Viruses and bacteria are both present at mucosal surfaces and it is not surprising that they interact in many ways. Sometimes members of the gut microbiome help viruses to infect cells, exemplified by poliovirus, reovirus, and mouse mammary tumor virus. The opposite also happens: segmented filamentous bacteria can prevent and cure rotavirus infection.
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The US Food and Drug Administration has updated its recommendations on both Rotarix and RotaTeq, vaccines for the prevention of rotavirus disease in infants:
Based on careful evaluation of a variety of scientific information, FDA has determined it is appropriate for clinicians and health care professionals to resume the use of Rotarix and to continue the use of RotaTeq.
In making its recommendation, the FDA considered the strong safety records of both vaccines, including clinical trials in tens of thousands of individuals and the use of the vaccines in millions of recipients. There is no evidence that either porcine circovirus type 1 or type 2 poses a safety risk to humans, and neither virus is known to infect humans or cause disease.
The FDA also noted that the benefits of the rotavirus vaccines are considerable and outweigh the small theoretical risk of the viral contaminant.
The product labels will be updated to reflect the fact that the vaccine contains a PCV contaminant. In addition, GlaxoSmithKline will rederive Rotarix so that it does not contain PCV. Merck has not yet made a decision about whether they should produce a PCV-free rotavirus vaccine. But if my suggestion carries any weight at Merck (I know it does not), they should not hesitate to follow GlaxoSmithKline’s lead.
The US Food and Drug Administration has recommended that administration of the Rotarix vaccine, which protects against rotavirus infection, be suspended. This action comes after an independent research group found that the vaccine contains DNA of porcine circovirus type 1.
Rotaviruses are the single leading cause of diarrhea in infants and young children. Each year rotavirus gastroenteritis causes over 1,250,000 episodes of diarrhea and 527,000 deaths, mainly in developing countries. Rotavirus vaccines are used to reduce the global burden of rotavirus disease. Rotarix, the vaccine manufactured by GlaxoSmithKline, is an infectious, attenuated vaccine that is administered orally to infants.
The genetic information of rotaviruses consists of 11 segments of double-stranded RNA. In contrast, porcine circoviruses are small viruses with a circular, single stranded DNA genome (pictured). At 1.7 kb in length, the DNA is among the smallest known viral genomes and encodes only two proteins. Porcine circovirus 1 was originally discovered as a contaminant of a pig kidney cell line. Later a second strain, porcine circovirus 2, was isolated and shown to be associated with postweaning multisystemic wasting disease, an emerging disease of swine.
Porcine circovirus 1 DNA was also found in the cells used to produce Rotarix. Therefore the contaminant has been present since the early days of vaccine development, including clinical trials. A different rotavirus vaccine produced by Merck, called RotaTeq, does not contain porcine circovirus DNA.
It’s not at all clear that the presence of porcine circovirus 1 DNA in Rotarix is a problem. Circoviruses have not been associated with human disease, and porcine circovirus 1 has not been found to cause disease in any animal. Many humans have antibodies to these viruses, including porcine circovirus, indicating that they were infected at one time. Furthermore, it’s not known if the vaccine contains infectious virus or DNA fragments. Nevertheless, it’s always preferable to err on the side of caution, as the FDA has done.
Because porcine circoviruses are widespread in commercial swine populations, there have been concerns about the use of porcine organs for xenotransplantation and for the production of products used in humans such as factor VIII, heparin, insulin and pepsin. It is therefore important to ensure that such products do not contain infectious circoviruses.
This incident will undoubtedly further increase public distrust of vaccines and vaccine manufacturers. I have not yet seen the journal article describing these findings, so I can’t comment on why the contaminant was not identified early in vaccine development. According to the FDA, “In four to six weeks, FDA will convene an expert advisory committee and make additional recommendations on the use of rotavirus vaccines.” If Rotarix is found to contain infectious porcine circovirus, then its use will certainly be discontinued. However, detection of small noninfectious fragments of porcine circovirus DNA Rotarix will likely lead to resumption of vaccine use. In either case, new lots of vaccine should be produced using circovirus-free cells.
Tischer I, Bode L, Apodaca J, Timm H, Peters D, Rasch R, Pociuli S, & Gerike E (1995). Presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle. Archives of virology, 140 (8), 1427-39 PMID: 7544971
Fenaux M, Opriessnig T, Halbur PG, Xu Y, Potts B, & Meng XJ (2004). Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product. The Journal of general virology, 85 (Pt 11), 3377-82 PMID: 15483254
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 explores the impact of the intestinal virome on seroconversion after rotavirus vaccination, and implications of the ability of the SARS-CoV-2 beta variant to infect wild-type laboratory mice.
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From Georgia State University, Vincent speaks with Chris, Andrew, Priya, and Richard about their careers and their work on Ebolaviruses, rotavirus, and antiviral drug development.
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Show notes at microbe.tv/twiv