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When Two Different Viruses Have Offspring

5 January 2023 by Gertrud U. Rey 6 Comments

by Gertrud U. Rey

This image is for illustrative purposes only and does not reflect the exact geographical locations of the IAV and RSV genomes and glycoproteins in the HVPs.

Have you ever wondered what would happen if you were infected with two different viruses at the same time? A recent study aimed at addressing this question has produced some astounding new findings.

The authors of the study wanted to observe the interactions between respiratory syncytial virus (RSV) and influenza A virus (IAV), so they infected lung cells with either virus or a mixture of both viruses. An initial experiment comparing the replication kinetics of each virus in co-infected cells to those infected with either virus showed that co-infection had no impact on the replication of IAV, but did lead to reduced replication of RSV.  

IAV and RSV each localize to distinct cellular regions during their individual courses of infection – RSV aggregates in cytoplasmic complexes known as inclusion bodies, and IAV scatters more diffusely throughout the cytoplasm. Analysis of infected cells by fluorescence microscopy using antibodies against both the RSV and IAV nucleoproteins revealed that co-infection did not alter this localization – RSV was still in inclusion bodies and IAV was diffuse. The authors then analyzed later stages of infection, when in single infections, these viruses assemble in structures called lipid rafts in the plasma membrane. Their results using antibodies against the IAV hemagglutinin (HA) protein or the RSV F protein (i.e., the viral surface glycoproteins) revealed that in co-infections, both viruses were also simultaneously in the same region around the plasma membrane, suggesting that viral particles budding from the cell surface could contain components of both RSV and IAV.  

Using high resolution confocal microscopy and a technique known as cryo-electron tomography, which reconstructs a series of image slices to generate a three-dimensional structure of a sample, the authors found that co-infection of cells produced two types of particles. The first type, called ‘pseudotyped viruses,’ consisted of RSV particles with IAV glycoproteins. The second type, designated ‘hybrid virus particles’ (HVPs), were true hybrids containing the genomes and surface glycoproteins of both viruses with distinct structural regions characteristic of each virus. Because glycoproteins determine which cells and cell surface proteins viruses can bind to (i.e., their “antigenicity”), it is reasonable to assume that HVPs would have a modified antigenicity relative to IAV and RSV. IAV entry into cells occurs via the viral HA protein, which binds the cell surface protein sialic acid. RSV entry is effected in part via its F protein, which mediates fusion of the virus with the host cell membrane. To determine whether the HA and F glycoproteins on the HVPs were altered in terms of their antigenicity, the authors carried out a neutralization assay, which reveals whether an antibody can bind a glycoprotein and inactivate the viral particle. Anti-HA antibodies neutralized HVPs about three-fold less efficiently than viruses collected from cells that had only been infected with IAV, suggesting that the HA on HVPs is different enough so that antibodies won’t recognize it. In contrast, anti-F antibodies neutralized HVPs about as well as they did viruses isolated from cells infected with RSV only, suggesting that the antigenicity of the F glycoprotein on HVPs was well preserved. These results also suggested that HVPs cannot enter host cells using the IAV HA protein, and likely enters via the RSV F protein instead.    

To test this hypothesis, the authors treated cells with neuraminidase, which binds sialic acid and sequesters it, thus leaving no receptor for IAV to bind to and enter the cell. Viruses isolated from singly or co-infected cells were then used to infect these neuraminidase-treated cells, and the cells were stained with IAV and RSV nucleoprotein-specific fluorescent antibodies and visualized by fluorescence microscopy to determine whether IAV, RSV, or HVPs had infected them. As expected, neuraminidase-treated cells infected with viruses isolated from RSV only- or IAV only-infected cells contained RSV nucleoprotein but not IAV nucleoprotein, suggesting that IAV was unable to infect these cells (because there was no sialic acid to bind to), while RSV infected these cells normally because RSV entry is not dependent on sialic acid. Interestingly, neuraminidase-treated cells infected with viruses isolated from co-infected cells contained an abundance of IAV nucleoproteins, further implying that HVPs containing IAV genomes entered these cells using RSV F protein.

To confirm that the RSV F protein mediated HVP entry into cells, viruses isolated from co-infected cells were treated with a monoclonal antibody against RSV F protein before they were used to infect neuraminidase-treated cells. The monoclonal antibody would presumably sequester any viruses having the F protein and prevent any F protein-mediated entry into cells. This treatment led to significantly reduced entry of HVPs into cells, confirming that the RSV F protein mediates entry of hybrid particles into cells.

Studies of virus-host interactions are extremely common, and scientists have made a lot of progress in understanding the mechanisms that drive these interactions. In stark contrast, we know very little about how viruses interact with each other. Some work has shown that not all co-infections are successful and often result in “competitive exclusion,” with one virus displacing the other, thereby preventing it from completing a replication cycle or establishing an infection in the first place. To my knowledge, this is the first study showing that two completely different viruses can coordinate their replication cycles to develop some kind of symbiosis in a clear display of co-evolution. And although this phenomenon may seem extraordinary, it is probably more common than we think.  

[For a more detailed discussion of this study, please check out TWiV 958.]

Filed Under: Basic virology, Gertrud Rey Tagged With: antigenicity, co-infection, HA, hemagglutinin, hybrid virus, hybrid virus particle, IAV, influenza A, neuraminidase, rsv, RSV F protein, sialic acid, virus-virus interaction

Wuhan spiny eel influenza virus

28 October 2021 by Vincent Racaniello

Influenza B viruses, unlike influenza A viruses, do not cause pandemics. There are many non-human animal reservoirs of influenza A viruses which provide gene segments that go towards making reassortant viruses that can infect humans. Influenza B viruses do not appear to have an animal reservoir other than humans – they have been isolated from seals but these appear to be human viruses. Influenza viruses discovered in fish and amphibians, however, are clearly not human viruses.

A sampling of viruses in amphibians, fish, and reptiles revealed influenza B virus sequences in the spiny eel, hagfish, and Asiatic toad. An important question to answer is whether these putative viruses have to potential to donate RNA segments to viruses that infect humans. To answer this question, functions of the HA and HA proteins of Wuhan spiny eel influenza virus (WSEIV) were studied. These two glycoproteins, found in the viral membrane, are important determinants of host range.

The HA protein of influenza A and B viruses binds the cell receptor, sialic acid, and catalyzes membrane fusion. These viruses can also hemagglutinate red blood cells. The HA protein of WSEIV does not hemagglutinate red blood cells. Influenza virus HA binds alpha2,3 and alpha2,6 linked silica acids, but the WSEIV HA binds alpha2,3 sialic acids only when they are present in a ganglioside – a sialic acid-containing lipid. HA cleavage by cell proteases is required for fusion, but none of the human airway proteases that cleave influenza A or B virus HA cleave WSEIV HA. Furthermore, the HA of WSEIV cannot catalyze fusion of cells in culture. It is therefore functionally divergent from other influenza virus HA proteins.

In contrast, the NA protein of WSEIV does have neuraminidase activity – the ability to cleave sialic acid from glycoproteins – as do other influenza virus NA proteins. Furthermore, the NA of WSEIV has similar biochemical properties as the NA of other influenza viruses, and its activity can be inhibited by the antiviral drug Tamiflu.

Both the HA and NA proteins of WSEIV are antigenically diverged from other influenza viruses – they do not react with a panel of monoclonal antibodies directed against influenza B viruses. Furthermore, human sera do not appear to contain antibodies that bind the WSEIV HA or NA, indicating that humans have not been infected with this virus.

These data show that WSEIV is a bona fide influenza virus; the HA is functionally diverged from that of other influenza viruses while the NA has similar properties. Based on these observations it seems unlikely that the WSEIV HA RNA segment could be part of a virus that infects humans. However, the similarity of the WSEIV NA with those of other influenza viruses suggest that it might be compatible with reproduction in humans. The fact that there is little antigenic conservation of the WSEIV glycoproteins with those of other influenza viruses means that an influenza virus carrying them would encounter an immunologically naive human population. Sound familiar?

An important lesson of this work is that we have woefully undersampled wildlife for viruses. Consequently, conclusions about what viruses are found in which hosts are likely to be wrong.

Filed Under: Basic virology Tagged With: HA, influenza B virus, influenza virus, pandemic, viral, virology, virus, viruses, Wuhan spiny eel virus

TWiV 571: Piwi koalas

27 October 2019 by Vincent Racaniello

The League of Extraordinary Virologists celebrate the eradication of wild poliovirus type 3, and consider the effectiveness of an influenza vaccine produced in insect cells, and how small RNAs are protecting the Koala germline from retroviral invasion.

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Show notes at microbe.tv/twiv

Filed Under: This Week in Virology Tagged With: baculovirus, endogenous retrovirus, Flu, Flublock, Flucelvax, germline, HA, influenza, influenza vaccine, insect cell, Koala, koala retrovirus, piRNA, transposon, viral, virology, virus, viruses

How a toupee compromised influenza vaccine

30 November 2017 by Vincent Racaniello

The influenza virus vaccine is frequently updated to ensure that it protects against infection with circulating virus strains. In some years the vaccine matches the circulating strains, but in others, there is a mismatch. The result is that the vaccine is less effective at protecting from infection. During the 2014-15 influenza season there was a mismatch due to growing the vaccine in embryonated chicken eggs.

[Read more…] about How a toupee compromised influenza vaccine

Filed Under: Basic virology, Information Tagged With: antibody, antigenic drift, embryonated chicken egg, glycan, glycosylation, HA, influenza virus, vaccine, vaccine escape, viral, virology, virus

Unusual mortality pattern of 1918 influenza A virus

1 May 2014 by Vincent Racaniello

1918 influenza mortalityThe 1918 influenza pandemic was particularly lethal, not only for the very young and the very old (as observed for typical influenza), but unexpectedly also for young adults, 20 to 40 years of age (pictured). It has been suggested that the increased lethality in young adults occurred because they lacked protective immunity that would be conferred by previous infection with a related virus. Reconstruction of the origins of the 1918 influenza virus provides support for this hypothesis.

Analysis of influenza virus genome sequences using a host-specific molecular clock together with seroarchaeology (analysis of stored sera for the presence of antibodies to influenza virus) indicates that the 1918 H1N1 virus arose ~1915 by reassortment of an avian influenza virus with an H1 virus that had previously emerged around 1907. The 1918 virus acquired the HA gene from the 1907 virus, and the NA gene and internal protein genes from an avian virus. This 1918 virus also infected pigs, in which descendants continue to circulate; however the human 1918 virus was displaced in 1922 by a reassortant with a distinct HA gene.

Seroarchaeology and mortality data indicate that an influenza pandemic in 1889-1893 was caused by an influenza H3N8 virus. This virus appears to have circulated until 1900, when it was replaced by a H1N8 virus (the N8 gene originating from the previously circulating H3N8 virus).

How do these events explain the unusual mortality pattern of the 1918 influenza A virus? High mortality among 20-40 year old adults might have been a consequence of their exposure to the H3N8 virus that circulated from 1889-1900. This infection provided no protection against the 1918 H1N1 virus. Protection of other age groups from lethal infection was likely a consequence of childhood exposure to N1 or H1 containing viruses (this may also have resulted in the lower than usual mortality in the elderly population). Influenza is typically highly lethal in very young children due to lack of immunologic memory.

These observations suggest that childhood exposure to influenza virus is a key predictor of virulence of a pandemic strain. Antibodies against the stalk of the HA protein protect against severe disease, but only within groups of HA subtypes (HA groups are determined by phylogenetic analysis). In 1918, antibodies against a group 2 HA subtype virus (H3) did not protect against severe disease caused by a group 1 HA subtype virus (H1). Childhood exposure might also determine mortality of seasonal influenza. For example, the high virulence of currently circulating H3N2 influenza viruses in those older than 65 years might be a consequence of infection with an H1N1 virus at a young age.

This logic can also explain mortality caused by influenza H5N1 and H7N9 viruses. Most fatalities caused by H5N1 viruses (the H5 is a group 1 HA) have been in individuals who were infected as children with an H3 virus (group 2 HA). Most fatalities caused by H7N9 viruses (group 2 HA) have occurred in individuals who were infected as children with H1N1 or H2N2 viruses (group 1 HA).

The practical consequence of this work are clearly stated by the authors:

Immunization strategies that mimic the apparently powerful lifetime protection afforded by initial childhood exposure might dramatically reduce mortality due to both seasonal and novel IAV strains.

Filed Under: Basic virology, Information Tagged With: 1918 pandemic, H1N1, H2N2, H3N8, HA, influenza, seroarchaeology, vaccine, viral, virology, virus

Yet another avian influenza virus, H10N8, infects humans

10 February 2014 by Vincent Racaniello

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.

Filed Under: Basic virology, Information Tagged With: avian influenza, China, H10N8, H5N1, h7n9, HA, NA, viral, virology, virus, zoonosis, zoonotic

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