Basic virology

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.]

“Paxlovid Rebound” Is Just COVID Rebound

1 December 2022 by Gertrud U. Rey

by Gertrud U. Rey

The antiviral drug Paxlovid is highly effective at inhibiting SARS-CoV-2 replication and reducing symptoms associated with COVID-19. Nevertheless, there have recently been numerous reports of recurrence of positive tests and symptoms after completing treatment with Paxlovid, leading some to infer that the drug triggers the recurrence. Is this inference actually correct, or would the recurrences happen regardless of treatment? In other words, is “Paxlovid rebound” really just COVID rebound?

Most studies aiming to address this question have been retrospective analyses, which use existing data collected from events that have already happened. A major disadvantage of examining data retrospectively is that it is impossible to randomly assign participants to experimental or control groups, or to even apply the proper controls as is typically done in a prospective study. These drawbacks often lead to a biased selection of participants such that they do not always represent the population that is intended to be analyzed, which leads to inaccurate results and false conclusions.

In an attempt to remedy this shortcoming, a group of investigators led by Michael Mina carried out a prospective study in which they compared the outcomes between two groups of COVID-19 patients: a group of 127 subjects who chose to be treated with Paxlovid and a control group of 43 subjects who chose not to be treated. The aim of the study was to determine whether Paxlovid recipients experience a higher incidence of rebounds than non-treated individuals.

To qualify for the study, all participants had to test positive for SARS-CoV-2 using a rapid antigen test. The day of the first test was then documented as day 0 and the participants continued testing themselves and recording their symptoms on days 2, 5, 7, 9, 11, 13, 15, and 17 of the study period. Any positive antigen test after a negative test within the 17-day period was defined as a viral rebound, and any recurrence of symptoms after initial symptom clearance within the same period was defined as a symptom rebound. At the 17-day time point, among the Paxlovid group, 14% of subjects had experienced a viral rebound and 19% had experienced a symptom rebound. In contrast, only 9% of subjects in the (untreated) control group had a viral rebound and only 7% had a symptom rebound. There were no noteworthy differences in the number of rebounds between the two groups at the one-month time point. Although the incidence of rebound was slightly higher in the Paxlovid group, this difference between the two groups was not statistically significant; it was likely due to random chance and the small sample sizes of the groups. In other words, the slightly higher incidence of viral and symptom rebounds in the Paxlovid group has no clinical meaning, and one can interpret the rate of rebounds between the Paxlovid and control groups to be similar, meaning that Paxlovid probably does not cause viral and/or symptom rebounds.

The authors thoughtfully note that the study has several limitations. First, the overall sample size of 170 participants is small and there was a large difference between the sizes of the two groups (i.e., 127 subjects in the Paxlovid group and 43 subjects in the control group). Large and balanced sample sizes are critical for reducing the margin of error and for obtaining results that are both accurate and clinically useful. Second, the participants tested themselves, which could have introduced unknown errors such as whether the tests were carried out properly or at the correct time. Third, participants were asked to only test every other day to ensure compliance; however, daily testing would have provided additional data points and more comprehensive findings. Larger surveys done under more controlled and standardized conditions are needed to validate the results obtained in this study.

In contrast to popular opinion, rebounds can happen after most viral infections, so there is nothing unique about SARS-CoV-2 in this regard. Even if Paxlovid does cause viral and/or symptom recurrence in a small subset of people, a preponderance of the evidence indicates that early treatment with Paxlovid results in an overwhelming reduction in hospitalization and death for COVID-19 patients. Understanding the underlying mechanisms leading to rebounds can help guide practitioners to modify timing and length of treatment with Paxlovid or other antiviral drugs to reduce the incidence of rebound.

Antibodies Against SARS-CoV-2 Nucleocapsid Protein May Not Be Reliable Markers for Infection in Vaccinated People

3 November 2022 by Gertrud U. Rey

by Gertrud U. Rey

You are fully vaccinated against SARS-CoV-2 and have presumably never been infected with the virus. But how can you know for sure? One way to find out is by testing your blood for the presence of antibodies against the viral nucleocapsid protein, which can only be encountered during natural infection. This is because all of the SARS-CoV-2 vaccines used in the U.S. only encode the viral spike protein (none encode nucleocapsid [N] protein), and thus they only stimulate production of antibodies against spike. This approach differentiates between vaccine- and virus infection-induced antibodies and allows one to accurately determine whether a vaccinated person was naturally infected. Or so we thought until now.

Two recent letters to the editor of the Journal of Infection note that not every natural infection induces production of anti-nucleocapsid (or, “anti-N”) antibodies. The letters cast doubt on whether these antibodies are reliable markers for a prior SARS-CoV-2 infection.

The authors of the first letter measured antibody responses in 4,111 vaccinated and 974 unvaccinated Irish healthcare workers. Only 23 of the vaccinated participants, all of whom had received two doses of the Pfizer mRNA vaccine, experienced a SARS-CoV-2 infection at some time after vaccination. As expected, each of the 23 individuals had antibodies against the spike protein, but surprisingly, only six (26%) had detectable anti-N antibodies. In contrast, 82% of unvaccinated participants with a previous PCR-confirmed infection had detectable anti-N antibodies. This result suggests that anti-N antibodies may not be the most accurate indicators of a prior natural infection in vaccinated people; and it further implies that vaccinated individuals may neutralize incoming viruses early during infection, thus preventing and/or limiting their ability to develop antibodies against nucleocapsid protein.

The second letter, which was written in response to the first letter, confirmed and further substantiated these results. Citing data from serosurveys done in Japan, the authors showed that patients who were infected within two months of a third dose of the Pfizer mRNA vaccine were less likely to experience COVID-19 symptoms than patients who were infected 4-8 months after the third dose. These findings are in line with our current understanding of sterilizing immunity, a type of immunity that prevents both disease and infection, which appears to occur most often during the months following vaccination, when high levels of vaccine-induced antibodies probably sequester an incoming virus before it has a chance to infect cells. The authors also showed that participants infected within two months of their third vaccine dose had significantly lower levels of anti-N antibodies than those infected several months later. Although this result seems surprising at first, it actually further supports the notion that vaccination only induces sterilizing immunity for a short time after vaccination, when existing vaccine-induced anti-spike antibodies neutralize incoming virus before the immune system has a chance to respond to the virus and produce antibodies specific to the nucleocapsid protein.

The authors of both letters further mention that COVID-19 patients who experienced symptoms were more likely to have detectable anti-N antibodies than were patients without symptoms, an observation that is in agreement with serological surveys done before vaccines became available. This finding suggests that patients who developed symptoms did not have sterilizing immunity and were subject to a productive viral infection that led to the development of symptoms and production of antibodies to nucleocapsid and other viral proteins.

These two studies provide an interesting perspective of antibody responses to SARS-CoV-2 infection in vaccinated people, and they may inform better strategies for gauging infection after vaccination.

Why do Some People Develop Long COVID?

6 October 2022 by Gertrud U. Rey

by Gertrud U. Rey

Long COVID is a chronic manifestation of SARS-CoV-2 infection, and it is most commonly characterized by lingering fatigue, brain fog, memory impairment, and confusion. Although it is unclear how the viral infection leads to long COVID, experts speculate that one or more of the following factors may contribute: an inability to successfully clear virus, a reactivation of latent viruses, a disturbance of the gut microbiome, continuing inflammation, and/or autoimmunity.

Yale University researcher Akiko Iwasaki and colleagues recently explored some of these hypotheses in an attempt to identify diagnostic biomarkers associated with long COVID. The study involved four groups of participants, with the experimental group consisting of both vaccinated and unvaccinated individuals with long COVID. The other three groups served as three separate types of controls:

healthy, vaccinated, uninfected individuals;
healthy, unvaccinated, previously infected individuals without long COVID; and
healthy, vaccinated, previously infected individuals without long COVID.

The investigators obtained blood samples from all participants and analyzed the samples for the presence of specific immune cells. They found that compared to control groups, long COVID participants had lower levels of conventional dendritic cells and memory T helper cells. Conventional dendritic cells typically activate cytotoxic T cells, which in turn kill infected cells. Memory T helper cells are a central element of the adaptive immune response, where they help orchestrate downstream immune functions upon recognition of antigen. Long COVID participants also had increased numbers of “exhausted” T cells, which are no longer functional or capable of eliminating virus. These results suggested that people with long COVID may have insufficient numbers of immune cells able to inactivate virus, thus allowing viruses to linger and continue replicating and circulating. Whether this assumption is true, and whether long COVID participants do indeed have persistently circulating virus, is subject to ongoing analysis.

Previous studies have shown that patients with severe COVID-19 can have increased levels of functional antibodies directed against self antigens that circulate outside of cells (i.e., extracellular autoantibodies), suggesting that SARS-CoV-2 infection can cause autoimmune disorders. To identify a possible connection between autoimmunity and long COVID, the authors screened the collected blood samples for autoantibodies using a technique called Rapid Extracellular Antigen Profiling (REAP). Among other applications, REAP allows one to assess antibody reactivity against a panel of extracellular human proteins known to contribute to well-studied autoimmune disorders. Interestingly, long COVID participants did not have increased levels of autoantibodies compared to control groups, suggesting that the autoantibodies detected in patients with severe COVID-19 may only be present during the acute phase of disease. However, REAP only assesses antibodies directed to extracellular self proteins and does not provide any information regarding intracellular or non-protein-specific autoantibodies. Therefore, one cannot rule out a role for autoimmunity in long COVID.

The authors also used REAP to detect antibodies against various viruses. They found that long COVID participants had antibodies against several other viruses in addition to SARS-CoV-2, including Epstein-Barr virus (EBV), a herpesvirus that is well known for causing infectious mononucleosis (i.e., “mono”), a condition that is often compared to encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID. However, because most adults have antibodies against EBV and there was no significant difference in the percentage of EBV antibody-positive participants between experimental and control groups, it is unlikely that a positive EBV antibody status contributes to the risk of developing long COVID.

The most interesting observation in this study was that compared to control groups, long COVID participants had about 50% lower levels of the steroid hormone cortisol. Because cortisol is a potent anti-inflammatory agent, it is feasible that a shortage of cortisol would lead to persistent inflammation and the long-term tissue damage associated with inflammation. In line with this reasoning, cortisol levels were highest in healthy, vaccinated, uninfected controls (group 1 controls); lower in healthy, unvaccinated, previously infected individuals without persistent symptoms (group 2 controls); and lowest in long COVID participants. Using machine learning methods, the authors determined that cortisol deficiency was the single most significant predictor of a long COVID diagnosis. Low cortisol has also been implicated in ME/CFS, and treatment with hydrocortisone can provide some relief from symptoms.

The authors are careful to note that the small sample size of 215 participants is a considerable limitation of this study. Nevertheless, the results offer some valuable insight that may apply to other chronic conditions. In contrast to public perception, long-term symptoms following an acute viral infection are not unique to COVID-19. Unexplained chronic syndromes involving similar symptoms to long COVID have also been reported after dengue virus, poliovirus, SARS-CoV, Chikungunya virus, West Nile virus, Ross River virus, Coxsackie virus, and influenza virus infections. Because these syndromes are associated with high public health and economic burdens, more work needs to be done to clarify their underlying mechanisms.

Transmission of Monkeypox Virus Through Contaminated Objects

1 September 2022 by Gertrud U. Rey

by Gertrud U. Rey

Recent news headlines are fueling public fears about possible transmission of monkeypox virus through contact with contaminated objects like bedding or clothing. However, data generated using environmental sampling methods indicate that the likelihood of this type of transmission is very low.

In a study involving a two-person household in Utah in which both residents were infected with monkeypox virus, the investigators entered the home to obtain samples while the patients were present. The investigators then identified and swabbed various objects that had been touched frequently; including furniture, toilet handles, light switches, and remote controls. They subsequently processed the swab samples and performed PCR and cell culture analyses to detect the presence of monkeypox virus DNA and infectious monkeypox virus. Out of 30 samples tested, 21 yielded positive results by PCR, confirming that these 21 objects were contaminated with monkeypox virus DNA. However, none of the PCR-positive samples yielded any detectable virus in cell culture, suggesting that there was no infectious virus on any of the tested surfaces in that household.

A second study involved a one-person houshold in Texas whose resident had been hospitalized with monkeypox virus infection. On day 15 after the patient had left the home, the investigators entered the house and obtained 31 swab samples from various household objects similar to those tested in the Utah home. PCR analysis revealed that 27 of the tested objects were contaminated with monkeypox virus DNA. Cell culture analysis showed that 7 of these 27 samples also contained viable virus, with six of the virus-positive samples originating from porous surfaces like bedding and clothing, and only one originating from a non-porous surface – the top of a coffee table. When the authors determined the concentration of virus in each of these seven samples using a standard titration assay, they found that only one of the samples produced a quantifiable amount of virus – a swab from an article of clothing that had been in prolonged, direct contact with active monkeypox lesions. In contrast, the quantities of virus isolated from the other six samples were all below the detectable limit of the assay, suggesting that even if an object is contaminated with monkeypox virus, the amount of virus is probably lower than the minimal infectious dose needed to establish a successful infection in a person.

A popular historical anecdote involving transmission of pox virus through inanimate objects describes how the US Army attempted to reduce the Native American population by gifting them smallpox virus-contaminated blankets. However, there are no reports or evidence indicating that this strategy actually worked, suggesting that it was probably ineffective. In fact, some historians cast doubt on whether this incident even took place.

It is possible for pox viruses to remain viable on surfaces for long periods of time, but the conditions have to be just right. Studies involving variola virus (which causes smallpox) have shown that viral particles can stay viable on contaminated surfaces for up to 13 years if they are maintained at low humidity, low temperature, and in the absence of UV radiation. The authors of the Utah home study note that the residents cleaned and disinfected their home routinely during their illness, a practice that likely resulted in reduced viability and/or inactivation of infectious virus on all of the tested objects. In contrast, no such cleaning activities were noted for the Texas home. In addition, all windows of the Texas home were covered with closed blinds, a condition that likely reduced exposure to UV radiation and probably contributed to the preservation of viral particles.

Although it is possible to become infected with monkeypox virus by touching contaminated surfaces, the potential for this type of transmission appears to be limited, and the risk of acquiring monkeypox from hotel sheets, towels, or plane seats is probably very low. Monkeypox virus is transmitted most effectively through direct (i.e., skin-to-skin) contact with lesion material or inhalation of respiratory droplets during prolonged face-to-face interaction with an infected person.

Transmission of Enteric Viruses through Saliva

4 August 2022 by Gertrud U. Rey

by Gertrud U. Rey

Norovirus and rotavirus are considered to be enteric pathogens because they are traditionally thought to be transmitted by the fecal-oral route; i.e., when consuming food prepared by someone who did not wash their hands properly after using the bathroom. Unlike rabies virus, which replicates in the salivary glands and transmits through saliva, the presence of noroviruses and rotaviruses in saliva is usually considered to result from contamination, for example by vomitus or gastroesophageal reflux.

It was therefore surprising when a recent study showed that neonatal mice (“pups”) can apparently transmit enteric viruses to their mothers during suckling. Following oral inoculation with mouse norovirus 1 (MNV-1) or epizootic diarrhea of infant mice (EDIM, a mouse rotavirus), pups had secretory IgA (sIgA) in their intestines that increased gradually over the course of two weeks. sIgA is a type of antibody that provides immunity in mucous membranes such as those of the mouth, nose, and gut, and sIgA is typically passed from mothers to infants during suckling. Interestingly, this sIgA increase in pups correlated with a similar increase in the sIgA in the milk of the mothers, even though the mothers were not infected and did not have any antibodies to either virus at the start of the experiment (i.e., they were seronegative). Considering that pups can’t produce their own sIgA, it is likely that the pups infected the mothers during lactation, who then produced the sIgA and passed it back to the pups through their milk.

To rule out the possibility that the mothers became infected by consuming feces-contaminated food (i.e., by the traditional fecal-oral route) because they shared a living area with their infected pups, the authors orally inoculated pup-free seronegative mothers with EDIM, treated them with oxytocin to induce milk production, and analyzed the milk for sIgA and the mammary glands for viral RNA. Although the mothers were successfully infected, as evidenced by the presence of EDIM RNA in their small intestines, there was no detectable EDIM RNA in their mammary glands or detectable increase of sIgA in their milk. This result confirmed that the infected pups passed the virus to the mothers during feeding.

To further substantiate this finding, the authors orally infected one set of pups (pups A) with EDIM and placed them back in the cage with their seronegative mothers (mothers A) for suckling. The next day, mothers A were replaced with “mothers B” from a cage of uninfected pups (pups B), and mothers A were placed with pups B. Two days after this switch, all mice were euthanized and analyzed for the presence of viral RNA. All mothers had EDIM RNA in their mammary glands, suggesting that both sets of mothers became infected by suckling pups A. All pups had EDIM RNA in their small intestines, suggesting that pups B became infected by feeding from mothers A, or by ingesting their fecal matter.

The next set of experiments aimed to determine whether saliva contains enteric viruses and if it could serve as a means for transmission. Infection of adult mice with MNV-1 or EDIM revealed that these mice produced and shed virus in their saliva for up to ten days post infection. To see whether this saliva was infectious, the authors fed it to pups as a means of inoculation. At three days after infection, the pups had significantly high viral genome levels in their intestines, comparable to those observed in pups inoculated with virus of fecal origin. This result suggested that the viruses replicated in the intestines and confirmed that saliva can be a conduit for transmission of these enteric viruses.

In an effort to see whether these viruses replicate in the salivary glands, the authors orally infected pups and adults with various strains of norovirus and then isolated their submandibular glands – the largest component of the salivary gland complex. They then measured the number of infectious viral particles inside the submandibular glands using an alternative to the classical plaque assay known as a TCID₅₀ assay, which quantifies the amount of virus needed to infect 50% of cells in culture. Each of the viruses increased in quantity by about 100,000-fold throughout the following three weeks compared to the input level, suggesting that noroviruses do replicate in the salivary glands. Treatment of mice with 2’-C-methylcytidine, an inhibitor of the norovirus polymerase enzyme, led to a decline of virus in the salivary glands, suggesting that this enzyme inhibited viral replication, and confirming that replication occurred inside the salivary glands.

TCID₅₀analysis of various cell populations isolated from the submandibular gland revealed that MNV-1 replicates in epithelial and immune cells, both of which express Cd300lf, the gene encoding the intestinal receptor for all known strains of mouse norovirus. MNV-1 infection of mice lacking the Cd300lf gene led to no detectable MNV-1 replication in the salivary glands, suggesting that this receptor is needed for infection of submandibular gland cells. Partial extraction of the salivary glands from adult mice before inoculation led to faster clearing of the intestinal infection, suggesting that the salivary glands may serve as reservoirs for replication of these viruses.

The results of this study challenge the notion that noroviruses and rotaviruses transmit primarily by the fecal-oral route and raise several interesting questions. Do human noroviruses replicate in salivary glands, and do humans transmit noroviruses through saliva? If so, would protective measures in addition to handwashing (like face masks) prevent transmission of noroviruses? Are other enteric viruses (like poliovirus) also transmitted through saliva? It will be interesting to see future studies that address these questions.

[This paper was discussed in detail on TWiV 915.]