Infectivity of GII.4 human norovirus does not differ between T-B-NK+ severe combined immunodeficiency (SCID) and non-SCID gnotobiotic pigs, implicating the role of NK cells in mediation of human norovirus infection.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. It is unclear which arm of the immune system regulates resistance to HuNoV infection. Thus, we studied the pathogenesis of human norovirus (HuNoV) in T-B-NK+ Severe Combined Immunodeficiency (SCID) gnotobiotic pigs to investigate the role of innate (especially, natural killer (NK) cells) immunity in HuNoV infection. Forty SCID and non-SCID pigs were randomly grouped: 1) SCID+HuNoV (n = 12); 2) non-SCID+HuNoV (n = 14); 3) SCID mock-inoculated (n = 6); and 4) non-SCID mock-inoculated (n = 8). Pigs (8-14-day-old) were inoculated orally with GII.4 HuNoV strain HS292 (mean 9.1 log10 genomic equivalents/pig) or mock. Daily fecal consistency and fecal viral RNA shedding, and histopathology (at euthanasia) were evaluated. Frequencies of blood and ileal T, B, and NK cells were analyzed by flow cytometry, and a NK cell cytotoxicity assay was performed at post-inoculation day (PID) 8. Unlike the increased infectivity of HuNoV observed previously in T-B-NK- SCID pigs (Lei et al., 2016. Sci. Rep. 6, 25,222), there was no significant difference in frequency of pigs with diarrhea and diarrhea days between T-B-NK+ SCID+HuNoV and non-SCID+HuNoV groups. Cumulative fecal HuNoV RNA shedding at PIDs 1-8, PIDs 9-27, and PIDs 1-27 also did not differ statistically. These observations coincided with the presence of NK cells and NK cell cytotoxicity in the ileum and blood of the SCID pigs. Based on our observations, innate immunity, including NK cell activity, may be critical to mediate or reduce HuNoV infection in T-B-NK+ SCID pigs, and potentially in immunocompetent patients.

Cross protective immune responses in nursing piglets infected with a US spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original US PEDV strain.

We investigated cross-protective immunity of a US spike-insertion deletion porcine epidemic diarrhea virus (PEDV) Iowa106 (S-INDEL) strain against the original US PEDV (PC21A) strain in nursing piglets. Piglets were inoculated orally with S-INDEL, PC21A or mock. At 20-29 days post-inoculation (dpi), all pigs were challenged with the PC21A strain. The S-INDEL-inoculated pigs had lower ileal IgA antibody secreting cells, serum IgA and neutralizing antibody titers compared with PC21A-inoculated pigs. No pigs in the PC21A-group developed diarrhea, whereas 81 and 100% of pigs in the S-INDEL and mock-groups had diarrhea post challenge, respectively. S-INDEL induced partial protective immunity against the original US PEDV strain.

Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain.

UNLABELLED: The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses. IMPORTANCE: The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection.

Although the original US porcine epidemic diarrhea virus (PEDV) was confirmed as highly virulent by multiple studies, the virulence of spike-insertion deletion (S-INDEL) PEDV strains is undefined. In this study, 3-4 day-old conventional suckling piglets were inoculated with S-INDEL PEDV Iowa106 (4 pig litters) to study its virulence. Two litters of age-matched piglets were inoculated with either the original US PEDV PC21A or mock as positive and negative controls, respectively. Subsequently, all pigs were challenged with the original US PEDV PC21A on 21-29 days post-inoculation (dpi) to assess cross-protection. All S-INDEL Iowa106- and the original US PC21A-inoculated piglets developed diarrhea. However, the severity of clinical signs, mortality (0-75%) and fecal PEDV RNA shedding titers varied among the four S-INDEL Iowa106-inoculated litters. Compared with the original PC21A, piglets euthanized/died acutely from S-INDEL Iowa106 infection had relatively milder villous atrophy, lower antigen scores and more limited intestinal infection. Two of four S-INDEL Iowa106-infected sows and the original PC21A-infected sow showed anorexia and watery diarrhea for 1-4 days. After the original PC21A challenge, a subset (13/16) of S-INDEL Iowa106-inoculated piglets developed diarrhea, whereas all (5/5) and no (0/4) pigs in the mock and original PC21A-inoculated pigs had diarrhea, respectively. Our results suggest that the virulence of S-INDEL PEDV Iowa106 was less than the original US PEDV PC21A in suckling pigs, with 100% morbidity and 18% (6/33) overall (0-75%) mortality in suckling pigs depending on factors such as the sow's health and lactation and the piglets' birth weight. Prior infection by S-INDEL Iowa106 provided partial cross-protection to piglets against the original PC21A challenge at 21-29 dpi.

Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs.

Porcine epidemic diarrhea (PED) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. Consequently, it causes significant economic losses to the pork industry. There are limited studies on the innate immune responses to PED virus (PEDV) in pigs. The aims of our study were to investigate differences in innate immune responses to PEDV infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. Weaned 26-day-old pigs (n=20) and 9-day-old nursing pigs (n=20) were assigned to PEDV inoculated or uninoculated control groups. The pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (PIDs) 1 and 5 to assay immune responses. Blood samples were collected at PIDs 1, 3 and 5. The natural killer (NK) cell frequencies, NK cell activities (lysis of target K562 tumor cells in vitro), CD3+CD4+ T cell and CD3+CD8+ T cell frequencies were measured in blood and ileum at PIDs 1 and 5. The PEDV infected suckling pigs showed severe diarrhea and vomiting at PID 1, whereas the PEDV infected weaned pigs showed milder clinical signs starting at PID 3. PEDV infected suckling pigs had significantly higher diarrhea scores, earlier fecal PEDV RNA shedding and significantly higher viremia (viral RNA in serum) compared to weaned pigs. There was no mortality in either infected suckling or infected weaned pigs. The control pigs not inoculated with PEDV did not show any clinical signs and no detectable fecal or serum PEDV RNA. Strikingly, PEDV infected suckling pigs had significantly lower NK cell frequencies, undetectable NK cell activity and lower IFNγ producing NK cells in blood and ileum compared to PEDV infected weaned pigs. Pro-inflammatory cytokine profiles of PEDV infected suckling pigs differed from those of PEDV infected weaned pigs and coincided with onset of fecal PEDV RNA shedding and serum PEDV RNA titers. The infected suckling pigs have higher and earlier increases in serum IFNα, but lower serum IL-8 and TNFα levels compared to infected weaned pigs. CD3+CD4+ T cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in CD3+CD8+ T cell frequencies. In conclusion, the observations of impaired lytic activity and IFN-γ production by NK cells in suckling pigs coincided with the increased severity of PEDV infection in the suckling pigs compared with the weaned pigs.

Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.

Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs.

Our study demonstrated potential mechanisms by which porcine epidemic diarrhea virus (PEDV) infection induces greater disease severity of nursing vs. weaned conventional pigs. Twenty-six-day-old weaned [PEDV-inoculated (n=11); mock (n=9)] and 9-day-old nursing pigs [PEDV-inoculated (n=9); mock (n=11)] were inoculated orally [8.9 log10 genomic equivalents (GE)/pig] with PC21A strain or mock (MEM). Pigs were monitored for clinical signs and PEDV RNA titers in feces and serum. For pathology and immunofluorescence staining for Ki67 (marker for crypt proliferation) and LGR5 (marker for crypt stem cell), 3-4 pigs were euthanized at postinoculation days (PIDs) 1, 3 and 5. Severe watery diarrhea and atrophic enteritis with moderate to high PEDV RNA titers in feces (7.5-12.2 log10 GE/ml) and low viral RNA titers in serum (5.6-8.6 log10 GE/ml) were observed in all inoculated nursing piglets at PIDs 1-5. In contrast, weaned pigs did not show evidence of PEDV infection at PID 1. Pigs exhibited high fecal shedding titers at PIDs 2-5 and mild to severe atrophic enteritis at PIDs 3-5, indicating a longer incubation for PEDV infection. While uninoculated or inoculated 27-31-day-old pigs showed large numbers of Ki67- or LGR5-positive cells in the intestinal crypts, there was a lack of LGR5-positive cells and low proliferation of crypts in jejunum of uninoculated 10-14-day-old piglets, possibly causing a slower turnover of enterocytes; however, the number of LGR5-positive cells and proliferation of intestinal crypts increased remarkably at 3-5 days after inoculation. Biologic mediators that promote crypt stem cell regeneration would be targets to improve the intestinal epithelium renewal during PEDV infection.

Structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus.

Integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. It is maintained mainly by tight junctions (TJs) and adherens junctions (AJs). We conducted immunofluorescence (IF) staining for in situ identification of zonula occludin (ZO)-1 proteins for TJ and E-Cadherin proteins for AJ in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (PEDV). Twenty 9-day-old piglets [PEDV-infected (n=9) and Mock (n=11)] from PEDV seronegative sows, were orally inoculated [8.9 log10 genomic equivalents/pig] with PEDV PC21A strain or mock. At post-inoculation days (PIDs) 1-5, infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. By immunohistochemistry, PEDV antigens were evident in enterocytes lining the villous epithelium. At PIDs 1-5, PEDV-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of ZO-1 or E-Cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. The structural destruction and disorganization of TJ and AJ were extensive in PEDV-infected pigs at PIDs 1-3, but then appeared to reversibly recover at PID 5, as evident by increased numbers of ZO-1-positive epithelial cells and markedly improved appearance of E-Cadherin-positive villous epithelium. Our results suggest a possible involvement of structurally impaired TJ and AJ in the pathogenesis of PEDV, potentially leading to secondary bacterial infections.

Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens.

The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue.