Understanding viruses

Virology lecturesIf you want to understand life on Earth, you need to know about viruses.

We have reached the halfway point in my 2016 Columbia University undergraduate virology course. So far we have learned the basics of virus replication: how viruses enter cells, how the genome is reproduced, and how proteins are made and assembled into new virus particles. In the second half of the course, we will consider how viruses cause disease, how immune responses prevent infection, vaccines, antivirals, emergence of new viruses, and much more.

All of my lectures are recorded as videos and available freely on YouTube. Below is a list of the first thirteen lectures, with links to the YouTube videos. You can also subscribe to the videos at iTunes University. If you would like copies of the lecture slides and study questions, go to virology.ws/course.

Lecture 1: What is a virus?
Lecture 2: The infectious cycle
Lecture 3: Genomes and genetics
Lecture 4: Structure
Lecture 5: Attachment and entry
Lecture 6: RNA directed RNA synthesis
Lecture 7: Transcription and RNA processing
Lecture 8: DNA replication
Lecture 9: Reverse transcription and integration
Lecture 10: Translation
Lecture 11: Assembly
Lecture 12: Infection basics
Lecture 13: Intrinsic and innate defenses


I was asked to write a commentary for the Proceedings of the National Academy of Sciences to accompany an article entitled SARS-like WIV1-CoV poised for human emergence. I’d like to explain why I wrote it and why I spent the last five paragraphs railing against regulating gain-of-function experiments.

Towards the end of 2014 the US government announced a pause of gain-of-function research involving research on influenza virus, SARS virus, and MERS virus that “may be reasonably anticipated to confer attributes to influenza, MERS, or SARS viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route.”

From the start I have opposed the gain-of-function pause. It’s a bad idea fostered by individuals who continue to believe, among other things, that influenza H5N1 virus adapted to transmit by aerosol among ferrets can also infect humans by the same route. Instead of stopping important research, a debate on the merits and risks of gain-of-function experiments should have been conducted while experiments were allowed to proceed.

Towards the end of last year a paper was published a paper on the potential of SARS-virus-like bat coronaviruses to cause human disease. The paper reawakened the debate on the risks and benefits of engineering viruses. Opponents of gain-of-function research began to make incorrect statements about this work. Richard Ebright said that ‘The only impact of this work is the creation, in a lab, of a new, non-natural risk”. Simon Wain-Hobson wrote that a novel virus was created that “grows remarkably well” in human cells; “if the virus escaped, nobody could predict the trajectory”. I have written extensively about why these are other similar statements ignore the value of the work. In my opinion these critics either did not read the paper, or if they did, did not understand it.

Several months later I was asked to write the commentary on a second paper examining the potential of SARS like viruses in bats to cause human disease. I agreed to write it because the science is excellent, the conclusions are important, and it would provide me with another venue for criticizing the gain-of-function pause.

In the PNAS paper, Menachery et al. describe a platform comprising metagenomics data, synthetic virology, transgenic mouse models, and monoclonal antibody therapy to assess the ability of SARS-CoV–like viruses to infect human cells and cause disease in mouse models. The results indicate that a bat SARS-like virus, WIV1-CoV, can infect human cells but is attenuated in mice. Additional changes in the WIV1-CoV genome are likely required to increase the pathogenesis of the virus for mice. The same experimental approaches could be used to examine the potential to infect humans of other animal viruses identified by metagenomics surveys. Unfortunately my commentary is behind a paywall, so for those who cannot read it, I’d like to quote from my final paragraphs on the gain-of-function issue:

The current government pause on these gain-of-function experiments was brought about in part by several vocal critics who feel that the risks of this work outweigh potential benefits. On multiple occasions these individuals have indicated that some of the SARS-CoV work discussed in the Menachery et al. article is of no merit. … These findings provide clear experimental paths for developing monoclonal antibodies and vaccines that could be used should another CoV begin to infect humans. The critics of gain-of-function experiments frequently cite apocalyptic scenarios involving the release of altered viruses and subsequent catastrophic effects on humans. Such statements represent personal opinions that are simply meant to scare the public and push us toward unneeded regulation. Virologists have been manipulating viruses for years—this author was the first to produce, 35 y ago, an infectious DNA clone of an animal virus—and no altered virus has gone on to cause an epidemic in humans. Although there have been recent lapses in high-containment biological facilities, none have resulted in harm, and work has gone on for years in many other facilities without incident. I understand that none of these arguments tell us what will happen in the future, but these are the data that we have to calculate risk, and it appears to be very low. As shown by Menacherry et al. in PNAS, the benefits are considerable.

A major goal of life science research is to improve human health, and prohibiting experiments because they may have some risk is contrary to this goal. Being overly cautious is not without its own risks, as we may not develop the advances needed to not only identify future pandemic viruses and develop methods to prevent and control disease, but to develop a basic understand- ing of pathogenesis that guides prevention. These are just some of the beneficial outcomes that we can predict. There are many examples of how science has progressed in areas that were never anticipated, the so-called serendipity of science. Examples abound, including the discovery of restriction enzymes that helped fuel the biotechnology revolution, and the development of the powerful CRISPR/Cas9 gene-editing technology from its obscure origins as a bacterial defense system.

Banning certain types of potentially risky experiments is short sighted and impedes the potential of science to improve human health. Rather than banning experiments, such as those described by Menachery et al., measures should be put in place to allow their safe conduct. In this way science’s full benefits for society can be realized, unfettered by artificial boundaries.

TWiVOn episode #380 of the science show This Week in Virology, the TWiVeroos deliver the weekly Zika Report, then talk about a cryoEM structure of a plant virus that reveals how the RNA genome is packaged in the capsid, and MIMIVIRE, a CRISPR-like defense system in giant eukaryotic viruses.

You can find TWiV at microbe.tv/twiv, or you can listen below.

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FlavivirusEvidence is mounting that Zika virus is neurotropic (able to infect cells of the nervous system) and neurovirulent (causes disease of the nervous system) in humans.

The most recent evidence comes from a case report of an 81 year old French man who developed meninogoencephalitis 10 days after returning from a 4 week cruise to New Caledonia, Vanuatu, Solomon Islands, and New Zealand (meningoencephalitis is infection of the meninges – the membranes that cover the brain – and the brain). His symptoms included fever, coma, paralysis, and a transient rash. A PCR test revealed Zika virus genomes in the cerebrospinal fluid, and infectious virus was recovered after applying the CSF to Vero cells in culture.

A second case report concerns a 15 year old girl in Guadeloupe who developed left hemiparesis (weakness of one side of the body), left arm pain, frontal headache, and acute lower back pain. After admission she developed dysuria (difficulty urinating) that required catheterization. PCR revealed the presence of Zika virus genomes in her serum, urine, and cerebrospinal fluid; other bacterial and viral infections were ruled out.

Until very recently Zika virus was believed to cause a benign infection comprising rash, fever, joint pain, red eyes, and headache. There is now strong evidence that the virus can cause congential birth defects, and growing evidence that the virus is neurotropic and neurovirulent. Previously the entire Zika virus genome was recovered from brain tissue of an aborted fetus.

Zika virus is classified in the family Flaviviridae, and other members are known to be neurotropic, including West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. West Nile virus infection may lead to acute flaccid paralysis, meningitis, encephalitis, and ocular manifestations. Examination of additional cases of Zika virus infection will be needed to document the full spectrum of illness caused by this virus.

Update: Neurotropism of Zika virus is also indicated by the findings that the virus infects human cortical neural progenitors.

FlavivirusData from several clinical studies in Brazil establish a strong link between infection of pregnant women with Zika virus and a variety of birth defects collectively called congenital Zika syndrome.

In the latest study conducted in Rio de Janeiro, the authors enrolled 88 pregnant women who had a rash in the previous 5 days. Of the 88 subjects, 72 tested positive for Zika virus by PCR. Fetal ultrasound was performed in 42 of the Zika virus positive women, and in all the Zika virus negative women.

The results are convincing: fetal abnormalities were detected in 12 of the 42 Zika virus positive women (29%) and in none of the Zika virus negative women.

The abnormalities include fetal death (2), microcephaly (5), ventricular calcification or other central nervous system lesions (7), and abnormal amniotic fluid volume or cerebral or umbilical artery flow (7). These observations show that Zika virus infection may lead to birth defects other than microcephaly.

The infections of these pregnant women with Zika virus took place throughout pregnancy, from week 8 to week 35. This window of susceptibility is in contrast to rubella virus which is more likely to cause birth defects when infection occurs in the first trimester.

Not all Zika virus infections seem to cause birth defects – 29% in this study. If this number holds outside of Rio de Janeiro, then birth defects should also be observed in other countries with high rates of infection. Only 20% of Zika virus infections are symptomatic, and it will be important to determine if these also lead to congenital Zika syndrome.

The increase in microcephaly associated with Zika virus infection was first noted in the northeast of Brazil. This study was done with women who live in Rio de Janeiro, in the southeast of Brazil, showing that the association is not geographically limited.

It has been suggested that fetal defects might be partly due to the presence of antibodies to dengue virus that cross-react with Zika virus and cause immune-mediated enhancement of disease. Thirty-one percent of the Zika virus positive women in this study were also positive for antibodies to dengue virus, but the paper does not report how these correlate with fetal defects.

These findings, together with results of previous studies showing recovery of the entire Zika virus genome from amniotic fluid or from fetal brain, demonstrate that this fast spreading and newly emerging virus infection is clearly a threat to the developing fetus.

We should not be surprised that a virus that had until recently only infected several thousand individuals, and which we believed caused a mild, self-limiting rash, suddenly is found to be extremely dangerous to the developing fetus. The potential for fetal damage was likely always present, but unobserved until the virus was introduced into a large population of susceptible individuals and hundreds of thousands of individuals were infected. The lesson to be learned, often easily forgotten, is that we should always expect more from viruses than we initially observe. Such was certainly the case for HIV-1; immunodeficiency was only the tip of the clinical syndrome caused by infection.

Given the pace at which Zika virus is racing through susceptible humans, it is likely to generate enough population immunity in the next five years to curtail this outbreak. However as susceptible individuals are born and accumulate, regular outbreaks will likely occur. Similarly, outbreaks of rubella virus in the US occurred every 5-6 years in the pre-vaccine era.

Not only do rubella and Zika viruses cause similar fetal and placental abnormalities, in the mother they both lead to rash, joint pain, skin itching, and lymphadenopathy without high fever.

Hopefully the similarities between rubella virus and Zika virus will stop there: it took nearly 30 years to develop a rubella virus vaccine after the discovery that infection caused birth defects.


TWiVOn episode #379 of the science show This Week in Virology, Scott Tibbetts joins the TWiVirate to describe his work on the role of a herpesviral nocoding RNA in establishment of peripheral latency, and then we visit two last minute additions to the Zika virus literature.

You can find TWiV #379 at microbe.tv/twiv, or listen below.

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ATP EBOV antiviralA small molecule antiviral compound has been shown to protect rhesus monkeys against lethal Ebolavirus disease, even when given up to three days after virus inoculation.

The compound, called GS-5734, is a nucleoside analog. After uptake into cells, GS-5734 is converted to a nucleoside triphosphate (illustrated, bottom panel) which is incorporated by the viral RNA dependent RNA polymerase as it copies the viral genome. However, the nucleoside is chemically different from ATP (illustrated, top) and no further nucleotides can be incorporated into the growing RNA strand. RNA synthesis ceases, blocking production of infectious virus particles.

In cell culture GS-5734 inhibits viral replication at micromolar concentrations, in a variety of human cell types including monocyte-derived macrophages, primary macrophages, endothelial cells, and a liver cell line. The drug inhibits replication of several strains of Zaire ebolavirus, including Kikwit and Makona (from the West African outbreak); Bundibugyo ebolavirus, and Sudan ebolavirus. It also inhibits replication of another filovirus, Marburg virus, as well as viruses of different families, including respiratory syncytial virus, Junin virus, Lassa fever virus, and MERS-coronavirus, but not chikungunya virus, Venezuelan equine encephalitis virus, or HIV-1.

The RNA dependent RNA polymerase of Ebolaviruses has not yet been produced in active form, so the authors determined whether GS-5734 inhibits a related polymerase from respiratory syncytial virus. As predicted, the compound was incorporated into growing RNA chains by the enzyme, and caused premature termination.

Typically tests of antiviral candidates begin in a small animal, and if the results are promising, proceed to nonhuman primates. While a mouse model of Ebolavirus infection is available, the serum from these animals degrades GS-5374. Consequently a rhesus monkey model of infection was used to test the compound.

After intravenous administration of GS-5374, the NTP derived from it was detected in peripheral blood mononuclear cells, testes, epididymis, eyes, and brain within 4 hours. All 12 monkeys inoculated intramuscularly with Zaire ebolavirus died by 9 days post-infection. In contrast, all animals survived after administration of GS-5374 2 or 3 days after virus inoculation. These animals also had reduced virus associated pathology as measured by liver enzymes in the blood and blod clotting. Viral RNA in serum reaches 109 copies per milliliter on days 5 and 7 in untreated animals, and was undetectable in 4 of 6 treated animals.

It is likely that resistant viruses can be obtained by passage in the presence of GS-5734; whether such mutant viruses emerge early in infection, and at high frequency, is an important question that will impact clinical efficacy of the drug. The authors did not detect changes in the viral RNA polymerase gene that might be assoicated with resistance, but further work is needed to address how readily such mutants arise.

These promising results have lead to the initiation of a phase I clinical trial to determine whether GS-5734 is safe to administer to humans, and if the drug reaches sites where Ebolaviruses are known to replicate. However, determining the efficacy of the compound requires treatment of acutely Ebolavirus infected humans, of which there are none. It might be of interest to determine the ability of GS-5734 to clear persistent virus from previously infected individuals.

You can bet that GS-5734 has already been tested for activity against Zika virus.

TWiVOn episode #378 of the science show This Week in Virology, Greg Smith joins the TWiVirate to reveal how his lab discovered a switch that controls herpesvirus neuroinvasion, and then we visit the week’s news about Zika virus.

You can find TWiV #378 at microbe.tv/twiv, or you may listen below.

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FlavivirusThe title of a Eurosurveillance article, “An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014” was written to make the headlines. The title should be “An autochthonous case of Zika due to person to person contact, Florence, Italy, 2014.”

An Italian man returns from a 10 day holiday in Thailand and a day later develops a rash with fever and headache. Within 6 days the rash has subsided. About two weeks later his girlfriend develops a similar disease. As this was 2014 no one looked for Zika virus and both were presumed to have dengue virus infection.

The serum samples taken from the patients were pulled from the freezer after Zika virus becomes a household word in 2015. Both patients’ sera are shown to contain neutralizing antibodies against Zika virus, with a clear rise between samples taken early in illness and after recovery.

Apparently the couple had sex between the time the man’s rash subsided, and the onset of the girlfriend’s symptoms. The authors of the paper conclude that transmission by semen is suggested.

Inexplicably, the authors write:

Other transmission modalities (i.e. direct contact with other bodily fluids) are unlikely to play a role but may not be completely ruled out.

Why is it unlikely that the man had a residual rash, possibly leaking virus, which he then transferred to the woman, perhaps on one or more mucus membranes? This mode of transmission is also known as ‘close contact’ between individuals. I am waiting for a similar case report in which the couple used condoms, yet Zika virus infection was still transmitted.

Like everyone else, the authors are seduced by the possibility of sexual transmission of Zika virus. I have yet to see any clear, convincing evidence of sexual transmission of Zika virus. At worst, the risk is extremely low, although probably not zero, given that Zika virus RNA (not virus) has been found in semen of one individual. Consider these facts and act accordingly.

Zika virus and the fetus

FlavivirusAn epidemic of Zika virus infection began in Brazil in April 2015, and six months later there was a surge in the number of infants born with microcephaly. Confirming that Zika virus causes microcephaly will require much more information than is currently available. So far there have been few isolations of Zika virus RNA from microcephalic fetuses or amniotic fluid.

A single case report revealed the entire Zika virus genome in fetal brain tissue from a 25 year old who developed fever, muscle and eye pain, and rash during the 13th week of gestation in Natal, Brazil. The fetus was aborted at 28 weeks of gestation when fetal abnormalities, including microcephaly, were detected. Virus particles 42 to 54 nm in diameter were detected in the brain by electron microscopy.

It is probably not normal to have Zika virus in the fetal brain. However, its presence there might be a consequence of microcephaly, not a cause. As Dr. Steven Seligman writes at ProMedMail, “It is possible that brain tissue in cases of microcephaly become susceptible to Zika virus infection by a mechanism such as diminution of he blood-brain barrier.”

The entire Zika virus genome has also been detected in amniotic fluid, which surrounds the developing fetus. Two pregnant women from the state of Paraíba in Brazil reported clinical symptoms early in pregnancy consistent with Zika virus infection (fever, myalgia, rash). Microcephaly was diagnosed at 21 weeks gestation by ultrasound, and 7 weeks later samples of amniotic fluid were obtained by amniocentesis.

Amniotic fluid was centrifuged to purify virus particles, and RNA was extracted, copied into DNA by reverse transcriptase, amplified by polymerase chain reaction and subjected to deep sequencing.

The complete Zika virus genome sequence was obtained from one sample, and two smaller genome fragments from the second. Sequence analyses revealed 97-100% similarity with Zika viruses isolated from French Polynesia in 2013.

IgM antibodies to Zika virus were detected in both amniotic fluid samples, indicating that the fetus was likely infected and mounting an immune response against the virus (this antibody does not cross the placenta). In contrast, serum and urine from both mothers was negative for Zika virus IgM. This antibody appears first during infection, then subsides as levels of IgG antibody rise. It is possible that the mothers were infected with Zika virus early in pregnancy and cleared the infection, but the virus entered the fetus where it persisted.

Even if Zika virus does cause birth defects, a vaccine will likely not be available for another two years. In the meantime it would be highly advisable to practice mosquito avoidance and control.

Update 2/24/16: I asked Carolyn Coyne how a virus might reach the amniotic fluid. Her reply:

Amniotic fluid is mainly urine from the baby (after month 4ish) so if the virus is being shed in the urine, that is one way. Cells from the baby are also shed into the fluid (these are usually skin cells, but I imagine could also be from the mouth as the baby is usually drinking amniotic fluid at later stages of gestation). I will note that this is usually in a normal pregnancy and I imagine is the fetus were dying/dead (a fetus can die in utero and not be miscarried for a shockingly long time sometimes), the virus might easily enter the amniotic fluid as the fetus begins to decompose (which also happens in utero).

The two routes of entry are hematogenous or ascending. In hematogenous infections, virus present in the maternal blood would have to cross the placenta across the villous trees. In an ascending infection, the virus would be introduced into the vagina, then would have to bypass the cervix and still have to cross the placenta to access the fetus. Usually ascending infections are associated with bacteria (from UTIs mainly, but can be other). In either case, the placenta is there and would have to be crossed.

Update 2/25/16: A report in PLoS Neglected Tropical Diseases describes finding Zika virus RNA by RT-PCR in neuronal tissues but not in heart, lung, liver or placenta, in a stillborn infant with microcephaly and hydrops fetalis. The 20 year old woman from Salvador, Brazil denied having any symptoms consistent with Zika virus infection, but only one in five infections are symptomatic. As in the case described above, we do not know if Zika virus caused the fetal defects, or if virus was able to invade the fetus as a consequence of severe developmental damage.