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Transmission of Enteric Viruses through Saliva

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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.   

TCID50 analysis 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.]

TWiV 915: Mouse mouth to mouse mom

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TWiV discusses the recent decision by an FDA advisory committee to update COVID vaccines for the fall, the monkeypox virus outbreak, and the finding that enteric viruses infect the salivary glands and are transmitted through saliva.

Hosts: Vincent Racaniello, Dickson Despommier, Rich Condit, Kathy Spindler, and Brianne Barker.

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Show notes at microbe.tv/twiv

How to End this Pandemic

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by Gertrud U. Rey

As of today, SARS-CoV-2 has infected 18.7 million people and caused 700,000 deaths worldwide. The most realistic way to quickly curb the spread of the virus would require daily identification and isolation of individuals who are contagious, a process that is hampered by cumbersome sampling and testing methods with slow turnaround times. 

The predominant test for diagnosing SARS-CoV-2 infection is a highly sensitive assay called quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). To carry out a SARS-CoV-2 qRT-PCR test, a mucus sample is processed to inactivate virus particles and extract the viral RNA. The RNA is converted to DNA (the reverse transcription step), which is then amplified during the polymerase chain reaction portion of the assay. For amplification to occur, a small piece of DNA (a primer) binds to a complementary target sequence in the SARS-CoV-2 DNA, while another piece of DNA (a probe) attaches to a sequence downstream of the primer binding site. Binding of the primer initiates amplification of the target DNA by an enzyme called polymerase, which copies the DNA in one direction towards the probe. Once the polymerase reaches the probe, it cleaves it, which activates a fluorescent marker attached to the probe. The use of this fluorescent probe allows for monitoring of the fluorescent signal quantitatively in real time rather than just detecting an accumulated end product. 

While the qRT-PCR test is very sensitive, it also has multiple limitations. It requires expensive laboratory instrumentation and trained technicians with an estimated cost of about $100 per test, meaning that most people probably only get tested once. Current testing capacities are limited and results often take days or weeks to return, meaning that individuals who don’t know they are infected can transmit the virus during this time. The high sensitivity of qRT-PCR may also be a drawback rather than an advantage, because the test often detects small fragments of RNA that don’t originate from whole virus particles and thus don’t represent transmissible virus. Such RNA fragments can persist in individuals for weeks and months. As illustrated in Figure 1, infection with SARS-CoV-2 usually results in high initial levels of viral replication that peak and begin to decline within a few days. Symptoms don’t usually appear until after that peak has already occurred, and, because most people don’t get tested until they experience symptoms, they are likely already on the downward slope of viral replication and no longer infectious at the time of testing. In the meantime, they have been unknowingly transmitting the virus to others for several days. Clearly, these people need to be identified and isolated during their period of high infectivity.

In late June, Harvard epidemiologist Michael Mina published a preprint that evaluates the effectiveness of current SARS-CoV-2 surveillance measures for reducing transmission when considering frequency of testing and delayed reporting of results. Mina and co-authors concluded that infrequent testing with an ultra-sensitive test like qRT-PCR often results in unnecessary quarantine of individuals who are no longer infectious. Notably, it also results in missing pre- or asymptomatic individuals who are at the beginning of their infection and thus highly contagious, allowing them to go about their daily routines and infect others. 

A few days after publication of the preprint, Mina co-authored an opinion article in the New York Times in which he discussed the potential for controlling the SARS-CoV-2 pandemic by widespread use of frequent, rapid at-home diagnostic tests. One example of such a diagnostic test is a lateral flow device, which is a paper strip that works similarly to a pregnancy test. The strip has a sample pad on one end and contains antibodies that recognize SARS-CoV-2 antigens. One would dip the sample pad portion of the strip into a sample of saliva and allow the saliva to wick across the strip. The presence of SARS-CoV-2 antigens in the saliva would be indicated by the appearance of a test line in addition to the control line, while a negative test would only indicate the control line (Figure 2). The test provides results in 10-15 minutes at a cost of about $1-2 per test and does not require any additional equipment. A positive result would indicate the need for self-quarantine and confirmation of test results through a doctor’s office. 

Although these rapid tests are only about half as sensitive as qRT-PCR tests, they detect the presence of viral antigen during the actual window of transmissibility when viral levels are very high. The highly sensitive qRT-PCR assays detect viral RNA for weeks after a patient is no longer transmitting virus, which is irrelevant for quarantine/isolation purposes and does nothing to curb transmission. A less sensitive test that is done on a daily basis and provides immediate results would be more valuable because it would identify individuals while they are actually infectious. This would also alleviate the need for costly contact tracing measures because most infected individuals would be aware of their status and would stay isolated during their period of transmission. 

Rapid lateral flow SARS-CoV-2 diagnostic tests are already available, but there is concern that the FDA may not approve these products because of their low sensitivity. You can help bring these products to market by writing to your elected officials (see sample letter templates here), contacting your local TV and radio stations, and telling your friends and family to do the same. Hopefully, with sufficient media attention, the FDA, CDC, and NIH will recognize the value of these tests and make them widely available to the public. This may be the ultimate solution for opening schools and workplaces, and for rebuilding the economy. 

[Michael Mina discussed rapid at-home SARS-CoV-2 testing options on TWiV 640. Tidbits of that episode were also reviewed on MedCram.]

Spit for SARS-CoV-2

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spitMight spitting in a tube be the solution to finding out who is infected with SARS-CoV-2? The results of a recent study suggest that might be the case.

To diagnose someone who is infected with SARS-CoV-2, a long swab is inserted deep into the nasal sinus to provide samples of the nasopharyngeal mucosa, a site of virus reproduction. The material collected is then subjected to RT-PCR to determine the number of copies of viral RNA. This procedure is not ideal: it is difficult to do properly, and may cause extreme discomfort to the patient. The sampling could induce coughing or sneezing, placing the healthcare worker at risk. Comparing serial samples is unreliable because different quantities of mucus can be sampled each time.

Saliva sampling has great appeal because it is not only non-invasive, but can provide consistent sample sizes and can be self administered (who hasn’t spit into a tube?). However we do not know whether assaying SARS-CoV-2 RNA in saliva is better than a nasopharyngeal swab.

To answer this question, the detection of SARS-CoV-2 RNA by nasopharyngeal swabs administered by healthcare workers was compared with self-collected saliva from 44 COVID-19 patients. Higher levels of viral RNA were found in saliva samples compared with nasopharyngeal swabs.

In another study of 22 participants administered multiple nasopharyngeal swabs or who provided multiple saliva samples, there were 5 cases in which a negative nasopharyngeal swab was followed by a positive result during the next collection. In contrast, there were no cases where saliva samples were first negative and then positive.

Finally, saliva was positive for SARS-CoV-2 in two healthcare workers who were asymptomatic and had tested negative by nasopharyngeal swab.

While these observations should be confirmed in a larger study, they suggest that saliva sampling for SARS-CoV-2 could be a sensitive, reproducible self-administered test for identifying both active and subclinical infections.

If the results of larger studies show that the saliva assay is a sensitive and reliable assay for detecting active infection with SARS-CoV-2, it could simplify testing in countries that lack nasopharyngeal swabs, personal protective equipment, and trained health care personnel.

If I were the COVID-19 Czar, I would have the saliva test sent to every individual in the US at the cost of the US government. Everyone would receive a kit consisting of a bar-coded tube into which they would spit, then record the barcode via cell phone to CDC. The tube of saliva would be mailed or dropped off at local collection boxes. The results of the RT-PCR assay would indicate who is currently infected in the US. This information will help to understand what fraction of the population might have immunity to infection.

TWiV 521: Spitting in the Allee

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Team TWiV cover the discovery of another giant virus from 30,000 year old Siberian permafrost, and how viral aggregation accelerates the production of new infectious viruses and increases fitness, demonstrating an Allee effect.

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Show notes at microbe.tv/twiv

Mosquito saliva enhances virus replication and disease

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Biting mosquitoMosquito saliva, which is injected into the host as a mosquito probes for a blood vessel, contains a collection of chemicals which include anticoagulants to prevent blood clotting, vasodilators to keep blood vessels wide, and anesthetics to prevent us from sensing the mosquito. Saliva also contains components that enhance viral replication, dissemination, and pathogenesis by inducing an inflammatory response that inadvertently promotes infection by providing new cell targets for infection (paper link).

To separate the bite from virus inoculation, mice were first exposed to Aedes aegyptii mosquitoes, and then infected at the bite site with two different mosquito transmitted viruses, Semliki Forest virus or Bunyamwera virus. Mosquito bites caused more virus replication at the inoculation site, greater dissemination of virus, and more lethality compared with control mice that received only virus.

How does mosquito saliva enhance virus replication and dissemination? Part of the story is that as the mosquito probes for a blood vessel, it causes damage that leads to vascular leakage and accumulation of fluid (edema) which inhibits movement of virus to draining lymph nodes.

But delaying dissemination of virus alone does not promote infection and disease. Mosquito bites cause an infiltration of neutrophils (a type of white blood cell) into the bite site. The edema at the bite site is enhanced by neutrophils, because depleting these cells from mice greatly reduced edema. This depletion also returned viremia to levels observed in unbitten control mice, and restored dissemination of virus to draining lymph nodes. Neutrophils are not susceptible to infection with Semliki Forest virus, and therefore cannot explain the increase in virus replication at the bite site.

Enhanced virus replication in the skin occurs because the neutrophils elaborate chemokines that attract macrophages, which can be infected by Semliki Forest virus and Bunyamwera virus. One of the chemokines produced by neutrophils that is a macrophage attractant – CCL2 – binds a receptor on macrophages. Mice lacking the gene encoding the CCL2 receptor are protected from bite enhancement of Semliki Forest virus enhancement.

When a mosquito bites a host, it delivers saliva along with a virus. The saliva induces an inflammatory response and attracts neutrophils into the bite site. The resulting edema holds virus at the bite site until chemokines produced by neutrophils attract macrophages, which are then infected. The virus produced disseminates widely, reaching secondary tissues and causing disease.

It seems likely that the ability to replicate in macrophages that are recruited to the bite site is a property that was selected during evolution of mosquito-transmitted viruses. By replicating in macrophages, the amount of virus in the blood is increased, as well as the likelihood that the virus will be picked up by another mosquito and transmitted to a new host – a powerful selection mechanism. The down side – increased disease in the mammalian host – is an accidental side effect.

Think about that the next time you are scratching that raised bump on your skin caused by a mosquito bite.