Respiratory syncytial virus co-infections might conspire to worsen disease

The respiratory tract is a rich ecological niche. In addition to the regular residents of the virome and microbiome, infectious agents such as influenza and respiratory syncytial virus (RSV) come and go, and sometimes overlap. RSV can cause acute illness and the virus can sometimes linger. Viruses that stick around can cause chronic conditions such as asthma¹, and could potentially change the course of future respiratory infections.

Today, polymerase chain reaction (PCR) diagnostics that can detect multiple pathogens are commonplace. Clinicians can swab a child’s nose, then send the sample to a hospital laboratory to test for 20 or more pathogens including SARS-CoV-2 and other coronaviruses, rhinovirus, various strains of influenza and RSV. Armed with multiplex PCR, which is able to detect multiple pathogens at once, clinicians now know that respiratory pathogens almost never appear alone.

Part of Nature Outlook: Respiratory syncytial virus

These multiplex diagnostic tests only came into broader use around 2015, says Carolina López, a molecular microbiologist who studies RSV and other respiratory viruses at the Washington University School of Medicine in St. Louis, Missouri. Because the diagnostic technology is relatively new, she says, co-infections have been understudied.

So far, most studies have focused on one virus at a time, says Pablo Murcia, a virologist at the University of Glasgow, UK. Now, scientists are learning more about how RSV can change the course of other respiratory infections and vice versa. And they’re trying to uncover the mechanisms behind these intricate interactions. Understanding these complex dynamics could help physicians to better predict an individual’s risk of infection and the adverse outcomes from viral infections. It will also help epidemiologists to forecast population-scale infection dynamics more precisely.

Virologists are beginning to ask questions about which pathogens tend to infect cells at the same time, and whether these affect clinical outcomes. “What co-infections are more common or more impactful in the clinic?” says López. Clinical samples give infectious-disease specialists clues about what kinds of co-infection might be significant.

Perfect pairing

The strongest evidence for the potency of RSV co-infections is in bacterial pneumonia caused by Streptococcus pneumoniae, also called pneumococcus. Many children have pneumococcus bacteria in their noses, but it doesn’t move into the lungs and make them unwell until provoked. But by what?

For a long time scientists have observed a correlation between RSV and pneumococcal-related diseases. Researchers suggested that RSV and pneumococcus can somehow make each other more virulent, on the basis of a few key pieces of evidence. RSV-related hospitalization rates decrease when children are vaccinated against the bacteria², for example. And the amount of nasal pneumococcus that children can carry increases during RSV infections. But establishing a causal relationship between the two was challenging — until the COVID-19 pandemic.

A group of researchers led by Ron Dagan at the Ben Gurion University of the Negev in Beersheba, Israel, had been studying a cohort of children under the age of five since 2016, taking note when participants developed pneumococcal pneumonia and other respiratory infections². In 2020, with social distancing measures in place, pneumococcal disease in his study “almost disappeared”, says Dagan, who specializes in paediatric respiratory diseases. So did respiratory viruses, including RSV. Yet, the number of children carrying the bacteria in their noses remained unchanged, but it didn’t make them sick. When social distancing was lifted, children became infected with RSV and then the bacteria did make them ill. This was evidenced when social-distancing measures were eased and viral respiratory infections roared back with a vengeance — as did pneumonia.

“There is a causative relationship between the two,” Dagan says. “These viruses are very strongly influencing the pneumococcus to become virulent.” RSV was the biggest contributor, causing 49% of pneumonia cases in the study.

Viral frenemies

For co-infections between RSV and other viruses, the clinical significance is murkier. In children, RSV “can cause severe infections no matter what”, says Tobias Tenenbaum, a paediatrician at Sana Hospital Lichtenberg in Berlin, who specializes in infectious diseases. Viral co-infections, however, don’t necessarily cause extra symptoms. Tenenbaum led a 2010 study³ that analysed a group of children for two years and found that the total amount of RSV a child had in their nose correlated with disease severity, but that co-infections with other viruses did not.

But these were early findings, and certainly not definitive. In fact, some studies directly contradict them, says Murcia. Other research has shown that when a viral infection occurs in the presence of RSV, disease severity is attenuated. The populations that were analysed differ between these studies, as do the mix of viruses and measures of severity, which might help to explain some of the discrepancies.

Murcia hopes in vitro and animal studies, the natural next step, will provide clarity. “Most studies of co-infection are based on clinical cases, and there is a lot of variability there,” says Murcia.

RSV and influenza A are a particularly vexing pair. Influenza A is one of two flu viruses that cause seasonal illness, capable of causing new influenza epidemics. Some research suggests that RSV and influenza A can exclude each other. On the basis of in vitro studies, researchers think that this might be because an immune response to one virus can trigger the release of signalling proteins called interferons that prevent the establishment of the second virus.

However, this simple, elegant explanation might not be playing out in the human population. A nine-year statistical study of 44,230 clinical cases of respiratory illness did not find any evidence of the viruses acting against each other between RSV and influenza⁴. The most recent flu season in Europe provides a good test case: during the winter of 2022–23 in Europe, says Tenenbaum, waves of RSV and influenza A closely overlapped. He’s currently studying whether co-infection with these two viruses caused more severe disease during that season.

Murcia says that in his study⁴, which corrected for the seasonality of viruses to uncover other connections, “the patterns of co-infections didn’t look random”. The study showed that influenza viruses and non-influenza viruses negatively affected each other on a population level. Murcia decided to take an in depth look at RSV and influenza in a lab study⁵, expecting to see a negative association.

But his team found the opposite. They infected human lung cells with influenza A and RSV. “What caught our eyes is that they didn’t seem to block each other,” he says. In fact, the two viruses seem to have combined forces, fusing to form what Murcia calls hybrid viral particles, which contain both genomes. What’s more, these hybrid particles can infect cells that lack surface receptors that are usually a baseline requirement for influenza infections.

“What we want to know now is whether this happens in real life,” he says. Murcia is the first to pose caveats and questions about the hybrid viral particles. His lab used research strains of the viruses; will hybrids form between viruses that are isolated from people? Does this happen with other viruses? Murcia notes that both RSV and influenza A are both enveloped in a lipid coat, which might enable them to fuse together.

“There’s been this debate in the field about whether you can package more than one viral genome in the same particle, even of the same virus,” says López. “I think a lot of people were impressed with the work, some people believe it, some people don’t, but now we have pictures,” she says. Murcia’s study included high-resolution microscopy images confirming the existence of the viral particles.

Making models

Virologists are now trying to work out how to move forwards out of the tangle of conflicting evidence about co-infections. Because controlled studies are hard to do in the clinic, researchers are pinning their hopes on better in vitro and animal models of co-infection. But many questions remain about how best to develop these models.

A researcher interested in mimicking co-infections in an animal or in vitro model faces a huge list of decisions, says López. First: choosing the right virus. “Are we really using the right viruses in animal models?” López wonders. “A human virus in a mouse may interact completely differently” than it does in its natural host, she says.

Molecular microbiologist Catherine Satzke and research fellow Sam Manna at the Murdoch Children’s Research Institute in Melbourne, Australia, decided to use the mouse equivalent of RSV for their work on RSV–pneumococcus co-infections⁶. Many mouse models have used human RSV, but the virus needs be given in unrealistically high doses and doesn’t replicate well in mice, says Manna. So the researchers used murine pneumonia virus (PVM), the rodent equivalent of RSV. Moreover, the team chose to work with infant mice, because RSV and its co-infections have the greatest affect on young children. Their research, published in 2022, found that the pneumococcal bacterial strain matters, too. Perhaps because some strains are already more virulent than others, and some benefit more from the presence of PVM.

López says that the unknown timing of viral co-infections makes them particularly difficult to study. Is it better to start by infecting cells with RSV, or should influenza come first? Another key set of questions relates to the length of time of infections. How long should the first virus be allowed to establish itself before the second is added?

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“We have a lot of viruses persisting for longer than we think,” López says. But most models that are now in use focus on acute viral respiratory infections. That might not match the clinical reality. “Viruses that seem acute could persist at low levels and change the environment for the next virus,” she says. “They do not function as a single entity, they are a community.”

As well as timing, there are questions of location. Scientists don’t know whether viruses need to infect not only the same part of the body but also the same types of cell to cause co-infections. Infecting the same cells is necessary to form the kind of hybrid particles Murcia saw in the lab, but it might not be needed to cause synergistic co-infections. López is developing an RSV co-infection model based on organoids — miniaturized versions of human tissues — that include multiple cell types from the human respiratory system. She hopes studying multiple viruses in these mixed groups of cells will help to answer some of these questions.

This research also raises queries about microbial and viral evolution. Viruses that infect the same part of the body at the same time — or prevent other viruses from infecting cells at the same time as or after them — are at the very least exerting evolutionary pressures on each other. RSV and influenza are, from a human point of view, a bad influence on pneumococcus. So, are these pathogens cooperating or is all this a coincidence? “It’s hard to differentiate what’s an evolutionary advantage and what’s happenstance,” says Satzke.

“We don’t have a lot of understanding of any of this,” says López. “This is the fun of science, right?”