Clinical Presentation

In all age-groups, the classical clinical presentation of rotaviral infection is fever and vomiting for 2-3 days, followed by non-bloody diarrhea. The diarrhea can be profuse, with patients commonly having 10-20 bowel movements each day. Especially when associated with vomiting, the diarrhea caused by rotavirus can lead to severe and potentially life-threatening dehydration. Though there is no immune state, secondary infections with rotavirus are generally less severe.

Diagnostic Tools

Because there are so many different causes of diarrheal diseases in children, it is a major challenge to distinguish rotavirus from other diseases when diagnosing the cause of illness. A major obstacle in proper diagnosis is multiplicity of infection, a situation which can be relatively common especially in endemic areas. [1] Identification is a necessary step to pursue, however, because it will allow for proper treatment. Whereas Giardia lamblia, and other parasitic infections may call for Metronidazole, such drugs would be superfluous for rotavirus infection. [2] Identification of the cause of infection is also necessary for the study of infectious diseases and vaccine development.

Diagnosis of rotavirus can be done by identifying the virus in the patient's stool. The most popular technique is enzyme immunoassay (EIA), and there are several commercial kits available for EIA of group A rotavirus. Other techniques include electron microscopy (EM), polyacrylamide gel electrophoresis (PAGE), and reverse transcription-polymerase chain reaction (RT-PCR). [3]

One study comparing the effectiveness of these techniques found that EIA, second round PCR, and EM were the most sensitive methods for identifying group A rotavirus. EIA has the advantage of being relatively quick, while PCR allows scientists to sequence nucleotides, which can be useful in studying the molecular epidemiology of the pathogen. EM is also relatively quick, and can be used to identify non-group A viruses, however access to electron microscopes is not usually available in developing nations. First round PCR, which can be used for groups A, B, and C, and latex agglutination (LA), which can be used for group A, were the least sensitive methods and not recommended for use in detection. [4]

Another study showed that enzyme-linked immunosorbent assay (ELISA) is an effective method for detecting rotavirus-specific antibodies, especially in current or recently acquired infections. High levels of rotavirus-specific IgM and IgA were present in patients' stools one day after the onset of disease, and remained so for about ten days, even after virus shedding was no longer observed. While ELISA is a sensitive means of detection, it is not a reasonable method after day 10 post-infection when antibody levels in the stool drop. ELISA has several advantages. It can be performed in-house without previous treatment of the sample, and it costs less than RT-PCR. ELISA was found to be as effective as RT-PCR, which is generally considered the standard in virus detection. It was estimated that ELISA required 10⁶ rotavirus particles per milliliter, RT-PCR required 10⁴, and PAGE required 10¹¹. [5]

Thus, there are several methods available for the detection and diagnosis of rotavirus. RT-PCR is generally considered the gold standard in detection, but ELISA, EIA, and EM provide sufficiently sensitive alternatives.

Pathology

Rotavirus spreads between people by fecal-oral transmission. After ingestion, the rotavirus particles are carried to the small intestine where they infect the mature enterocytes in the mid and upper part of the villi of the small intestine, leading to diarrhea. Rotavirus is thought to invade target cells in two possible ways, by direct entry or fusion with the enterocytes, and through Ca²⁺-dependent endocytosis. [6]

Rotavirus infection leads to structural changes in the intestinal epithelium. Within 24 hours of infection, the shape of the villus epithelium changes from columnar to cuboidal, and the villi become stunted and shortened. Changes are most severe in the upper portions of the small intestine, and there is little or no inflammation. The severity of these changes is correlated with the severity of the resulting illness. [6]

There are a multitude of possible mechanisms by which rotavirus might cause diarrhea. The following table, from Anderson and Weber [7], describes the possibilities.

Immune Response

Rotavirus infection of the intestinal enterocytes is thought to be controlled primarily by antibodies. In mice, the appearance of anti-rotaviral IgA in the intestine at 7 days post-infection correlates with the clearance of a primary rotaviral infection. [8] However, mice lacking in IgA still mount a successful immune response to the pathogen, thought to be mediated by IgG. [9]

CD4⁺ helper T (T_H) cells also play a vital role in the successful clearance of a rotaviral infection. [10] J.L. VanCott, et al. demonstrated that CD4⁺ T cells are vital to the induction of proper B cell response to rotavirus. Mice lacking CD4⁺ T cells chronically shed virus in their stool when infected with rotavirus, and produce only 5% of the amount of viral-specific IgA found in normal mice. In contrast, mice lacking CD8⁺ T cells responded normally to infection.

It has been shown by Franco and Greenberg at Stanford that a CD8⁺ cytotoxic T-lymphocyte (CTL) response is not necessary for clearance of a rotaviral infection. [11] In that experiment, they used mice knocked out for β₂ microglobulin (β₂m) to test the dependence of rotaviral clearance on CTLs. They found that though the β₂m knockout mice shed virus for an additional two days as compared to controls, they still completely resolved a primary infection. When the β₂m knockouts were then completely depleted of their CTL population using anti-CD8 antibodies, the mice shed virus for an additional day. Both the treated and untreated β₂m knockouts were compltely protected upon rechallenge. These findings demonstrated that CTLs likely play a role in rotavirus clearance, but that they are not required. They also demonstrated that CTLs are not neccessary for the development of protective immunity.

Though CTLs are not necessary, they still play an important role in the development of protective immunity. It was originally observed that severe combined immunodeficient (SCID) mice, which lack both functional B and T cells, develop a chronic rotavirus infection when innoculated with the virus. It was then demonstrated that these mice would clear the infection when CD8⁺ T cells were transferred from previously vaccinated mice. [12] This clearance occured in the absence of rotavirus-specific immunoglobulin. Additionally, has been observed that J_HD mice, which lack B cells, are still able to clear rotavirus infections. These mice develop chronic infections if depleting doses of anti-CD8 antibodies are administered. [11] Upon rechallenge with rotavirus, these mice still develop infections, but they shed virus at slightly lower levels, and for fewer days, than the naive mice, indicating a role for CD8⁺ T cells in the develop of a rotavirus-specific memory response.

In summary, it appears that both B and T cells play important roles in the immune response to rotavirus. B cells are involved in the T_H cell-dependent secretion of rotavirus-specific IgA and IgG, while CTLs play a role in the clearance of the virus. The immune system appears to be fully capable of both clearing a rotavirus infection in the absence of either one of these arms of the immune response. However, B cell-dervived Ig seems to play a more important role in the rotavirus-specific memory response, as demonstrated by the fact that CTL-depleted mice were resistant to reinfection, whereas mice lacking B cells were still succeptible, albeit with a less serious course of infection.

Correlates of Immunity

A natural immune state to rotavirus does not exist. Though primary infection by the virus induces production of rotavirus-specific memory B an T cells, these are not normally sufficient to prevent reinfection by the virus. However, they do serve to reduce the severity of secondary infections. It has shown that serum IgA antibody titers correlate with protection against reinfection. [13] It has been shown in mice that in the absense of IgA, IgG is also sufficient to protect mice. [14] However, in humans, high titers of IgG do not seem to be as protective as IgA against moderate to severe illness, so serum IgA is seen as the primary indicator of protective immunity to rotavirus. [13] One reason these antibody responses do not confer full proection is that they are serotype specific. Given the diversity of the various rotavirus serotypes, this prevents these antibodies from mediating full protection against infection by a different serotype. This is also why repeat infections are less severe, as each additional infection expands the population of B cells producing cross-reactive antibodies that can recognize multiple serotypes. Any vaccine effort would need to generate these cross-reactive antibodies to generate effective protection. It has been shown that high amounts of cross reactive secretory IgA, and serotype specific serum IgA and IgG seem to confer the most protection. [16]

As discussed above, CTLs also appear to play a role in the clearance of rotavirus infections. In the same B cell-deficient J_HD mice discussed above, it was later shown that the mice are completely protected against reinfection for 18 days after clearing the primary infection, and retain partial immunity 8 months after the primary infection. [15] Thus the correleates of immunity to rotavirus include both the presence of anti-rotaviral IgA, which requires a rotavirus-specific T_H cell response, as well as a rotavirus-specific CTL response.