Rotaviruses (RVs), a major cause of diarrhea in young animals and humans, are classified in eight groups (A-H) based on classifications of the inner viral capsid protein 6. Rotaviruses share similar features, such as appearance (a wheel-like structure as seen by electron microscopy), intestinal lesions that occur due to rotavirus infection and the arrangement of the viral genes. Reassortment commonly occurs within a group of rotaviruses, increasing the genetic diversity within each group. Fortunately, reassortment does not occur between groups. For example, group A rotaviruses (RVA) do not reassort with group C rotaviruses (RVC).
Five different groups of rotavirus infect swine: RVA, RVB, RVC, RVE and RVH. Historically, RVA was considered the most important rotavirus group because of the high prevalence and pathogenicity. Porcine RVB and RVC were first identified in 1980s. However, due to difficulties in isolating RVB and RVC in cell cultures and the lack of other diagnostic tools, the prevalence rates of RVB and RVC were unknown until recently. There are only single reports of RVE and RVH strains infecting pigs – one in the United Kingdom, another in Japan.
Traditionally, electron microscopy or RVA fecal enzyme-linked immunosorbent assay (ELISA) was used to diagnose rotavirus infection. Histopathological (tissue) examinations are very useful in rotavirus diagnosis. Diagnosis of new groups of rotavirus occurred when rotavirus-like lesions (villus atrophy, fusion and necrosis) as shown in Figures 1 and 2, were detected under light microscopy. Interestingly, rotavirus detection by electron microscopy or RVA fecal ELISA was negative, thus suggesting a problem with the current rotavirus diagnostic assays. Electron microscopy has a very low sensitivity, and the RVA fecal ELISA only detects the RVA virus. Therefore, both are unable to successfully detect RVB and RVC infections.
Initially, the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) developed a multiplex real-time polymerase chain reaction (RT-PCR) to detect RVA and RVC, which led to a better understanding of RVA and RVC in the swine population. Using histopathology and RT-PCR, the UMVDL was able to diagnose RVA in 60% and RVC in 52% of the diarrhea cases submitted during 2009-2011.
Samples negative for RVA and RVC by RT-PCR, but with tissue rotavirus-like lesions, revealed that another rotavirus species was causing enteritis in swine. This led to the development of a RVB RT-PCR to detect swine RVB. To investigate the ecology of RVA, RVB and RVC in swine diarrhea cases, pigs from three age groups (less than 21 days, 21-55 days and greater than 55 days) with swine diarrhea were tested. In addition to these detection assays, the RVB VP7 (an outer glycosylation protein utilized during cell attachment) was sequenced to further understand the genetic diversity of RVBs in the swine population.
Rotavirus Prevalence
During a one-month testing period, the most prevalent rotavirus group detected was RVA (63.58%), followed by RVC (57.8%). The prevalence of RVB was 46.8%, surprisingly higher than any other previously published articles. The highest number of RVB-positive samples was found in the 21-55 day age group (Figure 3). The majority of the RVB positive samples (86.4%) were part of a mixed rotavirus infection. Co-infections of RVA/RVB/RVC were detected at a high rate of 24.3% in the greater than 55-day-old age group. Single infections of RVB and RVC were not detected in this age group, meaning that every infection found in this age group also contained RVA. Further research on RVA infections in pigs greater than 55 days of age is still needed to fully understand these infections.
By sequencing RVB-positive samples, it was discovered that the genetic diversity of RVB was larger than initially thought. Currently, there are 16 distinct strains (genotypes) in the swine population; only nine genotypes have been found in the United States.
From our study, we detected four pig farms with more than one RVB strain, indicating that not only can different rotavirus groups be found in a single farm, but also different strains of a single RVB group can be found within a farm.
New Strain Significance
The initial prevalence and significance of RVB may have been overlooked due to the pathogenicity and high prevalence of RVA. However, previous diagnostic tools lacked the sensitivity and specificity to detect RVB in swine.
The newly developed RVA-RVB-RVC RT-PCR tests solved this problem and helped identify new strains, leading to a better understanding of rotavirus genetic diversity, especially the diversity of RVBs. Given the large genetic diversity of RVB in the swine population, RVB strains probably have been circulating for many decades in pigs. Like RVA and RVC, RVB is an important cause of enteritis in pigs. Identifying RVB as a causative agent of diarrhea will reduce the inappropriate use of antibiotics for viral enteritis.