On episode #304 of the science show This Week in Virology, the TWiV team consults an epidemiologist to forecast the future scope of the Ebola virus epidemic in West Africa.

You can find TWiV #304 at www.twiv.tv.

Transmission of Ebola virus

27 September 2014

jet nebulizerAs the West African epidemic of Ebola virus grows, so does misinformation about the virus, particularly how it is transmitted from person to person. Ebola virus is transmitted from human to human by close contact with infected patients and virus-containing body fluids. It does not spread among humans by respiratory aerosols, the route of transmission  of many other human viruses such as influenza virus, measles virus, or rhinovirus. Furthermore, the mode of human to human transmission of Ebola virus is not likely to change.

What is aerosol transmission? Here is a definition from Medscape:

Aerosol transmission has been defined as person-to-person transmission of pathogens through the air by means of inhalation of infectious particles. Particles up to 100 μm in size are considered inhalable (inspirable). These aerosolized particles are small enough to be inhaled into the oronasopharynx, with the smaller, respirable size ranges (eg, < 10 μm) penetrating deeper into the trachea and lung.

All of us emit aerosols when we speak, breathe, sneeze, or cough. If we are infected with a respiratory virus such as influenza virus, the aerosols contain virus particles. Depending on their size, aerosols may travel long distances, and when inhaled they lodge on mucosal surfaces of the respiratory tract, initiating an infection.

Viral transmission can also occur when virus-containing respiratory droplets travel from the respiratory tract of an infected person to mucosal surfaces of another person. Because these droplets are larger, they cannot travel long distances as do aerosols, and are considered a form of contact transmission. Ebola virus can certainly be transmitted from person to person by droplets.

Medical procedures, like intubation, can also generate aerosols. It is possible that a health care worker could be infected by performing these procedures on a patient with Ebola virus disease. But the health care worker will not transmit the virus by aerosol to another person. In other words, there is no chain of respiratory aerosol transmission among infected people, as there is with influenza virus.

In the laboratory, machines called nebulizers (which are used to administer medications to humans by inhalation) can be used to produce virus-containing aerosols for studies in animals. A human would likely be infected with an Ebola virus-containing aerosol generated by a nebulizer (theoretically; such an experiment would be unethical).

A variety of laboratory animals have been infected with Ebola virus (Zaire ebolavirus) using aerosols. In one study rhesus macaques were infected with aerosolized Ebola virus using a chamber placed over the animals’ heads. This procedure resulted in replication of the virus in the respiratory tract followed by death. Virus particles were detected in the respiratory tract, but no attempts were made to transmit infection from one animal to another by aerosol. In another study, cynomolgous macaques, rhesus macaques, and African Green monkeys could be infected with Ebola virus aerosols using a head-only chamber. Virus replicated in the respiratory tract, and moved from regional lymph nodes to the blood and then to other organs. Virus titers in the respiratory tract appeared to be lower than in the previous study. No animal to animal transmission experiments were done.

When rhesus macaques were inoculated intramuscularly with Ebola virus,  virus could be detected in oral and nasal swabs; however infection was not transmitted to animals housed in separate cages. The authors conclude that ‘Airborne transmission of EBOV between non-human primates does not occur readily’.

Pigs can also be infected with Ebola virus. In one study, after dripping virus into the nose, eyes, and mouth, replication to high titers was detected in the respiratory tract, accompanied by severe lung pathology. The infected pigs can transmit infection to uninfected pigs in the same cage, but this experimental setup does not allow distinguishing between aerosol, droplet, or contact spread.

In another porcine transmission experiment, animals were infected oronasally as above, and placed in a room with cynomolgous macaques. The pigs were allowed to roam the floor, while the macaques were housed in cages. All of the macaques became infected, but their lungs had minimal damage. However it is not known how the virus was transmitted from pigs to macaques. The authors write: ‘The design and size of the animal cubicle did not allow to distinguish whether the transmission was by aerosol, small or large droplets in the air, or droplets created during floor cleaning which landed inside the NHP cages’. The authors also indicate that transmission between macaques in similar housing conditions was never observed.

While these experimental findings show that animals can be infected with Ebola virus by aerosol, they do not provide definitive evidence for animal to animal transmission via this route. It is clear is that the virus does not transmit via respiratory aerosols among nonhuman primates.

We do not know why, in humans or non-human primates, Ebola virus does not transmit by respiratory aerosols. The virus might not reach sufficiently high titers in the respiratory tract, or be stable in respiratory secretions, to be efficiently transmitted by this route. There are many other possibilities. A careful study of Ebola virus titers in the human respiratory tract, and in respiratory secretions, would be valuable. However during Ebola virus outbreaks the main concern is to save people, not conduct experiments.

These experiments reveal the large gaps in our understanding about virus transmission in general, and specifically why Ebola virus is not transmitted among primates by respiratory aerosols.

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TWiV 303: Borna this way

21 September 2014

On episode #303 of the science show This Week in Virology, the TWiV team discusses transmission of Ebola virus, and inhibition of Borna disease virus replication by viral DNA in the ground squirrel genome.

You can find TWiV #303 at www.twiv.tv.

sneezeIn a recent New York Times OpEd entitled What We’re Afraid to Say About Ebola, Michael Osterholm wonders whether Ebola virus could go airborne:

You can now get Ebola only through direct contact with bodily fluids. If certain mutations occurred, it would mean that just breathing would put one at risk of contracting Ebola. Infections could spread quickly to every part of the globe, as the H1N1 influenza virus did in 2009, after its birth in Mexico.

Is there any truth to what Osterholm is saying?

Let’s start with his discussion of Ebola virus mutation:

But viruses like Ebola are notoriously sloppy in replicating, meaning the virus entering one person may be genetically different from the virus entering the next. The current Ebola virus’s hyper-evolution is unprecedented; there has been more human-to-human transmission in the past four months than most likely occurred in the last 500 to 1,000 years.

When viruses enter a cell, they make copies of their genetic information to assemble new virus particles. Viruses such as Ebola virus, which have genetic information in the form of RNA (not DNA as in other organisms), are notoriously bad at copying their genome. The viral enzyme that copies the RNA makes many errors, perhaps as many as one or two each time the viral genome is reproduced. There is no question that RNA viruses are the masters of mutation. This fact is in part why we need a new influenza virus vaccine every few years.

The more hosts infected by a virus, the more mutations will arise. Not all of these mutations will find their way into infectious virus particles because they cause lethal defects. But Osterholm’s statement that the evolution of Ebola virus is ‘unprecedented’ is simply not correct. It is only what we know. The virus was only discovered to infect humans in 1976, but it surely infected humans long before that. Furthermore, the virus has been replicating, probably for millions of years, in an animal reservoir, possibly bats. There has been ample opportunity for the virus to undergo mutation.

More problematic is Osterholm’s assumption that mutation of Ebola virus will give rise to viruses that can transmit via the airborne route:

If certain mutations occurred, it would mean that just breathing would put one at risk of contracting Ebola. Infections could spread quickly to every part of the globe, as the H1N1 influenza virus did in 2009, after its birth in Mexico.

The key phrase here is ‘certain mutations’. We simply don’t know how many mutations, in which viral genes, would be necessary to enable airborne transmission of Ebola virus, or if such mutations would even be compatible with the ability of the virus to propagate. What allows a virus to be transmitted through the air has until recently been unknown. We can’t simply compare viruses that do transmit via aerosols (e.g. influenza virus) with viruses that do not (e.g. HIV-1) because they are too different to allow meaningful conclusions.

One approach to this conundrum would be to take a virus that does not transmit among mammals by aerosols – such as avian influenza H5N1 virus – and endow it with that property. This experiment was done by Fouchier and Kawaoka several years ago, and revealed that multiple amino acid changes are required to allow airborne transmission of H5N1 virus among ferrets. These experiments were met with a storm of protest from individuals – among them Michael Osterholm – who thought they were too dangerous. Do you want us to think about airborne transmission, and do experiments to understand it – or not?

The other important message from the Fouchier-Kawaoka ferret experiments is that the H5N1 virus that could transmit through the air had lost its ability to kill. The message is clear: gain of function (airborne transmission) is accompanied by loss of function (virulence).

When it comes to viruses, it is always difficult to predict what they can or cannot do. It is instructive, however, to see what viruses have done in the past, and use that information to guide our thinking. Therefore we can ask: has any human virus ever changed its mode of transmission?

The answer is no. We have been studying viruses for over 100 years, and we’ve never seen a human virus change the way it is transmitted.

HIV-1 has infected millions of humans since the early 1900s. It is still transmitted among humans by introduction of the virus into the body by sex, contaminated needles, or during childbirth.

Hepatitis C virus has infected millions of humans since its discovery in the 1980s. It is still transmitted among humans by introduction of the virus into the body by contaminated needles, blood, and during birth.

There is no reason to believe that Ebola virus is any different from any of the viruses that infect humans and have not changed the way that they are spread.

I am fully aware that we can never rule out what a virus might or might not do. But the likelihood that Ebola virus will go airborne is so remote that we should not use it to frighten people. We need to focus on stopping the epidemic, which in itself is a huge job.

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TWiV 302: The sky is falling

14 September 2014

On episode #302 of the science show This Week in Virology, the TWiVers discuss the growing Ebola virus outbreak in West Africa, and an epidemic of respiratory disease in the US caused by enterovirus D68.

You can find TWiV #302 at www.twiv.tv.

Enterovirus

EV-A71 by Jason Roberts

During the winter of 1962 in California, a new virus was isolated from the oropharynx of 4 children who had been hospitalized with respiratory disease that included pneumonia and bronchiolitis. On the basis of its physical, chemical, and biological properties, the virus was classified as an enterovirus in the picornavirus family. Subsequently named enterovirus D68, it has been rarely reported in the United States (there were 79 isolations from 2009-2013). Towards the end of August 2014, an outbreak of severe respiratory disease associated with EV-D68 emerged in Kansas and Illinois.

Hospitals in Kansas City, Missouri, and Chicago, Illinois reported to the CDC an increase in the number of patients hospitalized with severe respiratory illness. EV-D68 was subsequently identified by polymerase chain reaction and nucleotide sequencing in 19/22 and 11/14 nasopharyngeal specimens from Kansas City and Chicago, respectively. Median ages of the patients were 4 and 5 years in the two cities, and most were admitted to the pediatric intensive care units due to respiratory distress. Other states have reported increases in cases of severe respiratory illness, and these are being investigated at CDC to determine if they are also associated with EV-D68.

There is no vaccine to prevent EV-D68 infection, nor is antiviral therapy available to treat infected patients. Current treatment is supportive to assist breathing; in a healthy individual the infection will resolve within a week. In the current outbreak no fatalities have been reported.

EV-D68 has been previously associated with mild to severe respiratory illness and is known to cause clusters of infections. It is not clear why there has been a sudden increase in the number of cases in the US. According to Mark Pallansch, Director of the Division of Viral Diseases at CDC, “our ability to find and detect the virus has improved to the point where we may now be recognizing more frequently what has always occurred in the past. So a lot of these techniques are now being applied more routinely both at the CDC but also at state health departments.” (Source: NPR).

I am sure that the nucleotide sequence of the EV-D68 virus isolated from these patients will reveal differences with previous strains. However whether or not those changes have anything to do with the increased number of isolations in the US will be very difficult to determine, especially as there is no animal model for EV-D68 respiratory disease.

Although how EV-D68 is transmitted has not been well studied, the virus can be detected in respiratory secretions (saliva, nasal mucus, sputum) and is therefore likely to spread from person to person by coughing, sneezing, or touching contaminated surfaces. The virus has been isolated from some of the children in California with acute flaccid paralysis, and there is at least one report of its association with central nervous system disease. In this case viral nucleic acids were detected in the cerebrospinal fluid. EV-D68 probably does not replicate in the human intestinal tract because the virus is inactivated by low pH.

Readers might wonder why a virus that causes respiratory illness is called an enterovirus. This nomenclature is largely historical: poliovirus, which replicates in the enteric tract, was the prototype member of this genus. Other viruses, including Coxsackieviruses and echoviruses, were added to the genus based on their physical and chemical properties. However soon it became apparent that many of these viruses could also replicate in the respiratory tract. Years later the rhinoviruses, which do not replicate in the enteric tract, were added to the enterovirus genus based on nucleotide sequence comparisons. While it was decided to keep the name ‘enterovirus’ for this group of viruses, it is certainly confusing and I would argue that it should be replaced by a more descriptive name.

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On episode #301 of the science show This Week in Virology, Vincent travels to the International Congress of Virology in Montreal and speaks with Carla Saleh and Curtis Suttle about their work on RNA interference and antiviral defense in fruit flies, and viruses in the sea, the greatest biodiversity on Earth.

You can find TWiV #301 at www.twiv.tv.

The Berlin patient

6 September 2014

HIV binding CD4 and ccrSince the beginning of the AIDS epidemic, an estimated 75 million people have been infected with HIV. Only one person, Timothy Ray Brown, has ever been cured of infection.

Brown was diagnosed with HIV while living in Berlin in 1995, and was treated with anti-retroviral drugs for more than ten years. In 2007 he was diagnosed with acute myeloid leukemia. When the disease did not respond to chemotherapy, Brown underwent stem cell transplantation, which involves treatment with cytotoxic drugs and whole-body irradiation to destroy leukemic and immune cells, followed by administration of donor stem cells to restore the immune system. When his leukemia relapsed, Brown was subjected to a second stem cell transplant.

The entry of HIV-1 into lymphocytes requires two cellular proteins, the receptor CD4, and a co-receptor, either CXCR4 or CCR5. Individuals who carry a mutation in the gene encoding CCR5, called delta 32, are resistant to HIV-1 infection. This information prompted Brown’s Berlin physician to screen 62 individuals to identify a stem cell donor who carried a homozygous CCR5∆32 mutation. Peripheral blood stem cells from the same donor were used for both transplants. 

Despite enduring complications and undergoing two transplants, Brown’s treatment was a success: he was cured both of his leukemia and HIV infection. Even though he had stopped taking antiviral drugs, there was no evidence of the virus in his blood following his treatment, and his immune system gradually recovered. Follow-up studies in 2011, including biopsies from his brain, intestine, and other organs, showed no signs of HIV RNA or DNA, and also provided evidence for the replacement of long-lived host tissue cells with donor-derived cells. Today Brown remains HIV-1 free.

Although Brown’s cure is somewhat of a medical miracle, and by no means a practical road map for treating AIDS, the example of the Berlin patient has galvanized research efforts and continues to inspire hope that a simpler and more general cure for infection may someday be achieved. Clinical trials have been conducted to test a variety of strategies in which CD4+ T or stem cells are obtained from a patient, the CCR5 gene is either mutated or its translation blocked by RNA interference, and then the resulting virus-resistant cells are returned to the patient. In one case zinc finger nucleases were used to delete the CCR5 gene in a patient’s cells, a procedure that we discussed in TWiV #278.

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Recording together for the first time, the hosts of the science show This Week in Virology celebrate their 300th recording at the American Society for Microbiology headquarters in Washington, DC, where Vincent  speaks with Dickson, Alan, Rich, and Kathy about their careers in science.

You can find TWiV #300 at www.twiv.tv.

Antibodies bound to poliovirus.

Antibodies (purple) bound to poliovirus. Image credit: Jason Roberts

Antigenic variation is a hallmark of influenza virus that allows the virus to evade host defenses. Consequently influenza vaccines need to be reformulated frequently to keep up with changing viruses. In contrast, antigenic variation is not a hallmark of poliovirus – the same poliovirus vaccines have been used for nearly 60 years to control infections by this virus. An exception is a poliovirus type 1 that caused a 2010 outbreak in the Republic of Congo.

The 2010 outbreak (445 paralytic cases) was unusual because the case fatality ratio of 47% was higher than typically observed (usually less than 10% of patients with confirmed disease die). The first clue that something was different in this outbreak was the finding that sera from some of the fatal cases failed to effectively block (neutralize) infection of cells by the strain of poliovirus isolated during this outbreak (the strain is called PV-RC2010). The same sera effectively neutralized the three Sabin vaccine viruses as well as wild type 1 polioviruses isolated from previous outbreaks. Therefore gaps in vaccination coverage were solely not responsible for this outbreak.

Examination of the nucleotide sequence of the genome of type I polioviruses isolated from 12 fatal cases revealed two amino acid changes within a site on surface of the viral capsid that is bound by neutralizing antibodies (illustration). The sequence of this site, called 2a, was changed from ser-ala-ala-leu to pro-ala-asp-leu. This particular combination of amino acid substitutions has never been seen before in poliovirus. Virus PV-RC2010, which also contains these two amino acid mutations, is completely resistant to neutralization with monoclonal antibodies that recognize antigenic site 2 (monoclonal antibodies recognize a single epitope, as opposed polyclonal antibodies which is a mixture of antibodies that recognize many epitopes. The antibodies in serum are typically polyclonal).

Poliovirus neutralization titers were determined using sera from Gabonese and German individuals who had been immunized with Sabin vaccine. These sera effectively neutralized the type I strain of Sabin poliovirus, as well as type 1 polioviruses isolated from recent outbreaks. However the sera had substantially lower neutralization activity against PV-RC2010. From 15-29% of these individuals would be considered not to be protected from infection with this strain.

Nucleotide sequence analysis of PV-RC2010 reveals that it is related to a poliovirus strain isolated in Angola in 2009, the year before the Republic of Congo outbreak. The Angolan virus had just one of the two amino acid changes in antigenic site 2a found in PV-RC2010.

It is possible that the relative resistance of the polioviruses to antibody neutralization might have been an important contributor to the high virulence observed during the Republic of Congo outbreak. The reduced ability of serum antibodies to neutralize virus would have lead to higher virus in the blood and a greater chance of entering the central nervous system. Another factor could also be that many of the cases of poliomyelitis were in adults, in which the disease is known to be more severe.

An important question is whether poliovirus strains such as PV-RC2010 pose a global threat. Typically the fitness of antigenically variant viruses is not the same as wild type, and therefore such viruses are not likely to spread in well immunized populations. Today some parts of the world have incomplete poliovirus immunization coverage, which together with the reduced circulation of wild type polioviruses leads to reduced population immunity. Such a situation could lead to the evolution of antigenic variants. This situation occurred in Finland in 1984, when an outbreak caused by type 3 poliovirus took place. The responsible strains were antigenic variants that evolved due to use of a sub-optimal poliovirus vaccine in that country.

The poliovirus outbreaks in the Republic of Congo and Finland were stopped by immunization with poliovirus vaccines, which boosted the population immunity. These experiences show that poliovirus antigenic variants such as PV-RC2010 will not cause outbreaks as long as we continue extensive immunization with poliovirus vaccines, coupled with environmental and clinical testing for the presence of such viruses.

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