viable in 53% of them. In addition, 20/44 (45%) saliva from 12 smokers compared to 0/16 saliva from 15 non-smokers tested positive for TMV RNA (mean titer, 3.8 log10 copies/ml) (p = 0.001). Conclusions: Our results indicate that the TMV genome may get access to the human body by direct exposure through smoking. Although plant viruses are considered not pathogenic for animals, these data prompt to study if TMV RNA is present and induces a modification of the transcriptional program in lung cells of cigarette smokers. © 2012 Elsevier B.V. All rights reserved.
1. Background Tobacco mosaic virus (TMV) is the first discovered virus.1 It belongs to the Tobamovirus genus and has a rod shape and a positive-sense single stranded RNA genome that is about 6400 nucleotides in length. TMV is a plant pathogen that infects plants of the Solanacae family, in particular tobacco (Nicotiana tabacum).2,3 This virus is known to be extraordinarily stable and heat resistant, and was described as viable in cigarettes or cigar tobacco.2–4 Recently, our team has reported the recovery of tobacco chloroplast DNA from the bronchoalveolar lavage of three patients with ventilator-associated pneumonia and who were smokers.5 In addition, TMV RNA has been recently recovered from the feces of
∗ Corresponding author at: Aix-Marseille Université, Unité des Rickettsies, URMITE UM63 CNRS 7278 IRD 198 INSERM U1095, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Tel.: +33 491 324 375; fax: +33 491 387 772. E-mail address: [email protected] (D. Raoult). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.08.012
non-diarrheic and diarrheic persons in two independent metagenomic studies.6,7 Notably, TMV RNA was found to be amongst the most abundant viral RNA recovered from three feces of two healthy patients.6 Interestingly, it had been also reported about 50 years ago that TMV was cultured from sputa and thoracentesis fluids of cigarette smokers with a history of pulmonary disease and from lung cancerous matter.8,9 Taken together, previous data suggest that tobacco and TMV can reach the lungs in humans. 2. Objectives We aimed to assess the prevalence, titers, and infectivity of TMV in tobacco cigarettes and in the saliva of smokers. 3. Study design We purchased 10 packs of tobacco cigarettes of different brands in tobacconists in the city of Marseille, France. Saliva was collected in sterile containers by self-sampling from 27 persons
F. Balique et al. / Journal of Clinical Virology 55 (2012) 374–376
375
Fig. 3. Tobacco mosaic virus (TMV) RNA detection by real-time reverse transcriptase-PCR in human saliva.
Fig. 1. Electron microscopy image of TMV-like viral particles in cigarette tobacco.
working at Marseille University hospitals. A total of 44 saliva samples were collected from 12 smokers before or just after smoking a cigarette and 16 saliva samples were collected from 15 non-smokers. This study has been approved by the institutional ethics committee of IFR48 (Aix-Marseille University; review no. 11-016). Cigarette tobacco was mixed with 15 ml of buffer 0.01 M Na2 HPO4 –KH2 PO4 pH 7.2. RNA extraction was performed from 200 l of the solution, or directly from saliva samples, using the EZ1 virus Mini kit v2.0 on the BioRobot EZ1 workstation (Qiagen, Courtaboeuf, France). TMV RNA testing was performed by a home-made quantitative real-time reverse transcriptase-PCR assay targeting the replicase gene of the TMV genome with SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen Life Technologies, Carlsbad, CA, USA) with primers TMV Rep Fwd: 5 -CCGTGATCAATCCGATCTCA (nucleotide (nt) 3079–3098 in reference to GenBank accession no. AB369276) and TMV Rep Rev: 5 -ATGAAGTGCAAGGCGAGACA (nt 3187–3206) and probe TMV Rep Pr: 5 -FAM-AAACCCTTGCATGGCAAGATCCTGATAMRA (nt 3099–3123). TMV RNA quantification was enabled by using dilutions of purified TMV produced by inoculating mechanically plants of Nicotiana tabacum Samsun with TMV strain tmj. Identification of TMV RNA in cigarette tobacco was performed by population sequencing of a fragment of the capsid gene using SuperScript One-Step RT-PCR System (Invitrogen
Life Technologies) then Big Dye Terminator cycle sequencing kit version 1.1 on the ABI Prism 3130 genetic analyzer (Applied Biosystems, Branchburg, NJ, USA) with primers TMV-Spe-Fwd: 5 -GGGCCGACCCAATAGAGTTA (nt 5764–5783) and TMV-SpeRev: 5 -AGTAGACGACGCRACGGTG (nt 6053–6071). Electron microscopy study of TMV in cigarette tobacco was performed by mixing tobacco in 1 ml of PBS 1× then observing the supernatant as described previously.10 TMV viability in cigarette tobacco was tested by mixing 200 l of the tobacco solution in buffer with carborundum then inoculating mechanically leaves of Nicotiana tabacum Xanthi (NtX), an hypersensitive host plant for TMV, and local necrotic lesions were searched for after one week. 4. Results All 47 purchased smoking cigarettes of six brands were TMV RNA-positive. The mean TMV RNA titer was 9.5 log10 copies/cigarette (range, 7.2–10.8). RNA sequences recovered from these tobacco cigarettes by population sequencing were identified as fragment of the TMV coat protein encoding gene with 95–100% nucleotide identity. TMV-like viral particles were observed by electron microscopy in a cigarette tobacco sample (Fig. 1). The viability of TMV in cigarettes, as indicated by the development of local lesions on leaves of Nicotiana tabacum Xanthi after inoculation, was shown for 18 (53%) of 34 processed cigarette tobacco samples (Fig. 2). In addition, 20 of 44 saliva samples (45%) from 12 smokers, compared to none of 16 saliva from 15 non-smokers tested positive for TMV RNA (p = 0.001) (Fig. 3). Overall, eight of the 12 (67%) smokers had positive saliva
Fig. 2. Leaves of Nicotiana tabacum Xanthi non-inoculated (left) or inoculated with processed TMV RNA-positive cigarette tobacco (middle and right (detail of lesions)).
376
F. Balique et al. / Journal of Clinical Virology 55 (2012) 374–376
and the mean TMV RNA titer was 3.8 log10 copies/ml (range, 2.3–4.9). Although differences were not statistically significant, the proportion of positive samples was higher (59% versus 32%) and mean TMV RNA titer was greater (3.9 log10 versus 2.8 log10 ) in saliva samples collected after compared to before smoking.
Competing interest
5. Discussion
This study has been approved by the institutional ethics committee of IFR48 (Aix-Marseille University; review no. 11-016).
Our study indicates that TMV is abundant and often infectious in currently commercialized tobacco cigarettes. As over 15 billion cigarettes are smoked worldwide every day (http://www.who.int/tobacco/en/atlas8.pdf), this means that humans are massively exposed to TMV RNA. Furthermore, the present work shows that TMV RNA can be recovered in saliva from cigarette smokers, albeit at low titers. Therefore, the viral genome can get access to the human body by direct exposure through smoking. Based on the current dogma, plant viruses are not pathogenic for animals.10 Nevertheless, several findings indicate that some plant pathogenic viruses can replicate in their insect vectors.11,12 Moreover, tomato spotted wilt virus, a plant bunyavirus that can infect arthropods has been shown to directly alter their feeding behavior.12 Besides, cowpea mosaic virus, a plant comovirus in the picornavirus superfamily, has been described to bind to and enter mammalian cells and persist for several days in a wide panel of body tissues in mice, including the lung and the liver, after oral or intravenous inoculation.13 In addition, it has been described that human cell lines (HeLa and diploid fibroblasts) could be turned permissive to Tomato spotted wilt virus, and that TMV RNA can be transfected and translated into various vertebrate animal cells, including in human epithelial carcinoma (HeLa) cells.14 Furthermore, the presence in human stools of pepper mild mottle virus, another tobamovirus, has been associated with specific immune responses and clinical symptoms including fever, abdominal pains and pruritus.10 Taken together, aforementioned data and our results incite in studying if TMV RNA is present and induces a modification of the transcriptional program in lung cells of cigarette smokers. Funding None for all authors.
None for all authors. Ethical approval
References 1. Ivanovski D. Über die Mosaikkrankheit der Tabakspflanze. St Petersb Acad Imp Sci Bull 1892;35:67–70. 2. Wahyuni WS, Hanapi M, Hartana I. The presence of tobacco mosaic virus in the compost extract of cigar tobacco debris. Hayati J Biosci 2008;15: 118–22. 3. Wetter C. Tobacco mosaic virus and para-tobacco mosaic virus in cigarettes. Naturwissenschaften 1975;62:533. 4. Bothwell PW. Lung cancer tobacco mosaic virus. Lancet 1960;275: 657–8. 5. Bousbia S, Papazian L, La Scola B, Raoult D. Detection of plant DNA in the bronchoalveolar lavage of patients with ventilator-associated pneumonia. PLoS ONE 2010;5:e11298. 6. Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, et al. RNA viral community in human faeces: prevalence of plant pathogenic viruses. PLoS Biol 2006; 4:e3. 7. Nakamura S, Yang CS, Sakon N, Ueda M, Tougan T, Yamashita A, et al. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach. PLoS ONE 2009;4: e4219. 8. Le Clair RA. Recovery of culturable tobacco mosaic virus from sputum and thoracentesis fluids obtained from cigarette smokers with a history of pulmonary disease. Am Rev Respir Dis 1967;95:510–1. 9. Katsilambros L. Tobacco mosaic virus lung cancer. Lancet 1960;2:934. 10. Colson P, Richet H, Desnues C, Balique F, Moal V, Grob JJ, et al. Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. PLoS ONE 2010;5:e10041. 11. Medeiros RB, Resende Rde O, de Avila AC. The plant virus Tomato Spotted Wilt Tospovirus activates the immune system of its main insect vector, Frankliniella occidentalis. J Virol 2004;78(10):4976–82. 12. Stafford CA, Walker GP, Ullman DE. Infection with a plant virus modifies vector feeding behavior. Proc Natl Acad Sci U S A 2011;108:9350–5. 13. Koudelka KJ, Destito G, Plummer EM, Trauger SA, Siuzdak G, Manchester M, et al. Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin. PLoS Pathog 2009; 5:e1000417. 14. Li L, Wang L, Xiao R, Zhu G, Li Y, Liu C, et al. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells. Biosci Rep 2012;32:171–86.