Linda J. Saif

Uniformity of Rotavirus Strain Nomenclature Proposed by the Rotavirus Classification Working Group (RCWG)

In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.

Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments.

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al. in J Virol 82:3204–3219, 2008). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the 11 gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.

The pig: a model for human infectious diseases

An animal model to study human infectious diseases should accurately reproduce the various aspects of disease. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of anatomy, genetics and physiology, and represent an excellent animal model to study various microbial infectious diseases. Indeed, experiments in pigs are much more likely to be predictive of therapeutic treatments in humans than experiments in rodents. In this review, we highlight the numerous advantages of the pig model for infectious disease research and vaccine development and document a few examples of human microbial infectious diseases for which the use of pigs as animal models has contributed to the acquisition of new knowledge to improve both animal and human health.

Pathogenesis of a Genogroup II Human Norovirus in Gnotobiotic Pigs

ABSTRACT We evaluated the gnotobiotic (Gn) pig as a model to study the pathogenesis of human norovirus (HuNoV) and to determine the target cells for viral replication. Sixty-five Gn pigs were inoculated with fecal filtrates of the NoV/GII/4/HS66/2001/US strain or with pig-passaged intestinal contents (IC) and euthanized acutely (n = 43) or after convalescence (n = 22). Age-matched Gn piglets (n = 14) served as mock-inoculated controls. Seventy-four percent (48/65) of the inoculated animals developed mild diarrhea compared to 0 of 14 controls. Pigs from postinoculation days (PID) 1 to 4 tested positive for HuNoV by reverse transcription-PCR of rectal swab fluids (29/65) and IC (9/43) and by antigen (Ag) enzyme-linked immunosorbent assay (ELISA) using antiserum to virus-like particles of HuNoV GII/4. No control pigs were positive. Histopathologic examination showed mild lesions in the proximal small intestine of only one pig (1/7). Seroconversion after PID 21 was detected by antibody ELISA in 13 of 22 virus-inoculated pigs (titers, 1:20 to 1:200) but not in controls. Immunofluorescent microscopy using a monoclonal antibody to HuNoV GII capsid revealed patchy infection of duodenal and jejunal enterocytes of 18 of 31 HuNoV-inoculated pigs with a few stained cells in the ileum and no immunofluorescence (IF) in mock-inoculated controls. Immunofluorescent detection of the viral nonstructural N-terminal protein antigen in enterocytes confirmed translation. Transmission electron microscopy of intestines from HuNoV-inoculated pigs showed disrupted enterocytes, with cytoplasmic membrane vesicles containing calicivirus-like particles of 25 to 40 nm in diameter. In summary, serial passage of HuNoV in pigs, with occurrence of mild diarrhea and shedding, and immunofluorescent detection of the HuNoV structural and nonstructural proteins in enterocytes confirm HuNoV replication in Gn pigs.

Porcine noroviruses related to human noroviruses.

Pigs may be reservoirs for human noroviruses, and porcine/human genogroup II recombinants could emerge.

Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis

Abstract Porcine epidemic diarrhea virus (PEDV), a member of the genera Alphacoronavirus in the family Coronaviridae, causes acute diarrhea/vomiting, dehydration and high mortality in seronegative neonatal piglets. For the last three decades, PEDV infection has resulted in significant economic losses in the European and Asian pig industries, but in 2013–2014 the disease was also reported in the US, Canada and Mexico. The PED epidemic in the US, from April 2013 to the present, has led to the loss of more than 10% of the US pig population. The disappearance and re-emergence of epidemic PED indicates that the virus is able to escape from current vaccination protocols, biosecurity and control systems. Endemic PED is a significant problem, which is exacerbated by the emergence (or potential importation) of multiple PEDV variants. Epidemic PEDV strains spread rapidly and cause a high number of pig deaths. These strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites. PEDV infections cause acute, severe atrophic enteritis accompanied by viremia that leads to profound diarrhea and vomiting, followed by extensive dehydration, which is the major cause of death in nursing piglets. A comprehensive understanding of the pathogenic characteristics of epidemic or endemic PEDV strains is needed to prevent and control the disease in affected regions and to develop an effective vaccine. This review focuses on the etiology, epidemiology, disease mechanisms and pathogenesis as well as immunoprophylaxis against PEDV infection.

PATHOGENESIS OF AN ATTENUATED AND A VIRULENT STRAIN OF GROUP A HUMAN ROTAVIRUS IN NEONATAL GNOTOBIOTIC PIGS

Gnotobiotic (Gn) pigs were orally inoculated with Wa strain (G1P1A[P8]) human rotavirus (Wa HRV) serially passaged in Gn pigs (virulent) or cell culture (attenuated) to determine the median virus infectious dose (ID50) and to assess the site of infection and type and progression of morphological lesions and clinical responses induced by these two strains in Gn pigs. The ID50 of virulent Wa HRV was = or < 1 f.f.u. whereas the infectivity of attenuated Wa HRV had to be determined by seroconversion and was approximately 1.3 x 1O(6) f.f.u. Diarrhoea developed at 13 h post-inoculation (p.i.) in pigs inoculated with approximately 1O(5) f.f.u. of virulent Wa HRV and correlated with the presence of viral antigen within villous epithelial cells; villous atrophy developed later at 24 h p.i. and correlated with peak faecal viral titres; recovery from disease correlated with the return of morphologically normal villi. Virus, diarrhoea and villous atrophy were not detected in pigs inoculated with approximately 2 x 10(8)f.f.u. attenuated Wa HRV although HRV-specific serum antibodies were present by 7 days p.i. These findings demonstrate that virulent Wa HRV infection in Gn pigs occurs primarily within intestinal villous epithelial cells with villous atrophy developing as a sequela to infection. However, factors other than villous atrophy appear to contribute to the early stages of HRV-associated disease expression in Gn pigs. The ability of the attenuated virus to elicit virus-neutralizing serum antibodies without disease or pathology indicates promise in the use of such strains for oral immunization.

Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.

ABSTRACT We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-α]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-γ]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-α and IFN-γ at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-α and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-γ and TNF-α CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the hosts local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

Cytokine Responses in Gnotobiotic Pigs after Infection with Virulent or Attenuated Human Rotavirus

ABSTRACT To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-γ) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor β (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-γ, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-α levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-γ responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-γ responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-γ-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-γ and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-γ (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.

Human and animal enteric caliciviruses in oysters from different coastal regions of the United States.

ABSTRACT Food-borne diseases are a major cause of morbidity and hospitalization worldwide. Enteric caliciviruses are capable of persisting in the environment and in the tissues of shellfish. Human noroviruses (HuNoVs) have been implicated in outbreaks linked to shellfish consumption. The genetic and antigenic relatedness between human and animal enteric caliciviruses suggests that interspecies transmission may occur. To determine the occurrence of human and animal enteric caliciviruses in United States market oysters, we surveyed regional markets. Oysters were collected from 45 bays along the United States coast during the summer and winter of 2002 and 2003. Samples were analyzed by reverse transcription-PCR, and results were confirmed by hybridization and sequence analysis. Nine samples (20%) were positive for HuNoV genogroup II after hybridization. Animal enteric caliciviruses were detected in 10 samples (22%). Seven of these samples were positive for porcine norovirus genogroup II, and one sample was positive for porcine sapovirus after hybridization and confirmation by sequencing. Bovine noroviruses were detected in two samples, and these results were confirmed by sequencing. Five HuNoV samples sequenced in the polymerase region were similar to the norovirus genogroup II US 95/96 subset (genogroup II-4) previously implicated in diarrhea outbreaks. Different seasonal and state distributions were detected. The presence of animal enteric caliciviruses was associated with states with high livestock production. Although the presence of human caliciviruses in raw oysters represents a potential risk for gastroenteritis, disease confirmation by investigation of outbreaks is required. The simultaneous detection of human and animal enteric caliciviruses raises concerns about human infection or coinfection with human and animal strains that could result in genomic recombination and the emergence of new strains.