TWiV 364: It’s not SARS 2.0

On episode #364 of the science show This Week in Virology, Vincent, Rich, and Kathy speak with Ralph Baric and Vineet Menachery about their research on the potential of SARS-like bat coronaviruses  to infect human cells and cause disease in mice.

You can find TWiV #364 at www.microbe.tv/twiv.

Lassa virus origin and evolution

arenavirusI have a soft spot in my heart for Lassa virus: a non-fictional account of its discovery in Africa in 1969 inspired me to become a virologist. Hence papers on this virus always catch my attention, such as one describing its origin and evolution.

Lassa virus, a member of the Arenavirus family, is very different from Ebolavirus (a filovirus), but both are zoonotic pathogens that may cause hemorrhagic fever. It is responsible for tens of thousands of hospitalizations, and thousands of deaths each year, mainly in Sierra Leone, Guinea, Liberia, and Nigeria. Most human Lassa virus outbreaks are caused by multiple exposures to urine or feces from the multimammate mouse, Mastomys natalensis, which is the reservoir of the virus in nature. In contrast, outbreaks of Ebolavirus infection typically originate with a crossover from an animal reservoir, followed by human to human transmission. Despite being studied for nearly 50 years, until recently the nucleotide sequences of only 12 Lassa virus genomes had been determined.

To remedy this lack of Lassa virus genome information, the authors collected clinical samples from patients in Sierra Leone and Nigeria between 2008 and 2013. From these and other sources they determined the sequences of 183 Lassa virus genomes from humans, 11 viral genomes from M. natalensis, and two viral genomes from laboratory stocks. All the data are publicly available at NCBI. Analysis of the data lead to the following conclusions:

  • Lassa virus forms four clades, three in Nigeria and one in Sierra Leona/Liberia (members of a clade evolved from a common ancestor).
  • Most Lassa virus infections are a consequence of multiple, independent transmissions from the rodent reservoir.
  • Modern-day Lassa virus  strains probably originated at least 1,000 years ago in Nigeria, then spread to Sierra Leone as recently as 150 years ago. The lineage is most likely much older, but how much cannot be calculated from the data.
  • The genetic diversity of Lassa virus in individual hosts is an order of magnitude greater than the diversity of Ebolavirus. Furthermore, Lassa virus diversity in the rodent host is greater than in humans, likely a consequence of the longer, persistent infections that take place in the mouse.
  • The gene encoding the Lassa virus glycoprotein is subject to high selection in hosts, leading to variants that interfere with antibody binding.
  • Genetic variants that arise in one rodent are not transmitted to another.

Perhaps the most important result from this work is the establishment of laboratories in Sierra Leone and Nigeria that can safely collect and process samples from patients infected with Lassa virus, a BSL-4 pathogen.

TWiV 347: Rose rosette and squirrel roulette

On episode #347 of the science show This Week in Virology, Vincent, Alan, and Rich discuss the virus behind rose rosette disease, and fatal human encephalitis caused by a variegated squirrel bornavirus.

You can find TWiV #347 at www.microbe.tv/twiv.

TWiV 327: Does a gorilla shift in the woods?

On episode #327 of the science show This Week in Virology, the eTWiVicators review evidence that the HIV-1 group O epidemic began with a single cross-species transmission of virus from western lowland gorillas.

You can find TWiV #327 at www.microbe.tv/twiv.

Yet another avian influenza virus, H10N8, infects humans

chicken market

To the collection of avian influenza viruses known to sporadically infect humans – H5N1, H7N9, H7N2, H7N3, H7N7, H9N2, and H10N7 – we can now add H10N8, recently found in two individuals in China.

Avian influenza virus H10N8 was first detected in tracheal aspirates from a 73 year old woman who was hospitalized in November 2013 for severe respiratory illness. The patient, who died, had previously visited a live poultry market. A second infection with this virus was detected in January 2014.

Virus isolated from tracheal aspirates on day 7 of illness was named A/Jiangxi-Donghu/346/2013(H10N8). Nucleotide sequence analysis of the viral genome reveals that it is a reassortant. The HA gene most closely resembles that of a virus isolated from a duck in Hunan in 2012, while the NA gene resembles that of a virus isolated from a mallard in Korea in 2010. All six other RNA segments resemble those from circulating H9N2 viruses in China. These viruses have also provided genes for H7N9 and H5N1 viruses.

Examination of the viral protein sequences provides some clues about virulence of the virus. The HA protein sequence reveals a single basic amino acid at the cleavage site, indicating that the virus is of low pathogenicity in poultry, like H7N9 virus. The sequence in the sialic acid binding pocket of the HA protein indicates a preference for alpha-2,3 linked sialic acids, typical  for avian influenza viruses (human influenza viruses prefer alpha-2,6 linked sialic acids). A lysine at amino acid 627 in the PB2 protein is known to enhance the ability of the virus to replicate at mammalian temperatures; the H10N8 virus has a mixture of lysine and glutamic acid, the residue associated with less efficient replication. The sequence of the M2 protein indicates that the virus is resistant to the antiviral adamantanes. In vitro testing indicated sensitivity to NA inhibitors Tamiflu and Relenza.

It is not known if this novel H10N8 virus will spread further in the human population. A novel influenza H7N9 virus was first detected in humans in early 2013 and has since caused 250 human infections with 70 deaths. Similar incursions of avian influenza viruses into humans have probably taken place for as long as humans have had contact with poultry. We are now adept at detecting viruses and therefore we are noticing these infections more frequently.

Live poultry markets are clearly a risk factor for humans to acquire infections with avian influenza viruses, as noted by Perez and Garcia-Sastre:

Live bird markets in Asia are undoubtedly the major contributor in the evolution of avian influenza viruses with zoonotic potential, a fact for which we seem to remain oblivious.

Given their role in transmitting new viruses from animals to humans, I wonder why live poultry markets are not permanently closed.

Update: George Gao agrees that the live poultry markets in China should be closed.

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.

Influenza A viruses in bats

A/bat/Peru/10 H18It is well known that aquatic birds are a major reservoir of influenza A viruses, and that pandemic human influenza virus strains of the past century derive viral genes from this pool. The recent discovery of two new influenza A viruses in bats suggests that this species may constitute another reservoir with even greater genetic diversity.

A new influenza virus had previously been isolated from little yellow-shouldered bats (Sturnira lilium) in Guatemala. Three of 316 rectal swabs were positive when tested by a pan-influenza polymerase chain reaction assay. Viral sequences were also detected in liver, intestine, lung, and kidney tissues, suggestive of viral replication and not passage of ingested material through the intestinal tract. Analysis of the viral genome sequence revealed that A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) is significantly diverged from all known influenza viruses.

When the same PCR approach was used to screen 114 rectal swabs from 18 different species of bats captured in Peru, a single flat-faced fruit bat (Artibeus planirostris) was positive. Viral sequences were also detected in liver, intestine, and spleen tissues from the same bat. Comparison of the sequences of all 8 genome RNA segments with those of the H17N10 Guatemalan isolate revealed sufficient divergence to justify naming it a new HA and NA subtype, A/flat-faced bat/Peru/033/2010 (H18N11).

Comparison of the nucleotide sequences of bat influenza A viruses from Peru and Guatemala with other influenza viruses leads to two amazing conclusions. First, 7 of the 8 viral RNAs of the bat influenza A viruses group separately from the RNAs of all other known influenza viruses. Second, the RNA sequences encoding four proteins, PB2, PB1, PA and NA, display greater genetic diversity than in all non-bat influenza virus sequences combined. The implication is that New World bats harbor a diverse pool of influenza viruses.

The H17 and H18 HA RNA sequences are, in contrast, far more related to known influenza virus HA and NA sequences. The implication of this observation is clear: some time after the bat and non-bat influenza A viruses diverged, a reassortment event occurred that introduced the HA of a non-bat influenza A virus into the genome of a bat influenza A virus.

Serological studies have revealed widespread circulation of these two new influenza viruses in bats. Sera from 55 of 110 (50%) Peruvian bats representing 13 different species were positive for antibodies against the viral HA or NA proteins. Twenty-one of these samples were positive for antibodies against both viral glycoproteins, while 30 were positive only for anti-HA18 antibodies and 4 were positive for only anti-N11 antibodies. These observations suggest that some bats are infected with reassortant viruses carrying the H18 or N11 genes. A study of sera from 8 different species of Guatemalan bats revealed antibodies to the H17 HA protein in 86 of 228 sera (38%).

A number of human viruses, such as SARS-coronavirus and Nipah and Hendra viruses, are known to have originated in bats. Can bat influenza A viruses infect humans and serve as a source of future pandemic strains? The answer to this question is not known, but the two new bat viruses cannot infect human cell lines in culture. However, it is possible that acquisition of other (e.g. avian or swine) influenza virus genes by reassortment could produce a virus with bat influenza virus genes that is capable of infection humans. The pathogenic and pandemic potential of such viruses is unknown. A first step to answering this question would be to determine if human populations with contact with bats have antibodies to the two new bat influenza A viruses.

The cell receptor for all known influenza A viruses is the carbohydrate molecule known as sialic acid  The cell receptor for the two new bat influenza A viruses is not known, but it is clearly not sialic acid, a conclusion reached by studying the crystal structures and binding properties of the H17 (paper one and two) and H18 HA (illustrated) molecules. Furthermore, the crystal structures of the N10 (paper one and two) and N11 proteins reveal that their substrate cannot be sialic acid (the function of the influenza A virus NA is to remove sialic acids from the cell surface, allowing newly synthesized virions to move away from the cell). For this reason the N10 and N11 proteins are called ‘NA-like’.

Bats also harbor many other kinds of viruses, including hepatitis B viruses, Marburg virus, hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses, and many more. They also contain parasites – specifically, malaria parasites. For more information, listen to these podcast episodes:

Hedging our bats (TWiV 258)
More bats out of hell (TWiP 62)
Hepaciviruses and pegiviruses in bats and rodents (TWiV 231)
Bats out of hell (TWiV 183)
Going to bat for flu research (TWiV 173)
Matt’s bats (TWiV 65)

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.

Hepatitis B viruses in bats

hepadnaviridae virionHepatitis B virus (HBV, illustrated) is a substantial human pathogen. WHO estimates that there are now 240,000,000 individuals chronically infected with HBV worldwide, of which 25% will die from chronic liver disease or hepatocellular carcinoma. The hepatitis B virus vaccine is highly effective at preventing infection. Because there are no known animal reservoirs of the virus, it is believed that HBV could be globally eradicated. The recent finding of HBV in bats raises the possibility of zoonotic introduction of the virus.

Serum and liver samples from 3,080 bats from Panama, Brazil, Gabon, Ghana, Germany, Papua New Guinea, and Australia were screened for HBV-like sequences by polymerase chain reaction (PCR). Ten positive specimens were found from three bat species: Uroderma bilobatum from Panama, and Hipposideros cf. ruber and Rhinolophus alcyone from Gabon. The complete viral genome sequence was determined for 9 of the positive specimens. Phylogenetic analysis revealed that the bat viruses form three different lineages, and that each virus differs by at least 35% from known hepadnaviruses.

The virus from Hipposideros cf. ruber has been named roundleaf bat HBV, while those from Rhinolophus and Uroderma have been named horshoe bat HBV, and tent-making bat HBV.

Viral DNA in the liver of Hipposideros bats was found to be higher than in other organs or serum. Some lymphocyte infiltration was observed in the liver of these animals, as well as deposits of viral DNA within hepatocytes. These observations indicate that the bat HBV viruses likely replicate in the bat liver and cause hepatitis.

Serological studies revealed that hepadnaviruses are widespread in Old World bats: antibodies against bat hepadnaviruses were detected in 18% of hipposiderid bats and 6.3% of rhinolophid bats.

An important question is whether these three bat hepadnaviruses can infect human cells. Only tent-making bat HBV could infect primary human hepatocytes, which occurred via the human HBV cell receptor, sodium taurocholate cotransporting polypeptide. However serum from humans that had been immunized with HBV vaccine did not block infection of human hepatocytes with this virus.

These observations show that viruses related to human HBV are replicating in the liver of bats. Earlier this year another hepadnavirus was identified in long-fingered bats (Miniopterus fuliginosus) in Myanmar. The complete genome sequence was obtained and virus particles were observed in bat liver tissues.

The finding of hepadnaviruses in bats raise many interesting questions. The first is whether human HBV originated by infection with bat HBV, either by consumption of bat meat or another mode of transmission. How long ago this occurred is not known. It has been suggested that HBV has been in humans for at least 15,000 years. Some avian species contain avihepadnaviral sequences integrated into their genome, indicating that these viruses originated at least 19 million years ago.

These findings also raise many questions about the pathogenesis of hepadnaviral infection in bats, including the mode of transmission (in humans, the virus is transmitted by exposure to blood, e.g. by injection or during childbirth), and whether chronic infections can occur as they do in humans.

Finally it is interesting to consider the zoonotic potential of tent-making bat HBV, which can infect human cells. Because bat hepadnaviruses are genetically distinct from HBV, current serological and nucleic acid screening programs would not detect human infections. The authors suggest that human and non-human primate sera from areas in which these bat viruses were isolated should be screened using assays that detect the bat hepadnaviruses. Without such information we do not know if these viruses currently infect humans.