Total RNA was isolated from approximately 5610 5 B cells with Trizol (Invitrogen). cDNA was generated and subjected to PCR to produce heavy chain fragments using 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems Inc.). PCR products were run on agarose gels, purified and cloned into the pCR2.1 TA cloning vector according to manufacturers' recommendations (Invitrogen). Sequence analysis was performed using BigDye Terminator chemistry (Applied Biosystems Inc.) and Vector-NTI software (Invitrogen). Consensus sequences were determined with Codon code (CodonCode Corporation). Figure S1 IL-21 induced Ig secretion in STAT5bER immortalized memory B cell cultures. A) STAT5bER transduced polyclonal memory B cells were cultured in the absence of tamoxifen (4HT) and with IL-2 and IL-4 or IL-21. IgG secretion was determined at day 7. Data from two representative donors out of 5 donors is shown. B) STAT5bER transduced monoclonal memory B cell lines were cultured in the absence of tamoxifen (4HT) and with IL-2 and IL-4 or IL-21. IgG secretion was determined on day 7. Data from two representative cell cultures is shown. All B cells in these cultures had lost surface immunoglobulin expression and were in culture for at least 2 months. (TIF) Figure S2 IgG and IgM secretion in time after removal of tamoxifen (4HT). IgG and IgM secretion was determined in cells cultured with CD40L and IL-21 for 7 days and for 14 days. Days 0 indicates the day when the culture conditions were changed from CD40L, IL-2, IL-4 plus tamoxifen (4HT) to CD40L and IL-21. The black and grey bars represents two different donors. (TIF) Figure S3 IgG and IgM expression in long term cultures. Multiple TT specific clones which were in culture for more then 5 months and subsequent frozen. They were then thawed and taken in culture again with CD40L, IL-2, IL-4 and tamoxifen (4HT). When a stable culture was obtained, the cells were cultured with CD40L and IL-21. An ELISA was performed to determine IgG and IgM concentrations. (TIF) Figure S4 rTT.C staining on freshly isolated memory B cells. Phycoerythrin (PE) labeled rTT.C was added to freshly isolated B cells. Cells were subsequently sorted using flow cytometry (FACSAria). Shown are the results of 5 different donors. (TIF) Figure S5 IgA and IgG expression on polyclonal CA-STAT5b transduced and non-transduced B cells. A polyclonal mixture of total CD27+ selected human memory B cells from two donors were transduced with caSTAT5b-IRES-NGFR and cultured for a maximum of two weeks before they were frozen. After thawing cells were stained immediately for the IgA and IgG isotype. Cells expressing NGFR indicates they were transduced with CA-STAT5b. (TIF) Genomic Characterization and High Prevalence of Bocaviruses in Swine Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ∼2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45–75% and 55–70% respectively, with 30–47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses. Members of the Parvoviridae family are small non-lipid enveloped viruses with a diameter of 18-26 nm, icosahedral symmetry (T = 1), encoded by a single-stranded linear DNA genome of approximately 4,000 to 6,000 nucleotides (nt) [1] . The family includes two subfamilies: Densovirinae, Parvovirinae. The subfamily of Densovirinae contains four genera: Densovirus, Iteravirus, Brevidensovirus and Pefudensovirus, which infect only invertebrates [1] . The Parvovirinae subfamily is currently subdivided into five genera: Parvovirus, Erythrovirus, Dependovirus (adeno-associated virus), Amdovirus, and Bocavirus, infecting vertebrates [1] .