Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotypespecific antibody-secreting cells (ASC) were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirusinoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease. . † Approved as Ohio Agricultural Research and Development Center manuscript 179-95. 3076 YUAN ET AL. J. VIROL. 3080 YUAN ET AL. J. VIROL.

Summary. Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets ov~r laboratory animal models is their prolonged susceptibility to human rotavirus-induced disease, permitting cross-protection studies and an analysis of active immunity. Major advances in rotavirus research resulting from gnotobiotic piglet studies include: 1) the adaptation of the first human rotavirus to cell culture after passage and amplification in piglets; 2) delineation of the independent roles of the two rotavirus outer capsid proteins (VP4 and VP7) in induction of neutralizing antibodies and cross-protection; and 3) recognition of a potential role for a nonstructural protein (NSP4) in addition to VP4 and VP7, in rotavirus virulence. Current studies of the pathogenesis of group A human rota virus infections in gnotobiotic piglets in our laboratory have confirmed that villous atrophy is induced in piglets given virulent but not cell culture attenuated human rotavirus (Gl, PIA, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive ~xamination of these factors, including a proposed role for NSP4 in viral-induced cytopathology, may reveal new mechanisms for induction of viral diarrhea. Finally, to facilitate and improve rotavirus vaccination strategies, our current emphasis is on the identification of correlates of protective active immunity in the piglet model of human rotavirus-induced diarrhea.Comparison of cell-mediated and antibody immune responses induced by infection with a virulent human rotavirus (to mimic host response to natural infection) with those induced by a live attenuated human rota virus (to mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Results of these studies have indicated that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the local site of viral replication (small intestine). Secondly, there was a direct correlation between the degree of protection induced and the level of the intestinal immune response, with significantly higher local immune responses and complete protection induced only after primary exposure to 154 L. J. Saif et al. virulent human rotavirus. These studies thus have established basic parameters related to immune protection in the piglet model of human rotavirus-induced disease, verifying the usefulness of this model to examine new strategies for the design and improvement of human rota virus vaccines.

We characterized isolates from porcine respiratory and reproductive syndrome virus epidemics in Vietnam and China in 2007. These isolates showed ≈99% identity at the genomic level. Genetic analysis indicated that they share a discontinuous deletion of 30 aa in nonstructural protein 2, which indicates that identical variants emerged in Vietnam and China.

To describe the spontaneous clearance rate of childhood hepatitis C virus (HCV) infection, to determine whether route of transmission affects the clearance rate and to identify other predictors of clearance. Children with chronic hepatitis C were identified between 1990 and 2001. The rate of spontaneous clearance (defined as >or=2 positive anti-HCV antibody test but negative HCV RNA) was calculated using survival analysis. Univariate and multivariate predictor variables [route of transmission, age at infection, age at last follow-up, alanine aminotransferase (ALT) and gender] for clearance were evaluated. Of 157 patients, 28% of children cleared infection (34 transfusional and 10 nontransfusional cases). The 123 transfusional cases were older at time of infection and at follow-up, compared with the 34 nontransfusional cases. Younger age at follow-up (p < 0.0001) and normal ALT levels (p < 0.0001) favoured clearance. Among cases of neonatal infection, 25% demonstrated spontaneous clearance by 7.3 years. The rate of spontaneous clearance of childhood HCV infection was comparable between transfusional and nontransfusional cases. If clearance occurs, it tends to occur early in infection, at a younger age.

Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075–3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.

SUMMARY 62Gnotobiotic piglets serve as a useful animal model for studies of rotavirus pathogenesis and immunity. An advantage over laboratory animal models is the prolonged susceptibility of piglets to . rotavirus-induced disease, permitting an analysis of cross-protection and active immunity. Studies from our laboratory of the pathogenesis of human rotavirus infections in gnotobiotic piglets have confirmed that villous atrophy is induced in piglets given virulent but not attenuated human rotavirus (Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. To facilitate and improve rotavirus vaccination strategies, it is important to identify correlates of protective immunity. Comparison of antibody immune responses induced by infection with virulent porcine and human rotaviruses (mimic host response to natural infection) with those induced by live attenuated human rotavirus (mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Our results indicate that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the site of viral replication (small intestine). Secondly there was a direct association between the degree of protection induced and the level of the intestinal immune response, with primary exposure to virulent rota viruses inducing significantly higher numbers of IgA ASC and complete protection against challenge. These studies thus have established basic parameters related to immune protection in the piglet model of rotavirus-induced disease, verifying the usefulness of this model to apply new strategies for the design and improvement of rota virus vaccines.

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Gnotobiotic pigs were orally inoculated with virulent Wa strain (G1P1A[8]) human rotavirus (group 1), attenuated Wa rotavirus (group 2) or diluent (controls) and were challenged with virulent Wa rotavirus 21 days later. On various postinoculation or postchallenge days, virus-specific responses of systemic (blood and spleen) and intestinal (mesenteric lymph node and ileal lamina propria) mononuclear cells (MNC) were assessed by lymphoproliferative assays (LPA). After inoculation, 100% of group 1 pigs and 6% of group 2 pigs shed virus. Diarrhea occurred in 95, 12, and 13% of group 1, group 2, and control pigs, respectively. Only groups 1 and 2 developed virus-specific LPA responses prior to challenge. Group 1 developed significantly greater mean virus-specific LPA responses prior to challenge and showed no significant changes in tissue mean LPA responses postchallenge, and 100% were protected against virulent virus challenge. By comparison, both group 2 and controls had significantly lower LPA responses at challenge and both groups showed significant increases in mean LPA responses postchallenge. Eighty-one percent of group 2 and 100% of control pigs shed challenge virus, and both groups developed diarrhea that was similar in severity postchallenge. The virusspecific LPA responses of blood MNC mirrored those of intestinal MNC, albeit at a reduced level and only at early times postinoculation or postchallenge in all pigs. In a separate study evaluating antibody-secreting-cell responses of these pigs (L.