Viral variation in single cells

QuasispeciesIt is well known that virus populations display phenomenal diversity. Virus populations are dynamic distributions of nonidentical but related members called a quasispecies. This diversity is restricted in single cells, but is restored within two infectious cycles.

Single cells infected with vesicular stomatitis virus (VSV) were isolated using a glass microcapillary, and incubated overnight to allow completion of virus replication. Replication in a single cell imposes a genetic bottleneck, as few viral genomes are present. Virus-containing culture fluids were then subjected to plaque assay, during which 2 viral replication cycles took place. For each infected cell, 7-10 plaques were picked and used for massive parallel genome sequencing. A total of 881 plaques from 90 individual cells were analyzed in this way. Of the 532 single nucleotide differences  identified, 36 were also present in the parental virus stock.

An interesting observation was that over half of the infected cells contained multiple parental variants. However, the multiplicity of infection (MOI) that was used should have only resulted in multiple infections in 15% of the cells. The results cannot be explained by RNA recombination as this process occurs at a very low rate in VSV-infected cells. The key is that MOI only describes the infectious virus particles that are delivered to cells.  Because the particle-to-pfu ratio of VSV is high, it seems likely that many cells received both infectious and non-infectious particles. Furthermore, it is known that some RNA viruses may be transmitted to other cells in groups, either by aggregation of particles or within a membrane vesicle.

The conclusion from these results is very important: a single plaque-forming unit can contain multiple, genetically diverse particles.  Plaque purification has been used for years in virology to produce clonal virus stocks, but at least for VSV, a plaque is not produced by a single viral genome.

The 496 single nucleotide changes that were not present in the parent virus arose after the bottleneck imposed by single cell replication. Between 0 and 17 changes were identified in the 7-10 plaques isolated from each cell. The single-cell bottleneck restricted the parental virus diversity to 36 nucleotide changes. In contrast, within 2 viral generations, the viral diversity was over ten times greater (496 changes). This observation illustrates the capacity of the RNA virus genome to restore diversity after a bottleneck.

The number of changes identified in the 7-10 plaques isolated from each cell, between 0 and 17, shows that some cells produce more diverse progeny than others. At least two sources of this variation were identified. The viral yield per cell varied greatly, from 0 to over 3000 PFU. Greater virus yields means more viral RNA replication, and more change for diversity. Indeed, greater virus yields per cell was associated with more mutations in the progeny.

Another explanation for the variation in single-cell diversity comes from analysis of cell #36. This infected cell produced viruses with 17 changes not found in the parental virus, more than any other cell. One of these changes lead to a single amino acid change in the viral RNA polymerase. This amino acid change appears to increase the mutation rate of the enzyme. Similar mutators – changes that increase the error frequency – have also been described in the poliovirus RNA polymerase.

RNA viruses must carry out error-prone replication to adapt to new environments. A consequence is that RNA virus populations exist close to an error threshold beyond which infectivity is lost. How the balance is maintained is not understood. The results of this study suggest that some infected cells may produce a highly diverse population, while in others a more conserved sequence is maintained. This distribution of diversity might permit the necessary evolvability without the lethality conferred by having too many mutations.

I would be very interested to know if the conclusions of this work would be changed by the ability to determine the sequences of all the viral genomes recovered from a single infected cell. The authors note that this is not technically possible, but surely will be in the future.

An Ebolavirus vaccine in Africa

filovirionAn Ebolavirus vaccine has shown promising results in a clinical trial in Guinea. This vaccine has been in development since 2004 and was made possible by advances in basic virology of the past 40 years.

The ability to produce the Ebolavirus vaccine, called rVSV-EBOV, originates in the 1970s with the discovery of the enzyme reverse transcriptase, the development of recombinant DNA technology, and the ability to rapidly and accurately determine the sequence of nucleic acids. These advances came together in 1981 when it was shown that cloned DNA copies of RNA viral genomes (a bacteriophage, a retrovirus, and poliovirus), carried in a bacterial plasmid, were infectious when introduced into mammalian cells. Production of an infectious DNA copy of the genome of vesicular stomatitis virus (VSV) was reported in 1995. In their paper the authors noted:

Because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

This technology was subsequently used in 2004 to produce replication competent VSV carrying the genes encoding the glycoproteins of filoviruses, which others had shown are the targets of neutralizing antibodies. When injected into mice, these recombinant viruses induced neutralizing antibodies that were protective against lethal disease after challenge with Ebolavirus.

In a series of experiments done over the next 10 years, rVSV-EBOV was shown to protect nonhuman primates from lethal disease. In these experiments, animals were injected intramuscularly with the vaccine and challenged with Ebolavirus. The vaccine induced protection against lethal disease and prevented viremia. Extensive studies of the VSV vector in ~80 nonhuman primates showed no serious side effects, and only transient vector viremia.

The rVSV-EBOV was originally developed by Public Health Agency of Canada, and subsequently licensed to NewLink Genetics. Financial support has been provided from Canadian and US governments and others. From 2005 to the present, the NIH Rocky Mountain Laboratory in Hamilton, Montana has also been involved in this work, particularly with nohuman primate challenge studies. In November 2014 Merck entered an agreement with NewLink to manufacture and distribute the vaccine.

In August 2014, well into West Africa Ebolavirus outbreak, Canada donated 800 vials of vaccine to WHO, which then established the VSV Ebola Consortium (VEBCON) to conduct human trials.

The results of Phase I trials of rVSV-EBOV in Africa (Gabon, Kenya) and Europe (Hamburg, Geneva) were published on 1 April 2015. These trials comprised three open-label, dose-escalation trials, and one randomized, double blind controlled trial in 158 adults. Each volunteer was given one injection of 300,000 to 50 million plaque-forming units of rVSV-EBOV or placebo. No serious vaccine related events were reported, but immunization was accompanied by fever, joint pain, and some vesicular dermatitis. A transient systemic infection was observed, followed by development of Ebolavirus-specific antibody responses in all participants, and neutralizing antibodies in most.

The interim results of a phase III trial of rVSV-EBOV, begun on 23 March 2015 in Guinea, have just been published. It is a cluster-randomized trial with a novel design that is modeled on the ring vaccination approach used for smallpox eradication in the 1970s. In ring vaccination, individuals in the area of an outbreak are immunized, in contrast to treating a larger segment of the population. During this trial, when a case of Ebolavirus infection was identified, all contacts and contacts-of-contacts were identified. Some of these individuals were immediately immunized intramuscularly with 2 x 107 PFU, and others (randomly chosen) were immunized three weeks later. The primary outcome was Ebolavirus disease confirmed by PCR. As new cases arose in other areas (clusters), these were treated in the same way, hence the name of cluster-randomized trial.

The press has widely reported that the vaccine was ‘100% protective’. This outcome sounds much better than is represented by the data, so let’s look at the numbers.

Zero cases of Ebolavirus disease were observed in 2,014 immediately vaccinated people, while 16 cases were identified in those given delayed vaccine (n=2,380). These numbers were used to calculate the vaccine efficacy of 100%. While statistically significant, the numbers are small.

More telling are the results obtained when we consider all individuals eligible for immunization, not just those who were immunized (some were excluded for a variety of reasons). Of 4,123 eligible individuals, 2,014 were immunized as noted above, but 2,109 did not receive vaccine. Eight cases of Ebola virus disease were noted in the non-immunized population. This number is small, a consequence of the fact that the outbreak is waning.

On the basis of these interim results, the data and safety monitoring board decided that the trial should continue. However because the board felt that the vaccine is a success, they decided to curtail randomization of subjects into immediately vaccinated and delayed vaccinated groups. Now all contacts and contacts-of-contacts will immediately receive vaccine. As a consequence of this change, it will not be possible to improve the accuracy of vaccine efficacy. For example, when many more individuals are immunized in the future, many fewer that 100% might be protected from disease.

There are two lessons I would like you to remember from this brief history of an Ebolavirus vaccine. Developing a vaccine takes a long time (minimum 11 years for rVSV-EBOV) and depends on advances made with both basic and clinical research.  Don’t believe anyone who says that this vaccine was made in a year. And always look at the numbers when you hear that a vaccine has 100% efficacy.

TWiV 311: Bulldogs go viral

On episode #311 of the science show This Week in Virology, Vincent visits the University of Georgia where he speaks with Zhen Fu and Biao He about their work on rabies virus and paramyxoviruses.

You can find TWiV #311, audio and video versions, at

A new rhabdovirus from a patient with hemorrhagic fever

Viral hemorrhagic fevers in AfricaHemorrhagic fevers are among the most graphic viral diseases, inspiring movies, novels, and a general fear of infection. They are characterized by an abrupt onset and a striking clinical course involving bleeding from the nose and mouth, vomiting with blood, and bloody diarrhea. The most famous hemorrhagic fevers are produced by infection with filoviruses like Ebola virus, but members of three other viral families – Arenaviridae, Bunyaviridae, and Flaviviridae – can also cause this syndrome. The isolation of a novel rhabdovirus from an African with hemorrhagic fever suggests that members of a fifth viral family can also cause this disease.

Three cases of hemorrhagic fever that occurred in the spring of 2009 were noteworthy because none of the typical viral suspects could be detected in one patient. Two were young (13, 15 year old) students in the village of Mangala, Bas-Congo province, Democratic Republic of Congo. They lived near each other and went to the same school. Both arrived at the local health center with typical symptoms of hemorrhagic fever, and both died 2-3 days later. The third case was a 32 year old male nurse at the health center who was involved in the care of the other two patients. He developed symptoms of hemorrhagic fever but recovered within a few days.

Deep sequence analysis of RNA extracted from the serum of patient #3 revealed the presence of a novel rhabdovirus, provisionally named Bas-Congo virus (BASV). Phylogenetic analyses reveal that BASV is substantially diverged from the two main human rhabdoviruses, rabies virus and Chandipura virus (ten of the 160 known species of rhabdoviruses have been isolated from humans). BASV is more related to viruses of the Tibrogargan group and the Ephemerovirus genus, which contain arthropod-borne viruses that infect cattle, but clusters separately in an independent branch of the phylogenetic tree.

Antibodies to BASV were detected in the serum of patient #3 and also in the serum of an asymptomatic nurse who had cared for this patient. However, no antibodies to this virus were found in 43 other serum samples from individuals with hemorrhagic fever of unknown origin. These samples came from individuals who lived in 9 of the 11 provinces of the DRC, including Bas-Congo. Nor were antiviral antibodies detected in plasma from 50 random blood donors in one DRC province.

Although the viral genome sequence was determined from RNA extracted from patient serum (where there were 1 million copies per ml of the viral RNA), the virus did not replicate in cell cultures from monkey, rabbit, and mosquito, or in suckling mice. These findings are in contrast to those obtained with a newly discovered coronavirus in humans. It is likely that the samples had not been kept sufficiently cold to maintain viral infectivity. It should be possible to recover virus from a cloned DNA copy of the viral genome.

These data suggest, but do not prove, that BASV caused hemorrhagic fever in the 3 patients. All three cases occurred in a 3 week period within the same small village. BASV nucleic acid and antibodies were detected in the third patient. Given that viruses of the closely related Tibrogargan group and the Ephemerovirus genus are transmitted to cattle by biting midges, it is possible that the initial infections were transmitted by such an arthropod vector. Human to human transmission of the virus could have taken place when the nurse was infected by one or both pediatric patients. However, it should be noted that infection with BASV was not confirmed in either of the first two cases as no clinical samples were available. Other etiologies for this outbreak of hemorrhagic fever should not be ruled out.

Rhabdoviruses are known to cause encephalitis, vesicular stomatitis, or flu-like illness in humans, not hemorrhagic fevers. But these viruses clearly have the potential to cause this disease: members of the Novirhabdovirus genus cause hemorrhagic septicemia in fish. As long as there are viruses to discover, any rules we make about them should be considered breakable.

G Gerard, JN Fair, D Lee, E Silkas, I Steffen, J Muyembe, T Sittler, N Veerarghavan, J Ruby, C Wang, M Makuwa, P Mulembakani, R Tesh, J Mazet, A Rimoin, T Taylor, B Schneider, G Simmons, E Delwart, N Wolfe, C Chiu, E Leroy. 2012. A novel rhabdovirus associated with acute hemorrhagic fever in central Africa. PLoS Pathogens  8.

VHSV – A Deadly Virus of Fish

vsv virionI came across an article in about VHSV, or viral hemorrhagic septicemia virus, which causes disease in fish. I have never worked on viruses of fish, but they’re fascinating, and economically very, very important. So let’s have a look at what this article is about.

VHSV causes a very serious disease of fresh and saltwater fish. Signs of VHSV infection include hemorrhaging, anemia, bulging eyes, and bloated abdomens, but not all fish display symptoms. The virus is lethal, causing outbreaks in which thousands of fish die.

VHSV is a member of the family Rhabdoviridae, which also includes the well known human pathogen rabies virus. However, VHSV is not known to infect humans. Members of the Rhabdovirus family have a enveloped capsid containing a single-stranded RNA genome of negative polarity. The diagram show a typical rhabdovirus, vesicular stomatitis virus.

VHSV was first isolated in the middle of the 20th century, when it became a disease of cultured rainbow trout. Since then the virus has been found to cause disease in many other fresh and saltwater fish. It first infected freshwater fish in the western hemisphere in 2005, when it was reported in Lake Ontario. The article in cited above suggests that the virus might be headed for Lake Michigan, because it has been detected in a northern region of Lake Huron only 20 miles away. The article discusses the potential effect of the virus on the $4 billion per year sport fishing industry in the Great Lakes.

How does the virus spread? In the water, of course: infected fish excrete the virus in their urine. The virus probably made its way into the Great Lakes in the ballast of ships – the water stored in the bottom to stabilize the vessels.