Archive for the ‘biotechnology’ Category

eBook on “Influenza Virus: Introduction to a Killer”

17 June, 2015

For some five years now, I have been simultaneously writing two ebooks on viruses. The one – originally part of a longer effort not yet finished – is “A Short History of the Discovery of Viruses” which is also advertised on Virology News; the other is a labour of love on influenza.

Labour of love for me because I got more into it the more I read, and because Russell Kightley’s images were so amazing.

Both were written using Apple’s iBooks Author app; both are designed to be read by Apple’s iBooks app on iPad, iPhone or Mac.

So here it is:

Influenza Virus: Introduction to a Killer

Enjoy. Buy!

Influenza_1-6-15_sample_iba

 

The double life of a geminivirus: Bean yellow dwarf virus

29 May, 2015

Every now and then I solicit contributions for this site – but this one came without coercion, or even prompting!  I thank Romana for being so enthusiastic B-)

Romana Yanez

Biopharming Research Unit, UCT

All material copyright Romana Yanez and UCT

***

I want to tell the story of a geminivirus called Bean yellow dwarf virus that has two very distinct “lives”: one as a crop pest, infecting bean plants in South Africa and the other as a powerful molecular tool as a viral vector for recombinant protein expression in plants. As if each one of the “heads” of the twinned capsids had a life of its own. The dark side and the bright side. The yin and yang…

Introduction

Geminiviruses are small, single-stranded, circular DNA plant viruses, so called because each particle is composed of two partially assembled icosahedra joined to form a twinned capsid [1], [2]. They infect plants and are carried by insect vectors such as leafhoppers and whiteflies [2]. They are divided into seven genera: Mastrevirus, Bogomovirus, Topocuvirus, Curtovirus, Becurtovirus, Eragrovirus and Turncurtovirus; according to their genomic organization, the hosts they infect, the insect vectors by which they are transmitted and by genome-wide pairwise sequence identities [3].

Geminiviruses belonging to the genus Mastrevirus are all monopartite viruses with genome sizes between 2.5 and 3.0 kb. They have as vectors different species of leafhoppers. They infect mostly monocotyledonous plants: Maize streak virus (MSV) causes devastating crop losses in African countries, Wheat dwarf virus (WDV), but also infect dicotyledonous plants: Bean yellow dwarf virus (BeYDV), Tobacco yellow dwarf virus (TYDV) and Chickpea chlorosis virus (CpCV) [2], [4], [5].

In 1997 the production of French beans (Phaseolus vulgaris cv. Bonus) was severely reduced  in South Africa, mainly in the Northern Province and Mpumalanga District [4]. Plants presented symptoms similar to a TYDV infection, which at the time was the only mastrevirus described to infect dicotyledonous plants. These symptoms included stunted growth, brittle and leathery leaves, and leaf curling. Investigating the aetiology of this disease were Liu and co-workers. They determined it was a geminiviral infection by identifying virus-like particles (VLPs) with the characteristic twinned morphology. They then sequenced DNA samples of the virus and found it to be most closely related to TYDV, both, in nucleotide sequence (65% identity) and in genomic organization. It was similar enough to be placed under the genus Mastrevirus but distinct enough to be considered a different virus. They Then called it Bean yellow dwarf virus – BeYDV [4].

In 2007 a mild strain of BeYDV (BeYDV-m) was described by Halley-Stott et al. also isolated from P. vulgaris (cv. Top Crop). It was phylogenetically similar to BeYDV having 97% nucleotide sequence identity, but presented sufficient phenotypic differences to be a different BeYDV strain. It contained 81 nucleotide differences compared to the BeYDV type, most of which (63 changes) were found in regions of the genome that directly influenced its replication. Thus, BeYDV-m produced typical symptoms that were less severe and temporally delayed when compared to BeYDV type. The authors also suggested that P. vulgaris is not the BeYDV natural host since it is a non-indigenous plant in South Africa. Furthermore, since both strains of BeYDV were isolated in the same region, it was also suggested that their natural host may show very mild or no symptoms upon infection [6]. Subsequently BeYDV-m was renamed as Chickpea chlorotic dwarf virus (CpCDV) [3]. [However, we like BeYDV, so we’re going to keep called it that – Ed]

Molecular Characteristics and Life Cycle of BeYDV

The genome of BeYDV (Figure 1) is 2,561 nucleotides long with an organization similar to that of other mastreviruses and that replicates by rolling circle mechanism [4], [7]. Its genome is bidirectional, consisting of virion-sense open reading frames (ORFs) V1 and V2, and complementary-sense ORFs C1, C2, C3 and C4. Of these, only C3 and C4 are non-functional and non-conserved between the mastreviruses; although they are also present in TYDV [4], [8]. Within the complementary sense ORFs C1 and C2 an intron is found which is also conserved in other mastreviruses. Virion sense and complementary sense ORFs are separated by a long intergenic region (LIR) and a small intergenic region (SIR) [4]. Liu and co-workers described the functions of each component of the BeYDV genome by mutational analysis.

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

Figure 1. Genomic organization of Bean yellow dwarf virus. CP, capsid protein. LIR, long intergenic region. MP, movement protein. Rep, replication associated protein. SIR, short intergenic region. [9]

The LIR contains a bidirectional promoter to which host factors can bind and a stem-loop structure within the origin of replication (ori) which is required for initiation of rolling circle replication. A binding site and nicking site for the replication associated protein (Rep) are also found in this region. The SIR in turn contains a primer binding region for initiation of complementary strand synthesis as well as transcription termination elements [8]. These are the only two cis-acting elements required for BeYDV replication [8], [10].

The V1 ORF encodes for the movement protein (MP) which is associated with plasmodesmata and is important for systemic spread of the virus. It was found to be a symptom inducer as transgenic plants expressing V1 developed wild type-like infection symptoms. The putative pathogen associated molecular pattern recognized by the host plant may be within the first 17 N-terminus amino acids as plants infected with a mutant  developed wild type-like symptoms as well [8]. 

The V2 ORF encodes for the capsid protein (CP) which is important for viral movement as well and therefore for systemic infection. Thus, intracellular movement or trafficking of the viral DNA may require encapsidation. This was suggested since V2 mutants did not infect plants systemically and also, a basic domain on the N-terminal of the CP was identified which putatively binds to DNA or is involved in nuclear localization [8], [11].

From the genome of BeYDV, the complementary sense ORFs C1 and C2 are the most interesting for me. These encode two regulatory proteins involved in the replication of the virus: Rep and RepA. Their expression is regulated by alternate splicing, where spliced C1 and C2 (C1C2) mRNA is translated into Rep and unspliced C1 mRNA is translated into RepA [8]. 

Rep is responsible for initiating rolling circle replication by nicking the stem-loop structure at the ori, and for releasing nascent virion sense single stranded DNA and later ligating it to form circular ssDNA molecules [8], [12]. Rep is the only protein required for BeYDV replication, but in the presence of RepA the replication is more efficient [8], [10], [11].

RepA is a multi-regulatory protein only found in mastreviruses [2]. Even though both Rep and RepA, have a retinoblastoma related protein (RBR)-binding motif, LeuXCysXGlu, in BeYDV only RepA is able to bind to RBR proteins [10]. In mammalian cells, the retinoblastoma protein is a tumor repressor that binds to and inactivates the transcription factor E2F. By binding to RBR proteins, RepA is thought to disrupt this interaction and force the plant cell cycle into the S-phase – where DNA is replicated just before cell division. RepA is thus acting like other viruses’ oncogenic proteins, such as the human papillomavirus E7 protein and the adenovirus E1A protein. Thus, keeping conditions favorable for enhanced viral replication and proliferation [10], [11]. This could be seen when Hefferon and Dugdale mutated the RBR binding-motif of Rep and RepA to LeuXCysXGln. Only the RepA mutant showed significantly decreased replication. While the Rep mutant showed wild type-like replication [11].

Having in mind what I just described, one can picture the life cycle of BeYDV as follows:

A leafhopper (which has not been identified yet) carrying the virus infects a host plant – this will be a dicotyledonous plant such as P. vulgaris, from which it was originally isolated. The virus releases its ssDNA genome into the cytoplasm. The ssDNA enters the nucleus where host’s replication machinery synthesizes the complementary strand from the primer located in the SIR region, generating a replicative double stranded circular DNA intermediate. At this point the dsDNA serves as template for gene expression, from which Rep and RepA are expressed. RepA transactivates virion-sense gene expression and interferes with plant cell’s life cycle to produce S-phase conditions. Rep nicks the stem-loop structure located at the ori and binds to the 5’ end of the nicked strand. The 3’ end acts as a primer for the synthesis of a new virion-sense strand displacing the previous virion-sense strand. When this new strand is complete, the ori is regenerated and Rep nicks it again. Subsequent release and recircularization of the nascent virion-sense strand is also mediated by Rep. The process continues on the new circular ssDNA molecules as well. Only later, when the amount of CP is high enough, ssDNA molecules are encapsidated. The CP and MP then mediate systemic spread of the viral genome [2], [8]–[12]. When another leafhopper visits the infected plant, the virus is transferred to other plants and all starts again (Figure 2).

gv fig 2

Figure 2. The life cycle of BeYDV. Black circle, BeYDV ssDNA with the stem-loop structure. Black and green circle, BeYDV dsDNA replicative intermediate. Orange spheres, plant host’s replication machinery. Yellow spheres, Rep protein. Black line, nascent ssDNA during rolling circle replication. Purple sphere, RepA. Green sphere, plant retinoblastoma-related protein. Red spheres, BeYDV movement protein. Geminal structures, BeYDV capsid proteins. Modified from [13], [14].

Liu et al. (1997) and Halley-Stott et al. (2007) showed that BeYDV is able to infect other dicotyledonous plants besides P. vulgaris, such as: Nicotiana tabacum, N. benthamiana, Datura stramonium and Arabidopsis thaliana [4], [6]. It has also been isolated from chickpeas in Pakistan [15]. It was noted by Liu and co-workers that the intron of BeYDV (and TYDV) is not as AU-rich as intron sequences present in dicotyledonous plants, which suggested that these viruses had evolved from monocotyledonous-infecting ancestors [8]. Other thing that suggests that BeYDV (and TYDV) evolved from monocotyledonous-infecting mastreviruses is that they encode for two variants of the Rep protein while other geminiviruses infecting dicotyledonous plants encode for only one Rep protein from a continuous ORF [11].

BeYDV as a Powerful Molecular Tool

I have talked about the relatively dark side of BeYDV as a crop pest and plant cell cycle manipulator. Now I would like to introduce you to the other face of this geminivirus.

The importance of recombinant proteins in pharmaceutical, medical and research fields makes them highly demanded, which in turn requires the use efficient production systems [16], [17]. Plants provide a cheaper, faster, more efficient and highly scalable platform for the production of proteins compared to other methods [18], [19]. Vectors based on DNA viruses can be used to express complex proteins without the limitations and complexity faced by RNA viruses such as the need to use more than one virus construct, size constraint imposed on the insert and genomic instability [2], [20]. BeYDV and other geminiviruses have small and simple DNA genomes which can be rapidly amplified to very high copy numbers using mainly host factors and that can be easily manipulated. These features make them attractive viruses for the design of plant vectors for the expression of recombinant proteins [21]. BeYDV has been extensively explored as a molecular tool for the expression of mainly pharmaceutically relevant proteins, such as vaccines, antibodies and enzymes [9], [21]. And recently it has also been used as a means to deliver reagents into plant cells to genetically engineer them [22].

Hefferon and co-workers were one of the first to design a vector derived from BeYDV. They expressed a synthetic version of Staphylococcus enterotoxin B (SEB) in tobacco NT-1 cells. The synthetic SEB sequence was placed under the control of a Cauliflower mosaic virus (CaMV) 35S constitutive promoter and flanked by the cis acting BeYDV LIR and SIR. The Rep encoding gene was provided in trans from a separate construct and also constitutively expressed from the CaMV 35S promoter. Constructs were co-delivered into NT-1 cells by bombardment [11]. They obtained expression levels of ≈0.025 mg SEB / kg of NT-1 cells. They showed that expression of SEB could be enhanced by 20 times by supplying Rep in trans compared to when no Rep was supplied. Overall they showed that BeYDV-based replicon systems promised enhancement of recombinant protein expression in plants [23].

In a more deconstructed approach, Mor et al. (2003) designed a replicon system similar to that of Hefferon and Dugdale (2003) in which the BeYDV MP and CP genes were replaced by the gene of interest (GUS), controlled by CaMV 35S promoter and flanked by the LIR and SIR sequences [24]. Since the CP can sequestrate viral ssDNA, preventing dsDNA to be formed [8], by removing the CP from the viral vector, expression levels can be increased. Removing non-essential features of the virus also gives more room for larger inserts and channels energy and building blocks that would be used to synthesize these proteins into expressing the recombinant protein [20]. Mor et al. obtained expression levels 40 times higher when supplying Rep as well as RepA than when no Rep/RepA was supplied. Showing that RepA also enhances expression levels, probably by making the cell environment more favorable for replication [24]. 

Regnard et al. (2010) designed a replicon vector, pRIC, based on the mild strain of BeYDV that contained the Rep/RepA coding regions in cis rather than in trans. This allowed the vector to autonomously replicate and thus generate high levels of gene copy number and in turn enhanced protein expression. They used N. benthamiana plants and Agrobacterium tumefaciens-mediated gene delivery. They obtained higher expression levels than previously described of three unrelated proteins: enhanced GFP, Human Papillomavirus type 16 major CP, L1, and a HIV-1 p24 antigen. Yields were higher when using the replicative vector than when compared to expression from a non-replicative A. tumefaciens expression vector: 550 mg ⁄ kg fresh leaf weight (FLW) vs. 337 mg L1 ⁄ kg FLW for L1 and 3.23 mg p24 ⁄ kg FLW vs. 0.95 mg p24 ⁄ kg FLW for p24. This study showed that autonomous replication of BeYDV-based vectors dramatically increases gene expression levels [25].

Huang et al. (2009) designed a three-component replicon system that consisted of a construct derived from a deconstructed version BeYDV similar to that described by Mor et al. (2003) containing the gene of interest expression cassette, a construct encoding for the Rep/RepA under CaMV 35S promoter control and a construct expressing the posttranscriptional gene silencing suppressor protein P19. They obtained 0.34 g of Norwalk virus CP (NVCP) / kg FLW and 0.8 g of hepatitis B core antigen (HBc) / kg FLW, which were able to form VLPs. In order to simplify the replicon system, they included the Rep/RepA sequences in cis. They obtained similar expression levels when using the simplified replicon, with or without P19 supplementation as when the three-component system was used [26].  Later they designed a single vector containing multiple replicon cassettes each flanked by a LIR and a SIR. The vector also contained the Rep/RepA sequences under LIR control. Co-delivering the single-vector replicon and a P19 expression vector, they expressed the light and heavy chain of an Ebola virus-targeting monoclonal antibody (mAB), 6D8. They obtained ≈0.5 g of 6D8 mAB / kg FLW which had been assembled correctly and could bind its antigen specifically. Expression levels were comparable to those obtained by Giritch et al. (2006) [27] using two vectors based on two non-competing RNA viruses. They speculated that using this single-vector multireplicon system, even four proteins could be expressed simultaneously using two vectors or placing expression cassettes in tandem  [28].

More recently, Moon et al. (2014) were able to express Brome mosaic virus (BMV) and Cucumber mosaic virus (CMV) VLPs at 0.5 and 1.0 g / kg FLW respectively, using a BeYDV-derived single-vector replicon system. This vector included the P19 coding sequence, the gene of interest as well as the Rep/RepA coding sequences in the same backbone. In this way enhanced expression of VLPs that can be used as carriers for nano-platforms with applications in material sciences and medicine was possible with only one agroinfiltration [29].

Finally, Baltes et al. (2014) demonstrated that BeYDV-based replicon system can be also used for plant genome engineering. They were able to deliver various nucleases (TALENs and CRISP/Cas system) as well as repair templates into tobacco cells and to regenerate plantlets with the desired DNA changes within 6 weeks. This highlighted the potential of vectors derived from BeYDV and other geminiviruses to be applied in the engineering of plants for, for example,  improvement of crop characteristics, crop resistance or in fundamental biology studies [22].

In conclusion, BeYDV is a small, dicotyledonous plant-infecting mastrevirus with apparently unlimited possible molecular applications.

References

[1] W. Zhang, N. H. Olson, T. S. Baker, L. Faulkner, M. Agbandje-McKenna, M. I. Boulton, J. W. Davies, and R. McKenna, “Structure of the Maize streak virus geminate particle.,” Virology, vol. 279, pp. 471–477, 2001.

[2] E. P. Rybicki and D. P. Martin, “Virus-derived ssDNA vectors for the expression of foreign proteins in plants,” Current Topics in Microbiology and Immunology, vol. 375, pp. 19–45, 2011.

[3] A. Varsani, J. Navas-Castillo, E. Moriones, C. Hernández-Zepeda, A. Idris, J. K. Brown, F. Murilo Zerbini, and D. P. Martin, “Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus,” Archives of Virology, vol. 159, pp. 2193–2203, 2014.

[4] L. Liu, T. Van Tonder, G. Pietersen, J. W. Davies, and J. Stanley, “Molecular characterization of a subgroup I geminivirus from a legume in South Africa,” Journal of General Virology, vol. 78, pp. 2113–2117, 1997.

[5] J. Hadfield, J. E. Thomas, M. W. Schwinghamer, S. Kraberger, D. Stainton, A. Dayaram, J. N. Parry, D. Pande, D. P. Martin, and A. Varsani, “Molecular characterisation of dicot-infecting mastreviruses from Australia,” Virus Research, vol. 166, no. 1–2, pp. 13–22, 2012.

[6] R. P. Halley-Stott, F. Tanzer, D. P. Martin, and E. P. Rybicki, “The complete nucleotide sequence of a mild strain of Bean yellow dwarf virus,” Archives of Virology, vol. 152, pp. 1237–1240, 2007.

[7] K. E. Palmer and E. P. Rybicki, “The molecular biology of mastreviruses.,” Advances in virus research, vol. 50, pp. 183–234, 1998.

[8] L. Liu, J. W. Davies, and J. Stanley, “Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants,” Journal of General Virology, vol. 79, pp. 2265–2274, 1998.

[9] Q. Chen, J. He, W. Phoolcharoen, and H. S. Mason, “Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants,” Human Vaccines, vol. 7, no. 3, pp. 331–338, Mar. 2011.

[10] L. Liu, K. Saunders, C. arole L. Thomas, J. W. Davies, and J. Stanley, “Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.,” Virology, vol. 256, pp. 270–279, 1999.

[11] K. L. Hefferon and B. Dugdale, “Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication,” Journal of General Virology, vol. 84, pp. 3465–3472, 2003.

[12] C. Gutierrez, “Geminivirus DNA replication,” Cellular and Molecular Life Sciences, vol. 56. pp. 313–329, 1999.

[13] “Adult drawing grape leafhopper,” Koppert Biological Systems 9103. [Online]. Available: https://www.flickr.com/photos/koppert/2400156751/. [Accessed: 09-Feb-2015].

[14] “Phaseolus vulgaris,” Belgium, Prelude – Royal Museum for Central Africa – Tervuren. [Online]. Available: http://www.africamuseum.be/collections/external/prelude/view_plant?pi=09910. [Accessed: 09-Feb-2015].

[15] N. Nahid, I. Amin, S. Mansoor, E. P. Rybicki, E. Van Der Walt, and R. W. Briddon, “Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan,” Archives of Virology, vol. 153, pp. 1441–1451, 2008.

[16] G. Pogue and S. Holzberg, “Transient Virus Expression Systems for Recombinant Protein Expression in Dicot-and Monocotyledonous Plants,” in Plant Science, N. K. Dhal and S. C. Sahu, Eds. InTech, 2012, pp. 191–216.

[17] F. Sainsbury, P.-O. Lavoie, M.-A. D’Aoust, L.-P. Vézina, and G. P. Lomonossoff, “Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2.,” Plant biotechnology journal, vol. 6, no. 1, pp. 82–92, Jan. 2008.

[18] E. P. Rybicki, “Plant-produced vaccines: promise and reality.,” Drug Discovery Today, vol. 14, no. 1–2, pp. 16–24, Jan. 2009.

[19] V. Yusibov, S. Rabindran, U. Commandeur, R. M. Twyman, and R. Fischer, “The potential of plant virus vectors for vaccine production.,” Drugs in R&D, vol. 7, no. 4, pp. 203–17, Jan. 2006.

[20] Y. Gleba, S. Marillonnet, and V. Klimyuk, “Plant Virus Vectors: Gene Expression Systems,” Encyclopedia of Virology, vol. 4, pp. 229–237, Apr. 2008.

[21] K. L. Hefferon, “DNA Virus Vectors for Vaccine Production in Plants: Spotlight on Geminiviruses,” Vaccines, vol. 2, no. 3, pp. 642–653, Aug. 2014.

[22] N. J. Baltes, J. Gil-Humanes, T. Cermak, P. a Atkins, and D. F. Voytas, “DNA replicons for plant genome engineering.,” The Plant cell, vol. 26, no. January, pp. 151–63, 2014.

[23] K. L. Hefferon and Y. Fan, “Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system,” Vaccine, vol. 23, pp. 404–410, 2004.

[24] T. S. Mor, Y.-S. Moon, K. E. Palmer, and H. S. Mason, “Geminivirus vectors for high-level expression of foreign proteins in plant cells.,” Biotechnology and bioengineering, vol. 81, pp. 430–437, 2003.

[25] G. L. Regnard, R. P. Halley-Stott, F. L. Tanzer, I. I. Hitzeroth, and E. P. Rybicki, “High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector.,” Plant biotechnology journal, vol. 8, no. 1, pp. 38–46, Jan. 2010.

[26] Z. Huang, Q. Chen, B. Hjelm, C. Arntzen, and H. Mason, “A DNA replicon system for rapid high-level production of virus-like particles in plants.,” Biotechnology and bioengineering, vol. 103, no. 4, pp. 706–14, Jul. 2009.

[27] A. Giritch, S. Marillonnet, C. Engler, G. van Eldik, J. Botterman, V. Klimyuk, and Y. Gleba, “Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 40, pp. 14701–6, Oct. 2006.

[28] Z. Huang, W. Phoolcharoen, H. Lai, K. Piensook, G. Cardineau, L. Zeitlin, K. J. Whaley, C. J. Arntzen, H. S. Mason, and Q. Chen, “High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system.,” Biotechnology and bioengineering, vol. 106, no. 1, pp. 9–17, May 2010.

[29] K. Moon, J. Lee, S. Kang, M. Kim, H. S. Mason, J. Jeon, and H. Kim, “Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.,” Applied microbiology and biotechnology, vol. 98, pp. 8281–90, 2014.

Tracing the bird flu outbreak in North American poultry flocks

14 May, 2015

(Reuters) – The United States is facing its worst outbreak on record of avian influenza as three deadly strains have hit North American poultry flocks since December, with the spread of infection picking

Source: www.reuters.com

Useful timeline!

See on Scoop.itVirology News

Versatile scanner / camera system for lab meetings

6 May, 2015

I don’t know about you, but if you have ten or so people round a table, and you’re all trying to look at a photo of a PA gel or western blot in a lab book, it can get a little frustrating.

So, if you’re like me, and don’t know how big a 54″ TV is when you order a screen for your meeting room / office…there is a very simple, and very elegant solution. Oh, and it helps to have bought an Apple TV unit so people can wirelessly do Powerpoint presentations.

So here it is:

Simple wireless lab book scanning device

Simple wireless lab book scanning device

Basically, a simple lab retort stand and clamp, holding an iPhone that is connected to the Apple TV via the wireless network – and instant blowup of anything you point it at.   It works even better with my iPad, except it’s harder to set up the stand, because the workshop has taken six months to NOT make me my custom iPad clamp modelled on my old Czech photographic enlarger – but we won’t go there B-)

It has not escaped our notice that we’ll be able to watch the Rugby World Cup in the meeting room…but we won’t speak of that either!

Viruses and Human Cancer: The Molecular Age

1 April, 2015

Hepatitis C virus

A suspicion that other viruses were involved in post-transfusion-(PTF) related hepatitis was first aired by Harvey J Alter and colleagues, after proof in 1972 that some PTF-related hepatitis cases had no HBV antigen associated with them.  By 1977 hepatitis A virus (HAV, infectious hepatitis) had been excluded as well, and the term “non-A, non-B” hepatitis (NANB) was coined.  By 1978, transmission studies using human serum injected into chimpanzees showed that

“Hepatitis was transmitted by serum derived from patients with chronic as well as acute hepatitis, strongly suggesting a chronic carrier state for the agent responsible for non-A, non-B hepatitis. Non-A, non-B hepatitis thus seems to be due to a transmissible agent which can persist and remain infectious for long periods”.

There was also evidence from Japan the same year that there might be a novel antigen – hepatitis C (HC) antigen – associated with NANB PTF hepatitis.  In 1979, it was suggested from ultrastructural studies in cells from infected chimpanzees that more than one NANB agent might exist; by 1980 Alter had concluded that that the NANB hepatitis agent(s) played a dominant role in the pathogenesis of PTF hepatitis. In 1987, in an interesting application of essentially the same technology used to characterise the first viruses, Alter’s group used polycarbonate membranes to filter the infectious agent, and showed it was  30-60 nm in diameter, and therefore highly unlikely to be a retrovirus, as had been suggested by some.

Also in 1987, Michael Houghton, Qui-Lim Choo, and George Kuo at Chiron Corporation collaborated with  DW Bradley at the CDC in using the much newer technology of constructing a random-primed cDNA clonal library from RNA extracted from human plasma in a lambda phage expression vector, and screening proteins expressed from the library against NANB hepatitis-infected patient serum.  They discovered one sequence that produced a protein fragment that bound antibodies, sequenced it, and used the sequence to “primer walk” through the entire genome by repeated cDNA generation, cloning and sequencing.  They published their finding in 1989 of

“…an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae”.

The agent was unique among viruses characterised until then, as no virus particle had yet been seen, let alone isolated. Alter and his team meanwhile tested for the presence of the virus in NANB patient samples, and in 1989 also published a paper – back-to-back with the previous – on

“An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis”.

The sequence of the putative agent allowed cloning and expression of a putative capsid protein in yeast, which allowed large-scale screening of patient samples and donated blood.  From their paper:

“These data indicate that HCV is a major cause of NANBH throughout the world”.

There was also already evidence that NANBH was associated with hepatocellular carcinoma (HCC) in Japan: M Sakamoto and colleagues showed that 90% of HBsAg-seronegative patients, who were also overwhelmingly HBV DNA-negative, showed evidence of chronic hepatitis in the non-cancerous liver, and 29% had a history of blood transfusion. This was followed as early as 1989 by evidence that 65% Italian HCC patients had antibodies to HCV, and again the same year by evidence from Spain that 75% of HCC patients had HCV antibodies, which

“…indicate[s] that HCV infection may have a role in the pathogenesis of hepatocellular carcinoma, even in patients with chronic liver disease apparently related to other agents such as alcohol”.

By 1990, K Kiyosawa and colleagues felt able to state that:

“These data suggest the slow, sequential progression from acute hepatitis C virus-related non-A, non-B hepatitis through chronic hepatitis and cirrhosis to hepatocellular carcinoma and support a causal association between hepatitis C virus and hepatocellular carcinoma”.

These findings rapidly led to the revelations that hepatitis C virus (HCV) was implicated in both an acute and relatively mild illness that lasts only a few weeks, and a chronic form that is usually more serious and can last lifelong.  Between 15–45% of infected persons spontaneously clear the virus within 6 months; however, the remaining 55–85% will develop chronic HCV infection.

Up to 150 million people globally are chronically infected with HCV.  Moreover – also from the WHO site – a significant number of these people will develop liver cirrhosis or liver cancer, and up to 500 000 people die worldwide every year from HCV-related liver disease.

There is as yet no vaccine against HCV infection, although trials are quite far advanced – and one candidate combination prime-boost strategy from Eleanor Barnes and coworkers seemed to show promise as of the end of 2014.  This consisted of

“…a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b” – which is using two well-characterised viruses as gene vectors to combat a third.

The authors make the point that the responses they achieved in human volunteers are similar to those seen in people who control natural infections.

Meanwhile, chemotherapy for chronic infections is both a realistic and well-established area: there are a number of treatments on the market already, and there have been significant recent developments which may make treatment even more effective.

HCV particles were finally characterised from cell culture-grown virus: both enveloped and non-enveloped pleomorphic spherical particles were found, of around 60 nm and 45 nm in diameter respectively.  This agrees well with the estimation by filtration of 30-60 nm previously determined in 1987.  The virus is classified as a ss(+)RNA genome flavivirus, similar to yellow fever virus, in the genus Hepacivirus, family Flaviviridae

Kaposi sarcoma herpesvirus

Moritz Kaposi in 1872 described what was originally called an “idiopathic multiple pigmented sarcoma of the skin”, which present as

“…disseminated blood- or bruise-coloured skin lesions (flat plaques or nodules) in the skin, usually on the lower extremities though sometimes on the hands and arms”.

What was subsequently called Kaposi Sarcoma, or KS, was at first thought to occur only among elderly men of Jewish, Arabic or Mediterranean origin; however by the 1950s it was realised it was quite common in sub-Saharan Africa, which led to the first suggestions that it might be caused by a virus.

In 1981-1982, however, the CDC received reports of KS occurring in otherwise healthy young homosexual men in California – often together with Pneumocystis carinii pneumonia, which was also previously very rare.  The disease was also much more aggressive, and spread beyond the skin into other tissues including bone, the mouth, gastrointestinal tract and lungs.

While there was at this time still no clue as to why this should be, the link to sexual acts as well as the previous observation that KS occasionally appeared in immune-suppressed organ transplant patients, led epidemiologists to discover the sexual transmission of immunodeficiency that led to the discovery of HIV and its causation of AIDS.

Valerie Beral and colleagues working at the CDC in the late 1980s used epidemiological data on KS in AIDS patients to build a compelling case for the tumour being caused by another sexually transmitted virus.  In a landmark paper in The Lancet, they announced in 1990, on the basis of painstaking and traditional-style investigation of 8 years’ worth of information from more than 90 000 people with AIDS collected in the US by the CDC since 1981, that:

“In the United States Kaposi’s sarcoma is at least 20000 times more common in persons with [AIDS] than in the general population and 300 times more common than in other immunosuppressed groups…Kaposi’s sarcoma was commoner among those who had acquired the human immunodeficiency virus (HIV) by sexual contact than parenterally, the percentage with Kaposi’s sarcoma ranging from 1% in men with haemophilia to 21% in homosexual or bisexual men. Women were more likely to have Kaposi’s sarcoma if their partners were bisexual men rather than intravenous drug users”.

The UK Cancer Research site on KS has an excellent account of the study as well as of its impact – one aspect of which was the proof in 1994 that indeed a virus was involved, using modern.  This was published by Y Chang and colleagues in Science, and detailed the use of the very modern technique of:

“Representational difference analysis … to isolate unique sequences present in more than 90 percent of Kaposi’s sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.”

This became human herpesvirus 8 (HHV-8), the newest of the seven viruses known to cause human cancers.

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Rinderpest and its eradication

30 March, 2015

Rinderpest virus infects cattle, buffalo and several species of antelope among other animals: it is a member of the genus Morbillivirus, family Paramyxoviridae, and is related to measles and mumps viruses in humans, distemper virus in dogs, and a variety of relatively newly-described viruses in marine mammals.  It also almost certainly gave rise to measles virus sometime around the 11th-12th centuries CE, as an originally zoonotic infection – sourced in domestic animals – took root in humans and began to be passed around (see here).

Wikipedia describes rinderpest virus as

“…an infectious viral disease of cattle, domestic buffalo, and some species of wildlife. The disease was characterized by fever, oral erosions, diarrhea, lymphoid necrosis, and high mortality.”  

And:

“The term Rinderpest is taken from German, and means cattle-plague.”

“Cattle plagues” have occurred throughout recorded history, often associated with the large-scale movement of animals accompanying military campaigns.  Europe was badly hit in the 1700s, with three epizootics in 1709–1720, 1742–1760, and 1768–1786, and a major British outbreak in 1865/66.

Of particular interest in South African folklore, an outbreak in the 1890s killed up to 90 percent of all cattle in southern and north-west Africa, and resulted in the deaths of many thousands of people who depended on them. It was devastating enough that people still remember it as a legendary time of hardship. Sir Arnold Theiler was instrumental in developing a vaccine that curbed the epidemic – by simultaneously injecting animals with blood from an infected animal and antiserum from a recovered animal. This protected animals for long enough to allow their immune systems to respond to the virus, but was rather risky, even though it was used for several decades.

In the 1920s J. T. Edwards in what is now the Indian Veterinary Research Institute serially passaged the virus in goats: after 600 passages it no longer caused disease, but elicited lifelong immunity. However, it could still cause disease in immunosuppressed cattle.

In 1924 the World Organisation for Animal Health (OIE) was formed, largely in response to rinderpest. This took on coordination of eradication efforts, which until then had been largely done on an individual country basis by means of vaccination.

This was followed by the Inter-African Bureau of Epizootic Diseases in 1950, with the aim of eliminating rinderpest from Africa. In 1962, Walter Plowright and R.D. Ferris used tissue culture to develop a live-attenuated vaccine grown in calf kidney cells.  Virus that had been passaged 90 times conferred immunity without disease even in immunosuppressed cattle, was stable, and did not spread between animals.  This vaccine was the one that allowed the prospect of eradicating the virus, and earned Plowright a World Food Prize in 1999.

Mass vaccination campaigns following outbreaks had, by 1972, eliminated rinderpest in all of Asia except for Lebanon and India. In the 1980s, a Sudan outbreak spread throughout Africa, killing millions of cattle, as well as much wildlife. The response was the initiation of the Pan-African Rinderpest Campaign in 1987, which made use of mass vaccination and surveillance to combat the disease. By the 1990s, all regions of Africa except for Sudan and Somalia were declared rinderpest-free.

By 1996, the complete nucleotide sequence of the virulent Kabete “O” strain of rinderpest had been obtained by Michael Baron and co-workers.  This could now be compared to that of the vaccine strain derived from it by Plowright and Ferris in 1962, that had been sequenced earlier, Despite the very different pathogenivities of the two viruses, there were only 87 base changes between them. It was interesting that  the Kabete strain – isolated in Kenya in 1910 – had been passaged by animal-to-animal transfer since then, and only 10 times since the derivation of the vaccine strain from it.  This provides a rare resource for determination of the determinants of pathogenicity.

The Food and Agriculture Organisation (FAO) has a Division of Animal Production and Health: their web site details a campaign known as the Global Rinderpest Eradication Programme (GREP), which has been going since 1994.  This had succeeded in reducing outbreaks to being small and infrequent by the late 1990s.  The last confirmed case of rinderpest was reported in Kenya in 2001. Final vaccinations were given in 2006; the last surveillance operations in 2009 failed to find any evidence of the disease.

By 14th October 2010, the BBC News site had this to say:

“The eradication of the virus has been described as the biggest achievement in veterinary history and one which will save the lives and livelihoods of millions of the poorest people in the world….”

If confirmed, rinderpest would become only the second viral disease – after smallpox – to have been eliminated by humans.”

A news item from the FAO site dated 25 May 2011, declared that:

“The national Delegates of Members of the World Organisation for Animal Health (OIE) declared today that rinderpest, one of the deadliest diseases of cattle and of several other animal species, is now eradicated from the surface of the earth.

At the organisation’s 79th annual General Session in Paris, France the national Delegates of OIE Members unanimously adopted Resolution 18/2011 which officially recognized, following thorough control by the OIE with the support of FAO, that all 198 countries and territories with rinderpest-susceptible animals in the world are free of the disease”.

When commenting on the significance of the achievement, John Anderson, the head of the FAO, described GREP’s announcement that rinderpest had been eradicated as:

“The biggest achievement of veterinary history“.

Like the smallpox eradication, even though much of the campaign happened in the era of modern virology, it was classical virological and disease control measures that were responsible for the success of the operation – with some assistance from molecular diagnostics towards the end.

This is only the second viral disease, ever, to have been eliminated.  And how was this possible?  Unlike smallpox, which has only humans as a natural and reservoir host (although it almost certainly also got into us from animals), rinderpest attacked a wider range of hosts.  However, it seemed mainly to have a reservoir in domesticated cattle, and it did not have an arthropod vector; moreover, the vaccine was cheap and effective.

This is momentous news: we may well have succeeded in ridding the planet of what has been a very significant disease of livestock and of wild animals, which has caused untold agricultural loss throughout recorded history, and which has resulted in enormous human hardship as well

We have also made a natural species go extinct – but it won’t be missed.  Like smallpox, it was completely sequenced some time ago, so we could theoretically recreate it if we ever needed to.

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Maize streak virus: the early history

30 March, 2015

The history of maize streak virus research is generally taken as starting in 1901, with the publication of the

The cover of the "Fuller Report"

The cover of the “Fuller Report”

by “Claude Fuller, Entomologist”. However, in the Report he does make reference to articles in the “Agricultural Journal” for August 3rd and 31st, 1900, and quotes personal sources as having noticed the disease of “mealie variegation” as early as the 1870s.  He comments that:

“…mealie growers…have been acquainted with variegated mealies…for at least 20 years…”, and “…Thomas Kirkman…has known the disorder for 30 years past…”.

His conclusions, although carefully arrived at, were very wrong. Fuller claimed the disease was due to soil deficiency or a “chemical enzyme” in soils, and could be combatted by intensive cultivation and “chemical manures”. However, his carefully-written account is still of great historical interest, and the observations are valuable as they are objective accounts of a skilled scientist.  The records of streaked grasses in particular are useful, as we still collect such samples to this day.  Fuller was later sadly a victim of one the first traffic accidents in what was then Lourenco Marques in Mozambique.

Streak symptoms in a maize leaf

The disease – now known as maize streak disease (MSD) – occurs only in Africa and adjacent Indian Ocean islands, where it is one of the worst occurring in maize.  The causal agent was discovered to be a virus by HH Storey in 1932, who termed it maize streak virus (MSV). The virus was found to be obligately transmitted by the leafhopper Cicadulina mbila, also by Storey, in 1928. In 1978, MSV was designated the type virus of the newly described group taxon Geminivirus.

Early studies indicated that there were several distinctly different African streak viruses adapted to different host ranges (Storey & McClean, 1930; McClean, 1947). These studies were based on the transmission of virus isolates between different host species and symptomatology.

In a subsequent study of streak virus transmission between maize, sugarcane, and Panicum maximum, the relatively new technique of immunodiffusion was employed, using antiserum to the maize isolate. From the results it was concluded that the maize, sugarcane, and Panicum isolates were strains of the same virus, MSV (Bock et al., 1974). The maize isolate was given as the type strain. The virus was only properly physically characterised in 1974, when the characteristic geminate or doubled particles were first seen by electron microscopy, and only found to be a single-stranded circular DNA virus in 1977 (Harrison et al., 1977).

Maize streak virus: photo from Robert G Milne in 1978

Maize streak virus: photo from Robert G Milne in 1978

The first isolates of MSV were sequenced in 1984 (Kenya, S Howell, 1984; Nigeria, P Mullineaux et al., 1984), and the virus was found to have a single component of single-stranded circular DNA (sscDNA), and to be about 2700 bases in size. The two isolates were about 98% identical in sequence. The second team took delight in noting that the first sequence was in fact of the complementary and not the virion strand.

A major advance in the field occurred in 1987, when Nigel Grimsley et al. showed that a tandem dimer clone of MSV-N in an Agrobacterium tumefaciens Ti plasmid-derived cloning vector, was infectious when the bacterium was injected into maize seedlings. Subsequently, Sondra Lazarowitz (1988) obtained the sequence of an infectious clone of a South African isolate (from Potchefstroom) – MSV-SA – and showed that it also shared about 98% identity with the first two sequences.

Since the early days other transmission tests and more sophisticated serological assays were performed on a wide range of streak isolates from different hosts and locales, and it was claimed that all forms of streak disease in the Gramineae in Africa were caused by strains of the same virus, MSV. This view changed as more and more viruses were characterised, however, and it became obvious that there were distinctly separate groupings of viruses that constituted different species: these were sugarcane streak viruses (SSV, see Hughes et al., 1993), the panicum streak viruses (PanSV, see Briddon et al., 1992), and the maize streak viruses. Together these viruses constituted an African streak virus group (see Hughes et al., 1992; Rybicki and Hughes, 1990), distinct from an Australasian striate mosaic virus group, and other more distantly related viruses (see here for the state of the art in 1997).  These studies together with a later one by Rybicki et al. in 1998 also pointed up the utility of the polymerase chain reaction (PCR) for amplification, detection and subsequent sequencing of DNA from diverse mastreviruses.

A more modern and comprehensive account can also be found here, in a recent review written for Molecular Plant Pathology.

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Human retroviruses and cancer

13 March, 2015

The very early discovery of avian viruses associated with cancer, and the subsequent failure for many years to isolate similar viruses from mammals, gave some researchers the idea that possibly birds were unique in this regard.  However, “RNA tumour viruses” or oncornaviruses, as they were known for a time, were first demonstrated to affect mammals when mouse mammary tumours were shown to be due to a virus by John Bittner in 1936, by transmission in milk. He also demonstrated vertical transmission, or inheritance of the virus. 

The nature of the agent was not known at the time, but by 1951 L Gross had shown that leukaemia could be passaged in mice using cell-free extracts.  In 1958 W Bernhard had proposed a classification of what were to become known as retroviruses on the basis of electron microscopy.  In 1964 a mouse sarcoma virus and a feline leukaemia virus had been isolated, and in 1969 bovine leukaemia was shown to be a viral disease.  1970 saw the description of reverse transcriptase from retroviruses, and in 1971 the first primate leukaemia virus – from gibbons – was described, and the first retrovirus (foamy virus) described from humans.  Bovine leukaemia virus was characterised as a retrovirus in 1976.

It is not surprising, therefore, that many labs tried to find cancer-causing disease agents in humans.  However, such effort had been put into finding oncornaviruses associated with human tumours, with such lack of success, that it led to people talking of “human rumour viruses” – a useful list of which can be seen here.  Nevertheless, by 1980 Robert Gallo’s group had succeeded in findingtype C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma”, which they called human T-cell leukaemia virus (HTLV).  The breakthrough was made possible by their prior discovery of “T cell growth factor”, now called interleukin 2 (IL-2), which meant human T cells could be successfully cultured for the first time.  A group of Japanese researchers described an “Adult T cell leukemia virus” (ATLV) in 1982: this proved to be the same as what became HTLV-1, given the description also in 1982 by Gallo’s group of another retrovirus associated with a T-cell variant of hairy cell leukaemia, which they dubbed HTLV-2. 

HTLV-1 is associated with the rare and genetically-linked adult T-cell leukaemia, found mainly in southern Japan, as well as with a demyelinating disease called “HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP)” and HTLV-associated uveitis and infective dermatitis.  The areas of highest prevalence are Japan, Africa, the Caribbean islands and South America.  HTLV-2 had a mainly Amerindian and African pygmy distribution, although it is now found worldwide, and causes a milder form of HAM/TSP, as well as arthritis, bronchitis, and pneumonia.  It is is also frequent among injecting drug users.  However, except for rare incidences of cutaneous lymphoma in people coinfected with HIV, and the fact of its origin in a hairy cell leukaemia, there is no good evidence that HTLV-2 causes lymphoproliferative disease.  The two viruses infect between 15 and 20 million people worldwide.  HTLV-1 infections can lead to an often rapidly fatal leukaemia.

By 2005 another two viruses had joined the family: HTLV-3 and HTLV-4 were described from samples from Cameroon that were presumably zoonoses – being associated with bushmeat hunters – and which are not associated with disease.  Interestingly, all the HTLVs have simian counterparts – indicating species cross-over at some point in their evolution.   Collectively they are known as the primate T-lymphotropic viruses (PTLVs) as they consitute an evolutionarily related group.  Another relative is bovine leukaemia virus.

The HTLV-1/STLV-1 and HTLV-2/STLV-2 relationships are relatively ancient, at more than 20 000 years since divergence.  However, their evolution differs markedly in that STLV-I occurs in Africa and Asia among at least 19 species of Old World primates, while STLV-2 has only been found in bonobos, or  Pan paniscus dwarf chimpanzees from DR Congo.  It is therefore quite possible that there are other HTLVs undiscovered in primates in Africa and elsewhere, that may yet emerge into the human population.

Human immunodeficiency virus type 1 (HIV-1) was for a time after its discovery in 1983 called HTLV-III by the Gallo group and lymphadenopathy virus (LAV) by the Montagnier group; however, evidence later obtained from sequencing and genome organisation showed by 1986 that it was in fact a lentivirus, related to viruses such as feline immunodeficiency virus (FIV) and the equine infectious anaemia virus discovered in 1904, and it was renamed.  Francoise Barre-Sinoussie and Luc Montagnier were awarded a half share in a 2008 Nobel Prize, commemorated here

HIV particle.  Russell Kightley Media

HIV particle. Russell Kightley Media

in Viroblogy.

HIV is indirectly implicated in cancer because it creates an environment through immunosuppression that allows the development of opportunistic tumours that would normally be controlled by the immune system: these include HPV-related cervical cancer, and Kaposi’s sarcoma caused by Human herpesvirus 8 (see later).  It is also possible that HIV may directly cause lymphoma development in AIDS patients by insertional activation of cellular oncogenes, although this appears to be rare.

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Epstein-Barr Virus and Hepatitis B Virus

13 March, 2015

Epstein-Barr Virus

While the early discoveries of Ellerman and Bang and Rous might have predicted that retroviruses would be found associated with human cancers, in fact it was a herpesvirus that was the first viral agent implicated in a human cancer.  This was what was called the Epstein-Barr virus – now Human herpesvirus 4 – that was discovered in 1964 by Michael Epstein, Bert Achong and Yvonne Barr in specimens from a  Burkitt lymphoma patient sent from Uganda by the surgeon Denis Burkitt.  The virus was later implicated in infectious mononucleosis or glandular fever, also known as the “kissing disease” or because it tends to get spread around by intimate contact between college students.

A human herpesvirus. Copyright Linda M Stannard

A human herpesvirus. Copyright Linda M Stannard

The virus is carried by up to 95% of adults worldwide, after mainly asymptomatic infections as children.  It is implicated in causing over 95% of nasopharyngeal carcinomas, nearly 50% of Hodgkin lymphoma, and about 10% of gastric carcinomas – for a total of nearly 200 000 cancers worldwide per year.  There is still no vaccine, although candidates are in clinical trial.

Hepatitis B virus

The next virus to be definitively linked to a human cancer was Hepatitis B virus (HBV), that had been discovered more or less accidentally during serological studies in the 1960s by Baruch Blumberg and colleagues.  However, a transmissible agent had been implicated in “serum hepatitis” as early as 1885, when A Lurman showed that contaminated lymph (serum) was to blame for an outbreak in a shipyard in Bremen after a smallpox prevention exercise.  Subsequently, reuse of hypodermic needles first introduced in 1909 was shown to be responsible for spreading the disease.

Blumberg’s “initial discoveries were based primarily on epidemiologic, clinical, and serological observations”; however, by 1968 the “Australia antigen” was seen to consist of 22 nm empty particles, now known to be composed of capsid protein or “surface antigen”, and by 1970 a 42 nm DNA-containing “Dane particle” was found which is now known to be the virion.  Blumberg had by 1972 patented a vaccine derived by purification of 22 nm particles composed of HBV surface antigen (HBsAg) from donor blood, a process pioneered by Maurice Hilleman.  By 1975 Blumberg and others had also implicated HBV in the causation of primary hepatic carcinoma, now known as hepatocellular carcinoma (HCC) and a serious complication of chronic infection with HBV, especially if acquired in early life.  The vaccine was licenced for use in 1982, meaning it was the first anti-cancer vaccine, and the first viral subunit vaccine.  Blumberg shared the 1976 Nobel Prize in Physiology or Medicine with D Carleton Gajdusek – who described the first prion-caused diseases – for “…their discoveries concerning new mechanisms for the origin and dissemination of infectious diseases”.

hbv particles

By 1979 the whole HBV genome had been cloned and sequenced, and molecular biology studies could start in earnest. A recombinant HBsAg produced in yeast was subsequently licenced in 1986, and has supplanted the earlier one.  It is being used in a many countries as part of the EPI (Extended Programme of Immunisation) bundle given to infants, as there is more risk of chronic infection the younger the person is that is infected.  Given that upwards of 2 billion people have been infected, and the over 300 million people that are chronically infected with HBV have a 15-25% risk of dying prematurely from HBV-related causes, there is the potential to make a very significant impact on liver disease.

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Papillomaviruses and human cancer

11 March, 2015

Human warts in all their forms – cutaneous, verrucous and genital growths and lesions – have been known since antiquity, and it was known since at least 1823 that at least some were infectious. Experiments done with human volunteers in the 1890s confirmed this, when it was shown that transplanting wart tissue resulted in typical disease.  As early as 1908, it was shown by a G Ciuffo that “verrucae volgare” – common warts – could be transmitted via a cell-free filtrate.  However, it was Richard E Shope who first showed that a papillomavirus was associated with animal tumours.  A useful review from 1931 on “Infectious oral papillomatosis of dogs” by DeMonbreun and the Ernest Goodpasture of egg culture fame covers the early history of the investigation of human disease as well as of animal papillomas very well, so we will not cover this further.

In light of later findings of the involvement of papillomaviruses, it was a prescient although premature observation by an Italian physician named Rigatoni-Stern in 1842 that cervical cancer appeared to be sexually transmitted, given that it occurred in married women, widows and prostitutes, but rarely in virgins and nuns.

Although papillomaviruses had been implicated as the first viruses known to cause a cancer in mammals as early as the 1930s, and the structurally very similar papovaviruses were similarly implicated in the late 1950s, it was only in 1972 that  Stefania Jabłońska proposed that a human papillomavirus (HPV; then called a papovavirus) was involved with the rare hereditary skin cancer called epidermodysplasia verruciformis.   

Meanwhile Harald zur Hausen had been investigating since 1974 the involvement of HPV in genital warts (condyloma accuminata) and squamous cell carcinomas, using DNA-based techniques such as hybridisation.  The rarely malignant condylomas had been shown to contain papillomavirus particles in some cases in 1968, with a better association in 1970; however, cross-hybridisation studies by zur Hausen’s group on DNA of these and common wart viruses showed no relationship despite their very similar morphologies. 

Virus particles from genital warts (6 &7) and a common skin wart (8).  Reproduced from Brit. J. vener. Dis., JD Oriel and JD Almeida, 46, 37-42, 1970 with permission from BMJ Publishing Group Ltd.

Virus particles from genital warts (6 &7) and a common skin wart (8). Reproduced from Brit. J. vener. Dis., JD Oriel and JD Almeida, 46, 37-42, 1970 with permission from BMJ Publishing Group Ltd.

Zur Hausen speculated on the role of HPVs in squamous cell carcinomas in 1977; Gérard Orth and Jabłońska and colleagues went on to define the “…Risk of Malignant Conversion Associated with the Type of Human Papillomavirus Involved in Epidermodysplasia Verruciformis” in 1979.

Because this was the new era of cloning and sequencing of DNA, the zur Hausen group and others went on to isolate and characterise a number of new HPVs associated with genital cancers and other lesions in the early 1980s.  In particular, they showed that HPV types 16 and 18 could be found both as free virus in cervical cell sample biopsies and integrated into the cell genomes of cell lines derived from cervical cancers.  A major finding in 1987 was that the legendary HeLa cell line – derived from a malignant cervical tumour from a Henrietta Lacks in 1951contains multiple copies of the HPV-18 genome.  The first HPV genome sequence (of type 1b) was obtained in 1982; the first genital type (6b, from condylomas) in 1983, and the first high-risk cancer virus (type 16) in 1985.

Later work involving large international surveys showed by 1995 that 99.7% of cervical cancers contained DNA from so-called “high risk” HPVs, leading to the conclusion that these were the necessary cause of cervical cancer, and that around 70% of these cancers were caused by HPVs 16 and 18.  Since then, HPVs have been found in more than 80% of anal cancers, 70% of vulval and 40% of vaginal cancers, around half of all penile cancers, and in roughly 20% of head and neck cancers.  If 16% of cancers are due to infection, and HPVs cause or are implicated in 30% of these, then they are a significant cause of cancers worldwide.

Harald zur Hausen was awarded a half share of the 2008 Nobel Prize in Physiology or Medicinefor his discovery of human papilloma viruses [sic] causing cervical cancer”.  I blogged on this at the time, here.

Work on vaccines against papillomaviruses (PVs) started early, after demonstrations presumably in the 1930s that domestic rabbits inoculated with the cottontail rabbit PV (CRPV) could become immune to reinoculation after recovery, and in 1962 that a “…formalin-treated suspension of bovine papilloma tissue” provided protection against challenge, but was not therapeutic.  However, progress was stymied by the fact that it proved impossible to culture any of the PVs, and challenge material had to be made from infected animal tissue, even though it had been shown that isolated viral DNA was infectious.

This changed after the advent of molecular cloning, when whole viral genomes could be prepared in bacteria.  Model systems for use in PV vaccine research by 1986 included cattle and bovine PVs, rabbits and CRPV and rabbit oral PV, and dogs and canine oral PV.  It had also been demonstrated that the L1 major structural protein of type 1 BPV produced in recombinant bacteria was protective against viral challenge in calves.  Jarrett and colleagues demonstrated, in 1991 and 1993 respectively, that they had achieved prophylactic and therapeutic immunisation against cutaneous (ie: skin; caused by BPV-2) and then mucosal (respiratory tract; BPV-4) bovine PVs, using E coli-produced proteins.  L1 and L2 proteins were protective against BPV-2, while L2 was protective against BPV-4 infection.  They suggested BPV-4 was a good model for HPV-16 given its mucosal tropism.

By the early 1990s several groups had demonstrated that it was possible to make PV virus-like particles (VLPs) by expression in eukaryotic systems such as yeast or animal cells of the L1 major virion protein either alone, or together with the minor protein L2.  In 1991 Ian Frazer’s group showed that expression of HPV-16 L1 and L2 together but not separately in animal cells via recombinant vaccinia virus, resulted in 40 nm particles resembling the virion being made.  In 1992 John Schiller’s lab showed VLP formation by L1 alone, with both BPV-1 and HPV-16 L1 genes expressed in insect cells via a baculovirus vector. In 1993 came the demonstration that expression of the plantar wart-causing HPV-1 L1 gene alone and L1 and L2 genes together in animal cells via vaccinia virus, as well as of the genital wart-causing HPV-11 L1 expressed in insect cells, resulted in VLP formation.  By 1995, it had been shown that immunisation of rabbits with CRPV L1-only or L1+L2 VLPs, and of dogs with canine oral PV L1 VLPs, protected completely against viral challenge.

hpv vlps

This groundwork made it possible for Merck and GlaxoSmithKline to develop and to push through to human trial and licensure, two independent VLP-based vaccines.  Merck’s vaccine – Gardasil – is quadrivalent, consisting of a mixture of VLPs made in recombinant yeasts from expression of L1 genes of HPV types 6 and 11, to protect against genital warts, and types 16 and 18, for cervical lesions and cancer.  GSK’s offering – Cervarix – is a bivalent HPV-16 and -18 vaccine only, consisting of VLPs made via recombinant baculoviruses in insect cell culture.  These are only the second anti-cancer vaccines on offer, and have gone on to blockbuster status within months of their release: Gardasil was licenced in June 2006, and Cervarix in October 2009.

Both appear to protect very well against infection with the types specified, but not to affect established infections.  Their long-term efficacy against cervical cancer is still to be established, although Gardasil has certainly lessened the incidence of genital warts in Australia post introduction in 2007.  There is now also a VLP-based vaccine for canine oral PV.

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