TWiV 285: Hokies go viral

On episode #285 of the science show This Week in Virology, Vincent meets up with XJ Meng and Sarah McDonald at Virginia Tech to talk about their work on viruses of swine and rotaviruses.

You can find TWiV #285 at www.microbe.tv/twiv.

TWiV 276: Ramblers go viral

On episode #276 of the science show This Week in Virology, Vincent meets up with Susan Baker and Tom Gallagher at Loyola University to talk about their work on coronaviruses.

You can find TWiV #276 at www.microbe.tv/twiv.

An epidemic of porcine diarrhea in North America

pig farmPorcine epidemic diarrhea arrived in the United States in the spring of 2013. The disease, caused by a coronavirus, was first identified in the United Kingdom in 1971, and has subsequently spread throughout Europe and Asia. The disease is a concern for the swine industry because it is associated with high case fatality ratios in suckling pigs.

Porcine epidemic diarrhea virus is a member of the coronavirus family, which also includes the SARS and MERS coronaviruses (CoV). Before 2013 the virus had not been isolated in North America. It was detected on a farm in Iowa in May and subsequently spread to 22 states. It is estimated that between 1-4 million pigs have died of the disease in the US.  Recently the virus was found on a Canadian pig farm in Middlesex County, where it most likely arrived from the US.

PEDV can infect pigs of all ages, but is most serious in nursing pigs in which the clinical symptoms are the most severe. The disease is characterized by acute vomiting and watery diarrhea which in nursing pigs leads to dehydration and frequently death. There is no treatment for the disease other than rehydration; no antiviral drugs are available, but a vaccine was developed in 2013. The virus does not infect humans.

Sequence analysis of genomes from three US isolates of PEDV indicate that they are most closely related to a virus isolated in 2012 in Anhui, China. These data suggest, but do not prove, that the US PEDV originated from China. How the virus might have arrived from that country is a matter of speculation. The virus is believed to move from farm to farm on trucks that are used to carry pigs, as well as on contaminated boots and clothing. Many farms observe strict biosecurity procedures in which trucks are properly washed, disinfected, and heated to inactivate the virus. However these procedures cost money and take time, and may be bypassed in some cases. If older pigs are asymptomatically infected, they might be transported to other farms and spread the virus. Once in a farm, stopping spread of the virus is difficult: it is spread by fecal-oral contamination.

The coronavirus family is divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Several human coronaviruses that cause common cold-like illness, as well as PEDV, are alphacoronaviruses. SARS-CoV and MERS-CoV are betacoronaviruses. Viruses in the alphacoronavirus and betacoronavirus genera are believed to have originated from bats, while birds might have been the origin of viruses in the other two genera. Analysis of the PEDV genome sequence indicate that the 5’-untranslated region is similar to that of a bat coronavirus. Based on this information it has been suggested that PEDV originated from a cross-species transfer of a bat alphacoronavirus into pigs.

National Hog Farmer is not usually on my reading list, but it has a good summary of porcine epidemic diarrhea.

MERS-CoV genome found in dromedary camels

coronavirusMiddle Eastern respiratory syndrome coronavirus (MERS-CoV), first identified in the fall of 2012 in a Saudi Arabian patient, has since infected over 160 individuals, causing 71 deaths. Identifying the source of infection is important for efforts to prevent further infections. Recently two studies revealed the presence of antibodies to the virus in dromedary camels in Jordan and Saudi Arabia, two countries where large clusters of infections have occurred. Detection of the viral RNA genome in clinical specimens by polymerase chain reaction (PCR) now provides additional evidence that MERS-CoV can infect camels.

Samples were obtained from all camels (n=14) on a farm where two individuals with laboratory confirmed MERS-CoV infection had been in contact with animals. Nasal swab specimens from three camels were positive when assayed by PCR using MERS-CoV specific primers. Nucleotide sequence analysis revealed that the virus from one camel clustered with sequences obtained from the two farm-associated MERS-CoV infections. Sera from all camels on the farm reacted with MERS-CoV in immunofluorescence and neutralizing assays.

These observations provide strong evidence that MERS-CoV can replicate in camels. However, the authors were not able to isolate infectious virus from camel specimens. MERS-CoV has been previously cultured from human clinical specimens, and it is known what types of cells should be used for virus isolation. Levels of virus in the camel specimens might be too low to detect by culturing, or alternatively only fragments of viral genomes might be present, especially if the infection is over.

Proof that infectious MERS-CoV virus is present in camels will require isolation of infectious virus in cultured cells. If PCR is routinely used to diagnose viral infections such as influenza, why is it not sufficient to conclude that MERS—CoV is present in camels? The answer is that this is not a routine case – the investigators are attempting to determine the origin of MERS-CoV and therefore demonstrating infectious virus is essential. You can bet that the investigators are hard at work attempting to isolate infectious virus from the camels.

The authors note that because of the nucleotide sequence similarity between the camel and human viruses, is not possible to determine if the camels were infected by humans, or if humans infected the camels. It is also possible that camels and humans were infected by a third source. Analysis of outbreaks in which the viruses have undergone more extensive sequence divergence should permit establishment of the chain of transmission. If I had to speculate, I would say the virus is going from camels to humans. So far there has been little evidence of seropositivity in humans outside of the known cases, while many camels have antibodies that react with the virus.

TWiV 258: Hedging our bats

On episode #258 of the science show This Week in Virology, Matt joins the TWiV team to discuss the discovery of a SARS-like coronavirus in bats that can infect human cells, and what is going on with MERS-coronavirus.

You can find TWiV #258 at www.microbe.tv/twiv.

Bat SARS-like coronavirus that infects human cells

Rhinolophus sinicusThe SARS pandemic of 2002-2003 is believed to have been caused by a bat coronavirus (CoV) that first infected a civet and then was passed on to humans. The isolation of a new SARS-like coronavirus from bats suggests that the virus could have directly infected humans.

A single colony of horseshoe bats (Rhinolophus sinicus) in Kunming, Yunnan Province, China, was sampled for CoV sequences over a one year period. Of a total of 117 anal swabs or fecal samples collected, 27 (23%) were positive for CoV sequences by polymerase chain reaction (PCR). Seven different SARS-like CoV sequences were identified, including two new ones. For the latter the complete genome sequence was determined, which showed a higher nucleotide sequence identity (95%) with SARS-CoV than had been previously observed before among bat viruses.

One of these new viruses was recovered by infecting monkey cell cultures with one of the PCR-positive samples. This virus could infect human cells and could utilize human angiotensin converting enzyme 2 (ACE2) as an entry receptor. The infectivity of this virus could also be neutralized with sera collected from seven different SARS patients.

None of the SARS-like coronaviruses previously isolated from bats are able to infect human cells. The reason for this block in replication is that the spike glycoprotein of these bat viruses do not recognize ACE2, the cell receptor for SARS-CoV. SARs-like CoVs isolated from palm civets during the 2002-2003 outbreak have amino acid changes in the viral spike glycoprotein that improve its interaction with ACE2. The civet was therefore believed to be an intermediate host for adaptation of SARS-CoV to humans. The isolation of bat SARS-like CoVs that can bind human ACE2 and replicate in human cells suggests that the virus might have spread directly from bats to humans.

This finding has implications for public health: if SARS-like CoVs that can infect human cells are currently circulating in bats, they have the potential to infect humans and cause another outbreak of disease. The authors believe that the diversity of bat CoVs is higher than we previously knew:

It would therefore not be surprising if further surveillance reveals a broad diversity of bat SL-CoVs that are able to use ACE2, some of which may have even closer homology to SARS-CoV than SL-CoV-WIV1.

Is there any implication of this work for the recently emerged MERS-CoV? Sequences related to MERS-CoV have been found in bats, and given that bats are known to be hosts of a number of viruses that infect humans, it is reasonable to postulate that MERS-CoV originated in bats. So far a 190 fragment of MERS-CoV nucleic acid has been found in a single bat from Saudi Arabia. Identification of the reservoir of MERS-CoV will require duplicating the methods reported in this paper: finding the complete viral genome, and infectious virus, in bats.

TWiV 247: Today’s weather in virology

On episode #247 of the science show This Week in Virology, Ian Lipkin joins Vincent, Alan, Rich, and Kathy to describe how his laboratory is searching for the origin of MERS-coronavirus.

You can find TWiV #247 at www.microbe.tv/twiv.

Part of MERS-CoV nucleotide sequence found in a bat

What is wrong with this paragraph from today’s New York Times that describes the origin of the Middle East Respiratory Syndrome (MERS) coronavirus:

Health officials confirmed Wednesday that bats in Saudi Arabia were the source of the mysterious virus that has sickened 96 people in the Middle East, killing 47 of them.

Here is the science behind that statement, which has been published in Emerging Infectious Diseases. Samples (fecal, fecal swab, throat swab, blood) were collected from bats in an area of Saudi Arabia where human infections with MERS-CoV have been identified. Total nucleic acids were extracted and analyzed for the presence of coronavirus sequences by polymerase chain reaction. Coronavirus sequences were amplified from 220 of 732 fecal samples and 7 of 91 rectal swab samples or fecal pellets. One PCR product obtained from a single bat sample (fecal pellet of a T. perforatus bat captured in October 2012 in Bisha) had 100% nucleotide identity to a human MERS-CoV isolate.

A single PCR product 190 nucleotides in length from one bat was a perfect match with the genome sequence of a MERS-CoV isolate.

No infectious MERS-CoV has yet been isolated from this single bat. Therefore it is not yet possible to say that bats are the source of virus causing the MERS-CoV outbreak. As I have written previously, a virus is very different from a viral sequence.

It is certainly possible that MERS-CoV originated in a bat. Bats are known to harbor many viruses, and of course the SARS coronavirus originated in bats. But there is more than one explanation for the presence of this short viral sequence in bats. Perhaps the virus (or viral sequence) was obtained when the bat ingested a meal. Perhaps the 190 nucleotides are from a recombinant virus that is not MERS-CoV. I can think of other reasons why bats might not be the source of MERS-CoV.

For these reasons I believe that it is inaccurate for ‘health officials’ and the New York Times to confirm that bats are the source of MERS-CoV. Additional work is clearly needed to show that T. perforatus is the source of MERS-CoV, including isolation of infectious virus from bats and demonstrating infection of bats by the presence of antibodies to the virus. The work is in clearly progress; indeed the results might even be known, but they are not included in the Emerging Infectious Diseases article on which the NY Times piece was based.

Update 1: The term ‘frag-virus’ was proposed in 2008 to indicate viruses known only from sequence data. Although the term never caught on, the short article points out the problems that arise when genomic fragments are used to identify new viruses :

Although unintentional, these reports may mislead the readership of scientific journals and the general press. Having no distinction between preliminary genome-based evidence and conclusive proof by biological isolation and characterization of a replication-competent virus blurs the meaning of new virus.

Update 2: A phylogenetic analysis of the DNA fragment amplified from T. perforatus has been carried out. The author writes that “although this fragment means a very close relative of the human MERS-CoV is found in a bat geographically close to the first case, the fact it is identical in this short region doesn’t mean that these bats are the direct source of the human case.”  I would add even more uncertainty because we have no evidence that the virus was replicating in this single bat.

Receptor for new coronavirus-EMC identified

Coronavirus virionViruses are obligate intracellular parasites, which means that they must enter a cell to reproduce. As virions are too large to diffuse passively across the plasma membrane, cellular pathways for uptake of extracellular materials provide entry routes. The first step in entry is adherence of virus particles to the membrane, an interaction mediated by binding to one or more receptor molecules on the cell surface. Identification of cell receptors for viruses is an important objective because their study may lead to information about how the virus enters the cell, how it is targeted to specific tissues, and how it causes disease. The cell receptor for the recently identified coronavirus-EMC, which has so far infected 15 humans with 9 deaths, has been identified as dipeptidyl peptidase 4 (DPP4).

Enveloped viruses such as CoVs typically attach to cell receptors via spike glycoproteins embedded in the viral envelope. To identify the CoV-EMC receptor, part of the viral spike (S) glycoprotein was expressed as a fusion protein with the Fc domain of IgG antibody. The protein was mixed with lysates of cells known to be infected with CoV-EMC, and bound proteins were isolated by using agarose beads bound to protein A (which binds the Fc domain). A single polypeptide bound to the CoV-EMC spike protein was identified as DPP4. Four different lines of evidence indicate that DPP4 is a bona fide receptor for CoV-EMC:

  • Soluble DPP4 blocks infection of susceptible cells with CoV-EMC
  • Expression of DPP4 in non-susceptible cells renders them susceptible to infection
  • Antibody to DPP4 blocks infection of cells with CoV-EMC
  • Purified DPP4 protein binds CoV-EMC and inhibits infection

Considering that CoV-EMC was isolated in November 2012 from a sick patient, the identification of the cell receptor is indeed rapid progress.

DPP4 protein is expressed in primary human bronchiolar lung tissue and on primary bronchiolar epithelial cell cultures, consistent with the ability of the virus to infect the respiratory tract. The protein is also present on the epithelium of kidney, small intestine, liver and prostate. CoV-EMC has been detected in the respiratory tract and in urine. Whether the virus replicates in the respiratory tract, and then disseminates and replicates elsewhere (e.g. kidney) remains to be determined.

CoV-EMC is believed to have originated in bats. Consistent with this hypothesis, expression of bat DPP4 confers susceptibility to the virus, although not to the same extent as human DPP4. Once the bat precursor of CoV-EMC is identified, it will be interesting to determine how the viral spike glycoprotein has evolved to enable more efficient usage of human DPP4.

DPP4 is a transmembrane protein that regulates the activity of hormones and chemokines through proteolytic cleavage. The cell receptors for two other CoVs are also membrane-bound peptidases, but proteolytic activity is not needed for infection. A soluble form of DPP4 is also present in blood. The authors speculate that reduction of DPP4 protein levels by CoV-EMC infection could result in higher virus-induced disease. If DPP4 is important in regulating the activity of cytokines – major components of immune responses – their removal from the circulation could result in greater virus replication and more tissue damage. It will be important to study the levels of DPP4 in humans infected with CoV-EMC, and to determine whether levels of the receptor affect viral disease.

TWiV 215: Illuminating rabies and unwrapping a SARI

On episode #215 of the science show This Week in Virology, Vincent, Alan, and Kathy review the finding that rabies virus infection alters but does not kill neurons, and provide an update on the novel coronavirus in the Middle East.

You can find TWiV #215 at www.microbe.tv/twiv.