Archive for the ‘Vaccines: General’ Category

A Short History of the Discovery of Viruses – Part 4

11 February, 2015

RNA as genetic material

While it had been known since Bawden and Pirie’s work in 1937 that TMV particles contained RNA, followed later by a number of other viruses, it must be remembered that DNA had only really been accepted as the genetic material of cells and viruses after the Hershey-Chase experiment in 1952 and the Watson-Crick demonstration of the nature of DNA in 1953.  Moreover, the way in which the information in DNA was used to make proteins was still very obscure in the 1950s, given that the proof that RNA was used as a template for the production of proteins was only provided in 1961 by Marshall Nirenberg.

It was hailed as a major development in molecular biology, therefore, when between 1955 and 1957, Heinz Fraenkel-Conrat, B Singer and Robley C Williams demonstrated that it was possible to reconstitute fully infectious TMV from separately-purified preparations of coat protein and RNA.  At the time it was assumed that neither of the two components was infectious on its own; however it was subsequently shown by Fraenkel-Conrat and Singer, and separately by A Gierer and G Schramm, that purified TMV RNA was in fact infectious – albeit several hundred times more weakly per unit mass than the native or reconstituted particles.

While this was revolutionary in itself, the clinching experiment was the proof that mixed reconstitution – or the reassembly of a RNA of one strain of TMV with the coat protein of another – followed by infection of plants resulted in particles made of protein specified by the RNA component rather than being determined by the protein donor.  This work possibly represents the birth of molecular virology as a sub-discipline within molecular biology, given that the molecular nature of viruses had so conclusively been shown – vindicating the prescient remark made by the virus pioneer Thomas Rivers in 1941, on the occasion of the presentation of a gold medal to Wendell Stanley, that:

“In fun, it has been said that we do not know whether to speak of the unit of this infectious agent [TMV] as an “organule” or a “molechism”” (p.7, CA Knight, Chemistry of Viruses 2nd Edn., 1975. Springer-Verlag, Wien)

Further important developments with TMV included the demonstration in 1958 by Gierer and KW Mundry that TMV mutants with altered genomes could be produced by treatment of virions with nitrous acid, which only alters nucleic acids, and the sequencing of the TMV coat protein in 1960 by two groups including Fraenkel-Conrat and Stanley and Knight in one, and Schramm in the other.

Between 1953 and 1954, an interesting class of new viruses was discovered in humans, birds, and later in other animals too.  These were dubbed “respiratory enteric orphans” based on where they were found, and the fact they were not associated with any disease – which gave rise to the name “reovirus”, and their description as a distinct group of viruses by Albert Sabin in 1959.  By 1962 the unique double-layered capsid morphology had been seen and the virions shown to contain RNA, and then in 1963 PJ Gomatos and I Tamm showed using physical and chemical techniques that the viruses as well as the similar wound tumour virus isolated from plants had a genome consisting of double-stranded (ds) RNA – a finding unprecedented in biology at the time.  Gomatos and W Stoeckenius  went on to show in 1964 – by electron microscopy – that the reovirus genome was also segmented – another unprecedented finding for viruses. In the 1963 paper the authors remark that “…all attempts to isolate the nucleic acid of reovirus in an infective form have failed” – which distinguished these viruses from the ssRNA viruses previously looked at – not surprisingly, given the requirement for a different replication method for dsRNA compared to viruses like TMV or poliovirus (see here).

A major highlight in molecular biology in 1961 was Marshall Nirenberg and Heinrich Matthaei’s 1961 demonstration of “…an assay system in which RNA serves as an activator of protein synthesis in E. coli extracts”, or the proof in an in vitro translation system that RNA was the “messenger” that conveyed genetic information into proteins.

In 1962, A Tsugita, Fraenkel-Conrat, Nirenberg and Matthaei used the still extremely novel in vitro translation system with purified TMV genomic RNA, and were able to show that:

“The addition of TMV-RNA to a cell-free amino acid incorporating system derived from E. coli caused up to 75-fold stimulation in protein synthesis (C14-incorporation). Part of the protein synthesized formed a specific precipitate with anti-TMV serum.”, indicating that TMV coat protein had been made.

This was the first demonstration of in vitro translation from any specific mRNA, and incidentally also direct proof that the single-stranded TMV genome was “messenger sense”.  They also concluded that their result showed that the newly-determined “genetic code” – the nucleotide triplets that code for individual amino acids – was universal, given that it was a tobacco virus RNA being translated by a bacterial system.

Later in 1962, D Nathans and colleagues used coliphage f2 RNA as template for translation in the same type of bacterial extract.  They showed that polypeptides corresponding to the coat as well as other proteins were made, showing that it was the input virion RNA that was responsible.

Modern virology

The proof that RNA was both the “messenger” that conveyed information from DNA to be made into protein, and was in fact a genetic material in its own right, made possible a revolution in virology that transformed it into the science we know today.  The new molecular biology together with well-established physical and biochemical techniques for molecular characterisation, coupled with the ability to reliably culture bacterial, plant and now animal viruses as well, enabled an explosion of discovery that continues to this day. 

A tour de force experiment in the modern molecular biological era was the in vitro synthesis of an infectious phage RNA genome by S Spiegelman and coworkers in 1965, using only purified Qbeta coliphage single-stranded virion RNA and the purified viral replicase.   They remarked:

“The successful synthesis of a biologically active nucleic acid with a purified enzyme is itself of obvious interest. However, the implication which is most pregnant with potential usefulness stems from the demonstration that the replicase is, in fact, generating identical copies of the viral RNA. For the first time, a system has been made available which permits the unambiguous analysis of the molecular basis underlying the replication of a self-propagating nucleic acid.”

Ribosomes translating protein from a messenger RNA molecule

Ribosomes translating protein from a messenger RNA molecule.  Russell Kightley Media

In 1967 there followed the demonstration that the same could be done for a single-stranded (ss)-DNA virus: M Goulian and colleagues reported in that they had successfully made a completely synthetic and infectious PhiX174 coliphage genome, by means of a series of syntheses using purified virion ssDNA, E coli DNA polymerase and a “polynucleotide-joining enzyme”, or DNA ligase.  It is instructive that the authors offer this as evidence for the involvement of the same enzymes in E coli chromosomal replication, the mechanism for which which was still obscure at the time.  Their justification for their work:

“If enzymatic synthesis of infectious bacteriophage DNA were achieved, it would be made clear at once that relatively few, if any, mistakes had been made in replicating a DNA sequence of several thousand nucleotides.”

- was undoubtedly borne out, in yet another example in the growing number of cases of the use of viruses to demonstrate important facets of cellular biology.

Naked nucleic acids as infectious agents: viroids

A potato disease that had been known in the New York and New Jersey state areas in the US since the 1920s was the source of an exciting discovery by Theodor (Ted) Diener and WB Raymer, reported in Science in 1967.  The potato spindle tuber disease agent had proved recalcitrant over many years to being characterised or isolated; all that was known was that it could be transmitted mechanically using sap, or via grafting, and that no fungi, bacteria or viruses could be isolated from diseased material.  Diener and Raymer showed that:

“Infectious entities, extractable, with phosphate buffer, from tissue infected with potato spindle tuber virus and inciting symptoms on tomato that are typical of this virus, have properties incompatible with those of conventional virus particles. …[Their properties] suggest that the extractable infectious agent may be a double-stranded RNA.”

By 1971 Diener had determined that

“…the infectious RNA occurs in the form of several species with molecular weights ranging from 2.5 × 104 to 1.1 × 105 daltons. No evidence for the presence in uninoculated plants of a latent helper virus was found. Thus, potato spindle tuber “virus” RNA, which is too small to contain the genetic information necessary for self-replication, must rely for its replication mainly on biosynthetic systems already operative in the uninoculated plant.”

This was a revolutionary concept: an infectious, pathogenic entity in the form of a naked RNA that was too small to encode a replicase or any other protein.  He proposed the term “viroid” to designate this and similar agents, a term that persists up to today.  By 1979, they were known to be single-stranded circular RNA molecules with a high degree of sequence self-complementarity, which results in them appearing as “highly base-paired rods”.

Reverse transcription and tumour viruses

While it was apparent in the 1960s that there were single-and double-stranded DNA and RNA viruses, it was only in 1970 that two back-to-back papers in Nature, by Howard Temin and S Mituzami, and David Baltimore respectively, revealed a highly novel viral replication strategy.  They showed that “RNA tumour viruses” such as the agents found by Ellerman and Bang and Peyton Rous contained an enzyme activity named reverse transcriptase – a colloquial term for RNA-dependent DNA polymerase – in their virions, which converted the single-stranded RNA genomes into double-stranded DNA. Later this was shown to result in resulted in insertion of the DNA into the host cell genome, vindicating Howard Temin’s 1960 proposal that “…a RNA tumor virus can give rise to a DNA copy which is incorporated into the genetic material of the cell”.

When Francis Crick formulated his ”Central Dogma” in 1956, it was indisputable that genetic information flowed from DNA to progeny DNA, from DNA to RNA, and from messenger RNA to protein – while he only postulated no return information flow from protein, it was generally assumed that this was also true for RNA

In the words of David Baltimore, in his Nature article:

“Two independent groups of investigators have found evidence of an enzyme in virions of RNA tumour viruses which synthesizes DNA from an RNA template. This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted.”

This gives rise to a modified Central Dogma, where information flows from DNA to DNA, from DNA to RNA, from RNA to RNA, from RNA to DNA, and from RNA to protein.  It is interesting that RNA seems central to this flow – which, incidentally, strengthens the proposal that RNA is the original genetic material.

Baltimore and Temin both received a share of the Nobel Prize in Physiology or Medicine 1975 for their discovery of reverse transcriptase – and shared it with Renato Dulbecco, who was credited with clarifying the process of infection and of cellular transformation by DNA tumour viruses.  He used the double-stranded (ds) DNA polyomavirus SV40: this was originally isolated from monkeys, but shown to cause a variety of tumours in a number of experimental animals, hence the name “poly-oma”. 

He and colleagues showed that polyomavirus grew and could be assayed normally in certain cell cultures, but caused tumour-like transformation of cells in others in which it did not grow.  They showed that transformed cell chromosomes contained covalently integrated viral DNA termed a provirus, which was active in producing mRNA which made virus-specific proteins.  Thus, his work was the first to show how DNA viruses might cause cancer, and he and his colleagues deserved their award “…for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell.

Viral genome cloning and sequencing: the new age

The techniques of recombinant DNA technology – or the artificial introduction of genetic material from one organism into the genome of another – were pioneered between 1971 and 1973 by Paul Berg, Herbert Boyer and Stanley Cohen.  In 1971 Berg performed an in vitro exercise in which a segment of the lambda phage genome was ligated into the purified DNA of SV40, which had been linearised using the then-new restriction endonuclease, EcoRI.  Cohen, Annie Chang, Boyer and Robert Helling took the technology further in 1973 by showing that:

“The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules.”

Cloning had arrived – made possible in part by use of viruses.  The fundamental nature of this advance of molecular biology was rewarded by a half share of the 1980 Nobel Prize in Chemistry to Paul Berg.

Nucleotide sequencing, or the determination of the order of bases in nucleic acids, started with laborious, difficult techniques such as the two-dimensional fractionation of enzyme digests of 32P-labelled for RNA described by Frederick Sanger and colleagues in 1965.  DNA sequencing followed in 1970: Ray Wu described the use of E coli DNA polymerase and radiolabelled nucleotides to sequence the single-stranded ends of phage lambda DNA. He and colleagues followed this with a more general method in 1973, using extension of synthetic oligonucleotide “primers” annealed to target DNA

Walter Gilbert and Allan Maxam published in February 1977 an immediately popular paper entitled “A new method for sequencing DNA”.  This became known as Maxam-Gilbert sequencing, or the chemical method, as it entailed sequencing by chemical degradation.  Also in 1977, however, Frederick Sanger and colleagues adapted the Wu technique to come up with the so-called Sanger method, or “DNA sequencing with chain-terminating inhibitors“: this soon became the industry standard for at least the next twenty years, because it was easier and cheaper than the chemical method.

Gilbert and Sanger were awarded a share of the Nobel Prize in Chemistry in 1980, for their contributions concerning the determination of base sequences in nucleic acids“.

MS2 phage sequencing

A highlight of Ed Rybicki’s introduction to the world of viruses was discovering during his Honours year in 1977, the paper in Nature in 1976 by Walter Fiers and his coworkers on completing the genome sequencing of the ssRNA E coli phage, MS2.  They had previously also been responsible for the first ever gene sequence, in 1972: this was of the coat protein gene from the same virus.  This was a landmark publication, because it completed the work of years by their group by sequencing the replicase gene, using the ribonuclease digestion and genome fragmentation and two-dimensional electrophoresis technique from Sanger.  Moreover, they proposed a secondary structure for the replicase gene based on intrasequence complementarity, and described it eloquently as follows:

“The secondary structure of the coat gene resembles a flower, and there are similar foldings in other parts of the molecule; the secondary structure of the whole viral RNA therefore constitutes a bouquet”.

Their achievement looks modest in retrospect, in this era of high-throughput sequencing – however, it is worth remembering that at this time in 1976,

“MS2 is the first living organism for which the entire primary chemical structure has been elucidated”. 

Depiction of the linear sequence of MS2 phage.  The maturation (M), coat (CP) and replicase (Rep) genes and proteins were known at the time of sequencing; the lysis gene that partially overlaps the Rep open reading frame was shown to be functional only in 1982

Depiction of the linear sequence of MS2 phage. The maturation (M), coat (CP) and replicase (Rep) genes and proteins were known at the time of sequencing; the lysis gene that partially overlaps the Rep open reading frame was shown to be functional only in 1982

While this comprised just 3569 nucleotides, encoding only three genes, this  is sufficient to constitute a self-replicating entity with an independent evolutionary history.

The immediate value of their work was that it provided a basis for understanding the biology of the interaction of the genome with the bacterial cell at the molecular level.  Moreover, the proposed secondary structures also helped explain how such a simple genome managed to temporally regulate its own expression – by means of long-distance interactions between different areas of the sequence.

PhiX174 phage sequencing

The next complete viral genome sequenced was that of the circular single-stranded DNA coliphage PhiX174, in 1977 by Sanger and his team in Cambridge, using the new sequencing technique invented by them.  The abstract of their paper reads:

“A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple ‘plus and minus’ method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.“

This was the first complete genome sequenced for any DNA-containing organism, and a satisfying conclusion to many decades of work on the virus.  One of the most interesting features of the sequence was the fact that several of the 11 genes  are highly overlapping: that is, the same DNA sequence is used to encode completely different genes in different open reading frames.  This represented an economy of use of genetic information that was hitherto unknown. 

Ed Rybicki was also able to greatly impress his Honours external examiner – one DR Woods – by launching into a detailed account of the sequencing and the genetic implications, when asked “What did you find interesting in the literature this year?”

SV40 sequencing

The simian vacuolating virus 40, or SV40, was discovered in 1960 by Ben Sweet and Maurice Hilleman as a contaminant of live attenuated polio vaccines made between 1955 and 1961: this was as a result of use of vervet or African green monkey cells that were inadvertently infected with SV40 to grow up the polioviruses.  As a consequence, between 1955 and 1963 up to 90% of children and 60% of adults – 98 million people – in the USA were inadvertently inoculated with live SV40.  Given the demonstration by  Bernice  Eddy and others in 1962 that hamsters inoculated with simian cells infected with SV40 developed sarcomas and ependymomas, the class of viruses including SV40 and MPyV described earlier became known as “polyomaviruses”, and DNA tumour viruses.  However, and despite considerable concern over many years, SV40 has not been shown to cause or to definitively be associated with any human cancers.

Still, it had become an object of considerable interest as mentioned earlier in connection with Renato Dulbecco, and it was accordingly the next virus to be completely sequenced.  This was by Walter Fier’s group: they determined by Maxam-Gilbert sequencing that the circular dsDNA genome comprised 5224 base pairs, and had an interesting organisation.  In their words:

“Particular points of interest revealed by the complete sequence are the initiation of the early t and T antigens at the same position and the fact that the T antigen is coded by two non-contiguous regions of the genome; the T antigen mRNA is spliced in the coding region. In the late region the gene for the major protein VP1 overlaps those for proteins VP2 and VP3 over 122 nucleotides but is read in a different frame.”

Linear depiction of the circular SV40 genome and its protein coding capacity.  Regions of RNA spliced out of of transcribed genomic sequence, and the direction of transcription, are shown as red arrows.  Genes shown are those depicted in the current Genbank sequence entry.

Linear depiction of the circular SV40 genome and its protein coding capacity. Regions of RNA spliced out of of transcribed genomic sequence, and the direction of transcription, are shown as red arrows. Genes shown are those depicted in the current Genbank sequence entry.

This was the first time that RNA splicing had been demonstrated for an entire genome; indeed, it had only been discovered in 1977 when two separate groups of researchers showed that adenovirus-specific mRNAs made late in the replication cycle in cell cultures were mosaics, being comprised of sequences from noncontiguous or separated sites in the viral genome.  This was subsequently found to be a common feature in eukaryotic but not prokaryotic mRNAs.

The SV40 genome showed major gene overlaps, as for the PhiX174, again demonstrating the effectiveness with which viruses could pack protein coding capability into a small genome

A Short History of the Discovery of Viruses – Part 3

29 January, 2015

The Phage Group and the birth of molecular biology

Some of the more fundamental discoveries in modern biology were facilitated either by the study of viruses, or by use of viruses as tools for exploring host cell mechanisms.  The foundations for this work were laid by Felix d’Hérelle and others, working after 1917 with bacterial viruses in cultured bacteria.  Indeed, Macfarlane Burnet’s first important work was in 1929, showing by use of plaque counting that a single bacterial cell infected with a single phage produced 20 – 100 progeny some 20 minutes following infection.  The fact that phages adsorbed irreversibly to their hosts as part of the infection process was shown by AP Krueger and M Schlesinger in 1930 – 1931.  Schlesinger later showed between 1934 and 1936 that the bacteriophage he worked with consisted of approximately equal amounts of protein and DNA, the first proof that viruses might be nucleoprotein in nature.

However, it took until 1939 for the former physicist Max Delbrück, working with the biologist Emory Ellis at Caltech, to elucidate the growth cycle of a sewage-isolated Escherichia coli bacteriophage in a now-classic paper simply entitled “The Growth of Bacteriophage”.  This used the simple technique of counting plaques in a bacterial lawn in a Petri dish, following infection of a standard bacterial inoculum with a dilution series of a phage preparation.

Their principal finding was that viruses multiply inside cells in one step, and not by division and exponential growth like cells.. This was determined using the so-called “one-step growth curve”, which allowed the accurate determination of the titres of viruses released from bacteria that had been synchronously infected.  This allowed calculation of not only the time of multiplication of the virus, but also the “burst size” from individual bacteria, or the number of viruses produced in one round of multiplication.  This was a fundamental discovery, and allowed the rapid progression of the field of bacterial and phage genetics

One important facet of this work was that it showed that infection could be caused by single phages: the power of the plaque assay meant that even dilutions of phage preparations that contained only a single particle could produce a detectable plaque.

The Phage Group was started in the 1940s after Delbrück and Salvador Luria – also famous for inventing the Luria broth used to this day to grow bacteria -  met at a conference.  They soon began to collaborate, and in 1943 published the famous Luria–Delbrück experiment or Fluctuation Test: this showed that resistance to phage infection in bacteria could arise spontaneously and without selection pressure.

Also in 1943, they added Alfred Hershey to the group.  An important early result of their joint work was the proof that co-infection of one bacterium with two different bacteriophages could lead to genetic recombination, or mixing of the phage genomes.  Hershey and his assistant Martha Chase subsequently went on in 1952 to perform the legendary Hershey-Chase experiment in order to prove whether or not DNA was the genetic material of the phage: this purportedly used a new high speed Waring blender Hershey had purchased for his wife, but which never made it to her.  They grew up preparations of the E coli bacteriophage T2 separately in the presence of the radioisotopes 35S and 32P, to label the protein and nucleic acid components of the phage respectively.  Their most exciting result was achieved by allowing adsorption of phages to bacteria in liquid suspension for different times, then shearing off adsorbed phage particles from the bacteria using the blender.  Pelleting the bacteria by centrifugation and assaying radioactivity allowed them to determine that over 75% of the 35S – incorporated into cysteine and methionine amino acids – remained in the liquid, or outside the bacteria, whereas over 75% of the 32P – incorporated into the phage DNA - was found inside the bacteria.  Subsequent production of phage from the bacteria showed that DNA was probably the genetic material, and that protein was not involved in phage heredity.

Aside from their ground-breaking discoveries, the main influence of the Phage Group was felt via their establishment of the yearly summer phage course at Cold Spring Harbor Laboratory. From 1945 through to the 1960s, Delbrück and colleagues taught the fundamentals of bacteriophage biology and experimentation to generations of biologists, which helped to instill a culture of rigorous mathematical and analytical techniques in attendees – many of whom went on to help establish the emerging field of molecular biology.

Indeed, not only did Delbrück, Luria and Hershey receive the 1969 Nobel Prize for Physiology or Medicine for their work on bacteriophages, but Luria’s first graduate student James Watson was also awarded the prize in 1962 for his work with Francis Crick on elucidating the structure of DNA.  It is a not particularly well known fact that Watson honed his analytical skills for 3-D reconstructions from X-ray data of DNA with data from TMV, which he helped to show had helical virions.

Animal cell culture

Possibly the most important development for the study of animal viruses since their discovery was the growing of poliovirus in cell culture: this was reported in 1949 by John Enders, Thomas Weller and Frederick Robbins from the USA, and was rewarded with a joint Nobel Prize to them in 1954.  They did this around the same time as David Bodian and Isabel Morgan identified three distinct types of poliovirus.

While both bacterial and plant viruses could be both grown and assayed in “culture” – bacterial cells for phages, and plants for viruses like TMV – it was very difficult to grow and work with animal viruses, and especially to assay them, or measure their concentration.  While the pock assay done on egg membranes for influenza virus was very useful, it was not applicable to many viruses.  Indeed, people working with animal and human viruses were envious of the advantages enjoyed by their colleagues working with bacteriophages and plant viruses, because their assay systems were far more generally useful, even if local lesion assays on leaves for plant virus were limited compared to the precision obtainable for bacteriophages using pure cultures of bacterial cells on Petri dishes.  Titration or assay of poliovirus, for example, required the injection of virus preparations into the brains of monkeys, or later, in the case of the Lansing or Type II poliovirus strain, into brains of mice.

The technological advances that led to the breakthrough were incremental, and in fact had occurred over a period of over sixty years: Wilhelm Roux is credited with creating the first “tissue culture” with animal cells, by maintaining extracts of chicken embryos in warmed saline in 1885.  Other early workers had used minced-up chick embryos as far back as the early 1900s; roller-tube cultures had been in use for some time for studying viruses; a number of human and other tissues had been used to culture viruses.  Part of the development was, however, the increased ease of making the necessary reagents, such as ultrafiltered bovine serum, and a greater understanding of the requirements of cells for successful growth in culture.  Another major enabling factor was the post-Second World War availability of antibiotics, which meant contaminating microorganisms could be killed in culture – which had been impossible previously.

Enders, Weller and Robbins started with a suspended cell culture of human embryo skin and muscle tissue – a technique first described in 1928 – with the idea of studying varicella zoster herpesvirus.  However, in a case of chance favouring the prepared mind(s), the proximity of these tissue cultures and the Lansing strain of poliovirus in the same lab led to them using this instead, as part of an effort to determine whether all polioviruses exclusively multiplied in human nervous tissue.

Their cultures were started by inoculation with a suspension of infected mouse brains, and re-inoculation of mice with tissue culture fluids demonstrated that the virus was multiplying.  Injection of fluid into monkey brains after three passages of tissue culture resulted in typical symptoms of paralysis.  Later, Types I and III poliovirus were also successfully cultured – and suspended cell cultures of intestine, liver, kidney, adrenals, brain, heart, spleen, lung and brain derived from human embryos were also found to support growth of various polioviruses.

Adaptation of the culture technique to roller-tubes allowed higher yields of virus – and the possibility of direct observation of the effects of virus multiplication on large sheets of cells, rather than in clumps and pieces of tissue from suspension cultures.  These effects were termed “cytopathogenic” (now generally cytopathic) for the direct damage and morphological changes to cells that could be seen and measured, and roller-tubes made it far easier and quicker to do this by simple staining of cultures with various reagents such as haemotoxylin and eosin.

The technique of looking at cells for cytopathic effects (also abbreviated as CPE) quickly found application in assays of infectivity – and therefore of concentration – of poliovirus preparations.  It was also possible to do neutralisation assays with immune human sera.  There was also the observation that passaging the Lansing strain through cell suspensions reduced its virulence in mice, and similar passage of Type I poliovirus significantly reduced virulence in rhesus macaques.  These developments together were part of the advances that led to the development of live poliovirus vaccines soon afterwards.

These observations also quickly found application with a wide variety of other human and animal viruses, which triggered an explosion in these fields that led to them rapidly overtaking plant and bacterial virology in terms of understanding how the viruses replicated, and developing assays and vaccines for them.  Indeed, the poliovirus work was rapidly followed in the same lab by the isolation of herpes zoster and herpes simplex viruses; the agent of measles was characterised by Thomas Peebles and Enders via tissue culture by 1954; adenoviruses were discovered in 1953 by Wallace Rowe and Robert Huebner and shown to be associated with acute respiratory disease soon afterwards, by Maurice Hilleman and others.

Click here for Part 1: Introduction

and here for Part 2: Egg Culture and EM

Copyright Edward P Rybicki and Russell Kightley, February 2015, except where otherwise noted.

Ebola virus mutating, scientists say

29 January, 2015

Scientists at the Institut Pasteur in France who are tracking the Ebola outbreak in Guinea say the virus has mutated.


I would be surprised it there weren’t evidence by now of adaptation to humans: never in any previous outbreak of EHD [Ebola haemorrhagic disease] has the person-person chain of transmission been sustained for so long, meaning never before has there been the opportunity for human-specific adaptations to become established.

The article points out that on consequence of mutation may be that the virus becomes less virulent, leading to a greater incidence of asymptomatic infection – of which there is already evidence from previous outbreaks, and which has been implicated in the lessening incidence of transmission because of increasing herd immunity.

However, this same property might lead to increased transmission to the non-exposed, because of a lack of signs that contacts with the infected person(s) should be avoided – and for a disease as lethal as EHD, even a reduced mortality rate still means you should avoid it at all costs.

The idea of developing a modified live measles virus vaccine as an Ebola virus vaccine vector, which is what the Institut Pasteur is apparently doing, seems to be a very good one.  Measles is still a major potential problem in that part of the world, necessitating regular infant immunisations, and coupling anti-measles with an anti-Ebola vaccine in those countries is probably very good use of both a proven vaccine and existing EPI infrastructure.


See on Scoop.itVirology News

More Surprises in the Development of an HIV Vaccine

14 November, 2014

More Surprises in the Development of an HIV Vaccine

In the current issue of Frontiers in Immunology, Jean-Marie Andrieu and collaborators, report results from non-human primate experiments designed to explore a new vaccine concept aimed at inducing tolerance to the simian immunodeficiency virus (SIV) (1). This approach, which is significantly different from other vaccine concepts tested to date, resulted in a surprisingly high level of protection. If the results are confirmed and extended to the human immunodeficiency virus (HIV), this approach may represent a game changing strategy, which should be welcomed by a field that has been marred by mostly disappointing results.


HIV Graphic from Russell Kightley Media



This is a commentary by two well-respected friends of mine on a very surprising result published by the Andrieu group recently, which seems to have been ignored by the mainstream HIV vaccine world.

This is not surprising, in that Andrieu is an outsider in this field – he is a cancer researcher – but is typical of the disappointing tendency in science to ignore contributions from outside the various "Golden Circles" that exist for various specialties.

Something that should elicit interest, though, is that this group has shown that a previously obscure 

"…population of non-cytolytic MHCIb/E-restricted CD8+ T regulatory cells [that] suppressed the activation of SIV positive CD4+ T-lymphocytes".

This is interesting because Louis Picker’s groups’ recent findings, announced at the recent HIVR4P conference in Cape Town, highlighted the involvement of MHC-E proteins in what amounted to a cure of SIV infection in macaques by a modified Rhesus cytomegalovirus (RhCMV) HIV vaccine vector (see here: 

I tweeted at the time:

"Universal MHC-E-restricted CD8+ T cells – break all the rules for epitope recognition"

Could this be a link between the two mechanisms – both from way outside the orthodoxy, I will point out?

It will be interesting to see.

See on Scoop.itVirology News

Ethical dilemma for Ebola drug trials

13 November, 2014

Public-health officials split on use of control groups in tests of experimental treatments.

With clinical trials of experimental Ebola treatments set to begin in December, public-health officials face a major ethical quandary: should some participants be placed in a control group that receives only standard symptomatic treatment, despite a mortality rate of around 70% for Ebola in West Africa?

Two groups planning trials in Guinea and Liberia are diverging on this point, and key decisions for both are likely to come this week. US researchers meet on 11 November at the National Institutes of Health (NIH) in Bethesda, Maryland, to discuss US-government sponsored trials. A separate group is gathering at the World Health Organization (WHO) in Geneva, Switzerland, on 11 and 12 November to confer on both the US effort and trials organized by the WHO with help from African and European researchers and funded by the Wellcome Trust and the European Union.


I have to say – faced with a deadly disease, I think it is UNethical to have control / placebo arms of any trial.

Seriously: what about comparing ZMapp and immune serum, for example, with historical records of previous standard of care outcomes rather than directly?

I know if I were an Ebola patient, and I saw someone else getting the experimental therapy and I didn’t, that I would have a few things to say.

It’s not as if these therapies have not been tested in primates, after all – in fact, both the ChAd3 and MVA-based vaccines and ZMapp have been thoroughly tested in macaques, as have the other therapeutics, with no adverse events there.

I say if people say clearly that they want an experimental intervention, that they should get one: after all, the first use of immune serum was not done in a clinical trial, but rather as a last-ditch let’s-see-if-this-works intervention – yet its use does not seem controversial?

See on Scoop.itVirology News

Virology Africa 2015: consider yourselves notified!

7 November, 2014

Dear ViroBlogy and Virology News followers:

Anna-Lise Williamson and I plan to have another in our irregular series of “Virology Africa” conferences in November-December 2015, in Cape Town.

As previously, the conference will run over 3 days or so, possibly with associated workshops, and while the venue is not decided, we would like to base it at least partially in the Victoria & Alfred Waterfront.

We also intend to cover the whole spectrum of virology, from human through animal to plant; clinical aspects and biotechnology.

We intend to make it as cheap as possible so that students can come. We will also not be inviting a slate of international speakers, as we have found that we always get quite an impressive slate without having to fund them fully.

It is also the intention to have a Plant Molecular Farming workshop – concentrating on plant-made vaccines – concurrently with the conference, in order to leverage existing bilateral travel grants with international partners. If anyone else has such grants that could be similarly leveraged, it would be greatly appreciated.

See you in Cape Town in 2015!

Ed + Anna-Lise

ZMapp in an HIV context

30 October, 2014

It was truly a pleasure to run into Kevin Whaley of Mapp BioPharmaceutical today, here at the HIVR4P inernational conferrence in Cape Town – so I made him come and have coffee with me and Anna-Lise, so we could chat about molecular farming.
Of course, it is the ZMapp plant-made therapeutic antibody that has set the molecular farming world alight, that was the main topic. Apparently Mapp is looking at a January 2015 date for a clinical trial in the affected West African countries, alongside the adenovirus and RSV-vectored vaccines. The plants for the production of the thousands of doses that will be needed – and recall, that’s a couple of grams per dose at 50 mg/kg – are already growing at Kentucky Bioprocessing in Louisville, so one imagines that a pile of work will be coming their way in the near future.
It’s also sobering to realise that even though plants ARE a more scalable and POTENTIALLY cheaper means of production of biologics, that therapeutic antibody production in particular, MAY be better suited right now to conventional technologies, such as CHO cell or even fungal production.
This is because large quantities of MAbs will be needed, and there is established capacity for production of hundreds of thousands of litres of cell culture right now, and yields and production costs have been driven right down to US$10 / gram for MAbs already, according to Kevin.
This partly answers a question I had during the HIVR4P sessions: if one is to use 20-50 mg/kg dosages for anti-HIV neutralising MAbs such as VRC01, how would it be remotely possible to make the amounts required for use in a developing country setting, where the patient can almost definitely NOT pay?
I still think there is a role for plants – but maybe this will be in the area of prophylactic use of MAbs, where much lower doses may be effective because there is not nearly as much virus to neutralise or inactivate.
And of course, Mapp is involved here too, with plant-made VRC01 in particular being incorporated into microbicides.
A great bunch of people, with really noble aims.

Rabies Vaccine Protects Nonhuman Primates against Deadly Ebola Virus

26 October, 2014

The research team is pursuing the inactivated rabies/Ebola vaccine for use in humans. The live vaccine is being developed for use in protecting wildlife at risk of Ebola virus infection in Africa, which could also serve to prevent transmission into the human population.


I missed this one at the time – and it is an interesting piece of news.  Basically, the research team cloned the Ebola envelope glycoprotein GP1 into the extant rabies virus vaccine strain genome, and tested a live version, a replication-deficient version, and a killed whole virion version in macaques.

Their results are interesting enough – 100% protection against challenge for live, 50% for the other two – that they plan to follow up to see whether or not additional doses could improve protection in the two non-replicating versions, and to make a “multivalent filovirus vaccine”.

This can only be welcome news against the backdrop of the still-ongoing epidemic in West Africa – where two other vaccines (recombinant vesicular stomatitis and chimpanzee adenovirus) are probably going to be trialled next year. The rabies version at least is based on a very well characterised vaccine that already protects against an extremely deadly disease – it remains to be seen how well the other two do.

I forgot to mention that I found reference to this article on “The Zombie Research Society”‘s blog site: A very apt place if one considers the parallels that are already being drawn between Ebola and a “zombie virus”.

And because I like zombies B-)

See on Scoop.itVirology News

Packs of wild dogs spread Ebola after eating corpses!! Or…not, maybe?

13 October, 2014

Packs of wild dogs spread Ebola after eating corpses

The ever-evolving Ebola narrative is broaching into ludicrous territory, with reports now claiming that wild dogs are going around digging up the rotting remains of deceased victims and eating their flesh in the streets. Special Ebola graveyards, where the dead are being buried in haste and at shallow depths, are reportedly feasting grounds for these dogs, which officials say are capable of spreading the disease to humans.

The Daily Mail says Liberian villagers first came across the dogs while going about their daily routines. Right in the middle of busy streets, they said, hungry hounds were allegedly seen ripping through rotting corpses, to the shock of onlookers. After determining the source of the bodies, it was revealed that shallow graves were to blame.


Stephen Korsman of the Division of Medical Virology at UCT just alerted me to this article, in some distress because they had misquoted him and used his comments out of context.  This is a rather wild, sensationalist and highly inaccurate piece from a fringe web site that seems to have blocked me from commenting, because of previous criticism.  So, I’ll just do it here.

They comment: "Logically speaking, it makes little sense that asymptomatic dogs are possible Ebola carriers while asymptomatic humans are not. There exists no credible science to substantiate this apparent inconsistency beyond the baseless claims made by government health officials."

Utter garbage: bats carry Nipah virus, SARS-CoV, Ebola, Marburg AND rabies essentially asymptomatically – and can transmit ALL of them to other mammals. So too can deer mice transmit Sin Nombre hantavirus in the south-western USA without showing symptoms.  Rodents transmit Lassa fever virus in West Africa every year, again without being symptomatic.  Mice can transmit various South American haemorrhagic fever viruses without obviously being sick. I wish they would get their facts straight: this is is very easily checked!

See on Scoop.itVirology News

Norway to get world’s last dose of ZMapp – update

8 October, 2014

The Norwegian woman, infected by the Ebola in Sierra Leone and currently receiving treatment in Oslo, will get the last dose of the virus treatment medicine ZMapp


…and yet again, the emphasis is on how slow it is to make it – when the whole point of biofarming and transient expression is that it is supposed to be QUICK to make things, and easy to scale up production!!

What is the problem here?  KBP has facilities – or says it does – for large-scale production of proteins via transient expression in N benthamiana via rTMV or even BeYDV-based vectors. SO why has it been so difficult to make more ZMapp??

Why, in fact, are we told via other reports that the US government is considering getting Caliber to make it, or even to make the cocktail in CHO cells, because of capacity, when KBP has the equipment?

It can’t be supply of plants, surely: if they’d planted out a big greenhouse or two of N benth the moment ZMapp hit the news, they’d have enough to make many grams of ZMapp right now – given that it takes just a few days of incubation post-infiltraiton to make the protein.

Surely it’s not a protein purification thing – because THAT’S pretty quick too, once the plants have been mushed.

So what IS the bottleneck? cGMP requirement? Lack of certified protocols / equipment? Can someone tell us??  Otherwise, a posterchild for biofarming will end up being made by good old stainless steel cell culture technology, and our favourite way of doing things will have been found to be wanting.

NOTE ADDED 10th October:

Never let it be said I was unwilling to get schooled by a former colleague…Kenneth Palmer just told me what the problem is:

“You may not be aware that the human dose of Zmapp is 12 grams per patient, 3 infusions of 4 grams each.  Check the dose in recent Nature paper. If yield of one antibody is 100 mg per kg and you have to produce three antibodies for Zmapp… If you do the arithmetic you will see why the process is “slow””.

So…. Doing just that, you end up with 30 kg N benthamiana per gm of ZMapp as a best-case yield – meaning 360 kg PER PATIENT.

That’s a LOT of N benth – and tooling up for that sort of plant production takes time. Thanks, Kenneth!

I would be VERY interested in a cost breakdown of ZMapp vs CHO cell-produced MAbs – because producing at that sort of scale MUST be prohibitively expensive in stainless steel?


See on Scoop.itPlant Molecular Farming


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