brincidofovirThe Liberian man who was diagnosed with Ebola virus infection after traveling to Dallas, Texas, was treated with an antiviral drug called brincidofovir. This drug had originally been developed to treat infections with DNA-containing viruses. Why was it used to treat an Ebola virus infection?

Brincidofovir (illustrated) is a modified version of an antiviral drug called cidofovir, which inhibits replication of a variety of DNA viruses including poxviruses and herpesviruses. When cidofovir enters a cell, two phosphates are added to the compound by a cellular enzyme, producing cidofovir diphosphate. Cidofovir is used by viral DNA polymerases because it looks very much like a normal building block of DNA, cytidine. For reasons that are not known, incorporation of phosphorylated cidofovir causes inefficient viral DNA synthesis. As a result, viral replication is inhibited.

Cidofovir was modified by the addition of a lipid chain to produce brincidofovir. This compound (pictured) is more potent, can be given orally, and does not have kidney toxicity, a problem with cidofovir. When brincidofovir enters a cell, the lipid is removed, giving rise to cidofovir. Brincidofovir inhibits poxviruses, herpesviruses, and adenoviruses, and has been tested in phase 2 and 3 clinical trials. The antiviral drug is being stockpiled by the US for use in the event of a bioterrorism attack with smallpox virus.

Ebola virus is an RNA virus, so why was brincidofovir used to treat the Dallas patient? According to the drug’s manufacturer, Chimerix,  with the onset of the Ebola virus outbreak in early 2014, the company provided brincidofovir, and other compounds, to the CDC and NIH to determine if they could inhibit virus replication. Apparently brincidofovir was found to be a potent inhibitor of Ebola virus replication in cell culture. Based on this finding, and the fact that the compound had been tested for safety in humans, the US FDA authorized its emergency use in the Dallas patient.

Unfortunately the Dallas patient passed away on 8 October. Even if he had survived, we would not have known if the compound had any effect. Furthermore, the drug is not without side effects and these might not be tolerated in Ebola virus-infected patients. It seems likely that the drug will also be used if other individuals in the US are infected.

Looking at the compound, one could not predict that it would inhibit Ebola virus, which has an RNA genome. RNA polymerases use different substrates than DNA polymerases – NTPs versus dNTPs. NTPs have two hydroxyls on the ribose sugar, while dNTPs have just one (pictured). The ribose is not present in cidofovir, although several hydroxyls are available for chain extension. I suspect that the company was simply taking a chance on whether any of its antiviral compounds in development, which had gone through clinical trials, would be effective. This procedure is standard in emergency situations, and might financially benefit the company.

Update: The NBC news cameraman is being treated with brincidofovir in Nebraska.

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Poliovirus by Jason Roberts

Poliovirus by Jason Roberts

Vaccine-associated poliomyelitis caused by the oral poliovirus vaccine is rare, but its occurrence in a healthy, immunocompetent 6-month old child was highly unusual because the child had been previously immunized with two doses of the injected, inactivated poliovirus vaccine (IPV).

The three poliovirus vaccine strains developed by Albert Sabin (OPV, oral poliovirus vaccine) contain mutations which prevent them from causing paralytic disease. When the vaccine is ingested, the viruses replicate in the intestine, and immunity to infection develops. While replicating in the intestinal tract, the vaccine viruses undergo mutation, and OPV recipients excrete neurovirulent polioviruses. These so-called vaccine-derived polioviruses (VDPV) can cause poliomyelitis in the recipient of the vaccine or in a contact. During the years that the Sabin poliovirus vaccines were used in the US, cases of poliomyelitis caused by VDPV occurred at a rate of about 1 per 1.4 million vaccine doses, or 7-8 per year. Once the disease was eradicated from the US in 1979, the only cases of polio were caused by the Sabin vaccine.

To prevent vaccine-associated poliomyelitis, in 1997 the US switched to an immunization schedule consisting of two doses of IPV followed by one dose of OPV. The US then switched to using IPV exclusively in 2000. The child in this case essentially had a polio immunization course similar to that utilized in the US from 1997-2000: two doses of IPV, one dose of OPV. Why did the child develop poliomyelitis?

One clue comes from the fact that after the switch to an IPV-OPV schedule in 1997, there were still three cases of VAPP in 1998 and three in 1999. Another hint comes from a study of immune responses in children given multiple doses of IPV. Most of the children receiving two doses of IPV produced antibodies against types 1 and 2 poliovirus (92 and 94%), but only 74% of children produced antibodies against type 3 poliovirus.

The final piece of information needed to solve this puzzle is that the child in this case had vaccine-associated poliovirus caused by the type 3 strain, which was isolated from his feces.

Therefore, the child in this case most likely did not produce sufficient antibodies to type 3 poliovirus after receiving the two doses of IPV. As a consequence, when he was given OPV, he developed type 3 vaccine-associated poliomyelitis.

This case of VAPP could have been prevented: the child was born in Canada, and as customary in that country since 1995, he received two doses of IPV. At 5 months of age the child and his family visited China, where his parents decided to continue his immunizations according to the local schedule. China still uses OPV, so that is what the child received.

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Incubation periodThe time before the symptoms of a viral infection appear is called the incubation period. During this time, viral genomes are replicating and the host is responding, producing cytokines such as interferon that can have global effects, leading to the classical symptoms of an acute infection (e.g., fever, malaise, aches, pains, and nausea). These symptoms are called the prodrome, to distinguish them from those characteristic of infection (e.g. paralysis for poliovirus, hemorrhagic fever for Ebolaviruses, rash for measles virus).

Whether or not an infected person is contagious (i.e. is shedding virus) during the incubation period depends on the virus. For example, Ebola virus infected patients do not pass the virus on to others during the incubation period. This fact explains why Tom Frieden said there was ‘zero chance’ that the passenger from Liberia who was diagnosed with Ebola virus infection in Dallas would have infected others while on an airplane. He had no symptoms of infection because he was still in the incubation period of the disease.

In contrast to Ebolaviruses, poliovirus and norovirus are shed during the incubation period – in the feces, where they can infect others.

Remarkably, viral incubation periods can vary from 1 or 2 days to years (Table; click to magnify). Short incubation times usually indicate that actions at the primary site of infection produce the characteristic symptoms of the disease. Longer incubation times indicate that the host response, or the tissue damage required to reveal the symptoms of infection, take place away from the primary site of infection.

The table was taken from the third edition of Principles of Virology. Missing from the table (which will be corrected in the next edition) is the incubation period of Ebola virus, which is 2 to 21 days. I would also argue that the incubation period of HIV is not 1-10 years, but 2-4 weeks, the time until the prodromal symptoms occur. The characteristic symptom of HIV-1 infection, immunosuppression, occurs much later.

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EV-D68

Enterovirus D68 by Jason Roberts

In February 2014 I wrote about children in California who developed a poliomyelitis-like paralysis, also called acute flaccid paralysis or AFP. However, the cause of this paralysis was not known. The CDC has released its study of these cases and concludes “The etiology of AFP with anterior myelitis in the cases described in this report remains undetermined.”

A total of 23 cases of AFP* in California were reported to CDC during the period June 2012 through June 2014. These cases were from diverse geographic regions of the state. Specimens from 19 of the patients were available and tested for poliovirus, aroboviruses, herpesviruses, parechoviruses, adenoviruses, rabies virus, influenza virus, metapneumovirus, respiratory syncytial virus, parainfluenza viruses, Mycoplasma pneumoniae, Rickettsia, and amoebas. Rhinovirus was detected in one patient, and enterovirus D68 in two patients; all others were negative for potential etiologic agents.

All 23 patients with AFP also had anterior myelitis, inflammation of the grey matter of the spinal cord, which is characteristic of poliomyelitis. While the rate of AFP in California betweeen 1992-1998 was 1.4 cases per 100,000 children per year,  anterior myelitis was not described in any of 245 cases reviewed by CDC. However, poliovirus was ruled out as a cause in the 19 individuals who could be tested.

The cause of AFP is often difficult to determine because there infectious and non-infectious etiologies. Only 2 of the 19 clinical specimens met CDC guidelines for poliovirus detection (two stool specimens collected ≥24 hours apart and <14 days after symptom onset) and the others were likely taken too late to detect the presence of virus. The finding of enterovirus D68 in two of the samples is difficult to interpret, as the virus was detected in respiratory specimens and could have been a coincidental infection.

This investigation began with a request from a San Francisco area physician to the California State Department of Public Health to determine whether poliovirus was present in a 29 year old male with AFP and anterior myelitis. Subsequently this department posted alerts for AFP with anterior myelitis to  local health departments, and it is from the cases submitted that the 23 were drawn. Therefore the number of cases of AFP with anterior myelitis might be a consequence of this surveillance.

We are left with the unsatisfying conclusion that these 23 cases of AFP with anterior myelitis were either caused by an undetected infectious agent, or by something else.

*Defined by CDC as “at least one limb consistent with anterior myelitis, as indicated by neuroimaging of the spine or electrodiagnostic studies (e.g., nerve conduction studies and electromyography), and with no known alternative etiology”.

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The World Health Organization has issued a situation assessment entitled ‘What we know about transmission of the Ebola virus among humans‘. WHO is rather late entering the transmission discussion which began on 12 September 2014 with the suggestion that Ebola virus transmission could go airborne. WHO is a big organization and moves slowly; nevertheless their voice may reassure those who are not convinced by what virologists have to say. Here are the salient points (voiced here and by many others in the past few weeks).

The Ebola virus is transmitted among humans through close and direct physical contact with infected bodily fluids, the most infectious being blood, faeces and vomit.

Ebola virus disease is not an airborne infection. Airborne spread among humans implies inhalation of an infectious dose of virus from a suspended cloud of small dried droplets.

This mode of transmission has not been observed during extensive studies of the Ebola virus over several decades.

Moreover, scientists are unaware of any virus that has dramatically changed its mode of transmission*. For example, the H5N1 avian influenza virus, which has caused sporadic human cases since 1997, is now endemic in chickens and ducks in large parts of Asia.

That virus has probably circulated through many billions of birds for at least two decades. Its mode of transmission remains basically unchanged.

Speculation that Ebola virus disease might mutate into a form that could easily spread among humans through the air is just that: speculation, unsubstantiated by any evidence.

The last sentence is the key point:

To stop this outbreak, more needs to be done to implement – on a much larger scale – well-known protective and preventive measures. Abundant evidence has documented their effectiveness

*Sounds familiar?

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On episode #305 of the science show This Week in Virology, Vincent, Alan, and Kathy continue their coverage of the Ebola virus outbreak in West Africa, with a discussion of case fatality ratio, reproductive index, a conspiracy theory, and spread of the virus to the United States.

You can find TWiV #305 at www.twiv.tv.

Filovirus virion

Image credit: ViralZone

Given the extent of the Ebola virus outbreak in West Africa, transport of an infected individual to the US was bound to happen. The case is an adult who had contact with an Ebola virus-infected woman in Liberia, then traveled to Dallas. He had no symptoms before arriving in the US and therefore did not likely transmit the infection to airplane passengers. He sought medical care on 26 Sep 2014 and was admitted to Texas Health Presbyterian Hospital 28 Sep 2014 where he is currently under isolation. Samples sent to the CDC tested positive for Ebola virus. There are excellent summaries of the events at ProMedMail and the CDC website.

Apparently the Dallas patient told a healthcare worker during his first hospital visit that he had been in Liberia. This information was not transmitted to his physician. The word ‘Liberia’ should have set off alarm bells. Furthermore, if the physician did not receive the patient’s recent travel history, he/she should have requested it. There is no room for error when dealing with Ebola virus infection.

It is puzzling that travel (excluding healthcare workers) out of the affected West African countries is still permitted. As moderator JW notes on ProMedMail: “This chain of events illustrates the danger that anybody arriving from Liberia, even without symptoms on departure from there or on arrival in the USA (or anywhere else in Africa or overseas) may be incubating Ebola — but not international volunteers who have only been in contact with Liberians while wearing adequate PPE (personal protection equipment).”

Why is it important to stop travel out of the affected countries? While I’m confident that the US can detect and properly contain imported Ebola virus infections, not all countries will be able to do so. There are dozens of other countries that are unprepared to deal with an infected case, from diagnosis to isolation to treatment. I can easily imagine infection quickly getting out of control in such countries: millions are at risk. While the economics of stopping air travel out of Liberia, Sierra Leone, and Guinea will be severe, they cannot approach the devastation of having outbreaks burning simultaneously in multiple countries.

Update: NPR has a good explanation of the reproductive index, or the number of persons who can be infected by another infected person during an outbreak. For Ebola virus, this number is 1-2. This low number is why quarantine can be effective.

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Ebola is comingThe cover of this week’s issue of Businessweek declares that ‘Ebola is coming’ in letters colored like blood, with the subtitle ‘The US had a chance to stop the virus in its tracks. It missed’. Although the article presents a good analysis of the hurdles in developing antibody therapy for Ebola virus infection, the cover is overstated. Why does Businessweek think that Ebola virus is coming to the US? (there is no mention of this topic in the article). Are we sure that antibody therapy would have stopped the outbreak? (no, as stated in the article).

How the U.S. Screwed Up in the Fight Against Ebola is an analysis of why ZMapp, the cocktail of monoclonal antibodies that block infection with Ebola virus, has not yet been approved for use in humans. ZMapp was given to two American workers who had become infected with the virus while working in Africa. The two workers recovered, but the role of ZMapp in their recovery is unknown – as the authors of the article note. Although ZMapp can prevent lethal infection of nonhuman primates with Ebola virus, it is not known if it would work in humans. Answering that question requires a clinical trial, and the article explores why this phase was not done years ago. Only after the large Ebola virus outbreak in west Africa did the US provide funds to conduct a phase I trial of the drug.

The article discusses how development of ZMapp languished for years, because the US government did not consider the Ebolaviruses to be a pressing problem. In hindsight they were wrong, and now anyone can seem smart by saying we should have pushed development of Ebola virus vaccines and therapeutics.

The real question is whether we will learn from this experience, and be better prepared for the next viral outbreak. Just because infections are rare or geographically localized should not lessen their importance, as these features can change. Knowing the animal source of a viral infection may also lead to developing ways to prevent infections. For example, because people acquire Hendra virus from horses, immunization of these animals should prevent human infections.

What other antiviral vaccines and drugs should we be developing? This question is difficult to answer because we discover new viruses regularly and making therapeutics for all of them is not possible. Testing an antiviral drug or vaccine against rare viruses is difficult because identifying populations that are at risk for infection may be a hit or miss proposition.

Influenza viruses are at the top of the list for vaccine and drug development, because nearly everyone gets infected. Other viruses we should be ready for include SARS and MERS coronaviruses, dengue virus, chikungunya virus, Lassa virus, Nipah and Hendra viruses. I’m sure you can think of other viruses that belong on this list.

Developing antiviral vaccines and drugs is expensive. For some of the viruses on my list (dengue, chikungunya) there are currently large enough markets that permit involvement of for-profit pharmaceutical companies. Development of therapeutics against viruses that cause rare infections must be supported largely by governments.

The US does not spend enough money on basic life sciences research. We do spend a great deal of money on the military. President Obama recently declared Ebola virus to be a top national security priority. Why not view all infectious diseases in this way, to ensure that they receive the funding for research that they deserve?

While the Businessweek cover is misleading, intended to stimulate sales, the article does make us think about the problems we confront when dealing with rare but lethal diseases. No one should conclude that Ebola virus outbreak in Africa could have been prevented, because antiviral therapies have not yet been tested in humans. But we won’t know if we never do the research.

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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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