EVALUATION OF PORCINE ENTERIC CALICIVIRUSES (NOROVIRSES) IN PIG AND HUMAN DIARRHEA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0205772
• Project No.: KS481846
• Project Start Date: Oct 15, 2005
• Project End Date: Sep 30, 2007

Project Director
Chang, K.

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
DIAGNOSTIC MEDICINE AND PATHOBIOLOGY

Non Technical Summary
Caliciviruses are important veterinary and human pathogens associated with a wide range of economically important diseases in their respective hosts. Noroviruses and sapoviruses are enteric caliciviruses which cause acute gastroenteritis in humans and animals. The human enteric caliciviruses (noroviruses and sapoviruses) are currently leading cause of food- and water-borne viral infections. Swine diarrhea is a significant factor contributing to economic losses in the swine industry. The importance of porcine enteric caliciviruses is likely underestimated because of a lack of routine diagnostic reagents and sensitive tests for detection. Thus development of reliable and sensitive diagnostic reagents and assays such as RT-PCR is needed for diagnosis of PEC infections in swine. Because of the diversity of PEC, designing primers for detecting the genetically different PEC should be the key to screen the viruses in the field. Understanding the prevalence of diarrhea associated with PEC will provide new information about the epidemiology and impact of PEC infections in swine. We will develop RT-PCR/hybridization for routine detection of the PEC genome in feces. The recent identification of porcine noroviruses and sapoviruses worldwide has raised questions about PEC as potential zoonotic agents. We need to clarify if pigs are possible reservoirs for enteric caliciviruses potentially transmissible to humans. The extensive genetic analysis of field strains of porcine noroviruses and sapoviruses would provide important answers for the questions.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4030	1040	50%
311	4030	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology; 1040 - Molecular biology;

Keywords

pig, calicivirus, gastroenteritis, diagnosis, rt-pcr, sequence

analysis, zoonosis. ;

Goals / Objectives
1. Develop sensitive and specific diagnostic methods for detecting PEC genome in stools. 2. Study the genetic correlations between PEC and human enteric calicivirus using sequence analysis to access PEC as the source of human infection.

Project Methods
Design multiplex primer sets for porcine norovirus and sapovirus and establish optimal RT-PCR / hybridization method using established PEC in the lab. Because there are at least 2 different PEC (noroviruses and spoviruses) in pigs, we plan to use multiple primer sets targeting the polymerase region which is relatively conserved among caliciviruses. There are several porcine norovirus and sapovirus sequences available in the GenBank (AB074892, AB074893, AF194184 for porcine norovirus; AF182760 for porcine sapovirus). To increase the sensitivity for detecting PEC, we plan to do second round PCR using nested primers which are located within the RT-PCR target sequences. The specificity and sensitivity of the RT-PCR assay will be evaluated using a panel of calicivirus positive and several negative fecal samples. In addition, we will test fecal samples containing common pig enteric pathogens such as rotavirus, TGE virus, E.coli, etc. to confirm the specificity of RT-PCR for PEC. The RT-PCR will be optimized by using various strategies developed in our lab for detecting viral genomes in specimens. To confirm the RT-PCR products for PEC-specificity, we will use the hybridization method (Southern blots) using PEC specific probes. For Southern blots, the RT-PCR products in the agarose gel will be denatured and transferred to a positively charged nylon membrane. Hybridization of RT-PCR products will be performed using PEC specific biotinylated probes and NorthernMax kit (Ambion). Perform sequence analysis of the full genomes of PEC, and compare them with human norovirus and sapoviruses. We have obtained several porcine sapoviruses and noroviruses from Dr. Saif, and we will sequence the full-length genome of PEC after amplifying several fragments overlapping whole genomes by RT-PCR. For the RT-PCR and sequencing, we will use primers based on the strains in the same genogroups or conserved motifs of caliciviruses. It is important to sequence full-length genome of the PEC because there have been reports regarding the recombination events between different genogroups of caliciviruses. The 5 foot and 3 foot end of the genome will be determined using 5 foot- or 3 foot RACE kit (Invitrogen). The capsid gene is important to compare genetic relationships because it determines antigenicity of caliciviruses. The multiple alignments of nucleotide and deduced amino acid sequences will be performed using the DNASTAR and GCG program. The PEC genomic sequences will be compared with those of the porcine norovirus, sapovirus (including Cowden strain), and HuCV, as well as other animal caliciviruses. The genetic comparisons of human and porcine enteric caliciviruses with field strains should provide clues for whether porcine enteric caliciviruses are the causes of gastroenteritis outbreaks in humans.

Progress 10/15/05 to 09/30/07

Outputs
Interferons and ribavirin are non-specific antiviral agents with FDA approval for various viral diseases such as chronic hepatitis C virus infection and respiratory syncytial virus infection. Both agents were very effective in inhibiting Norwalk virus replication and when they were combined, we observed an additive effect. Without any commercially available drugs against noroviral gastroenteritis, these could be developed as antiviral drugs. As a continual study of the effects of bile acids on the calicivirus replication, we have studied the role of the farnesoid X receptor (FXR), a bile acid receptor, in the replication of norovirus. We have found that the activation of FXR promoted the replication of noroviruses in cells, demonstrated the importance of bile acids in norovirus replication.

Impacts
Our study could lead to the isolation of fastidious noroviruses in cell culture and vaccine development.

Publications

• Chang, K.O., George, D.W. 2007. Interferons and Ribavirin Effectively Inhibit Norwalk Virus Replication in the Replicon-Bearing cells. J Virol. Nov;81(22):12111-8.
• Chang, K.O., George, D.W. 2007. Bile acids promote the replication of hepatitis C virus in the replicon harboring cells. J Virol. 81(18):9633-40.
• Kim, D.Y., J.G. Calvert, K.O. Chang, R.R. Rowland. 2007. The cysteine protease, nsp2, of porcine reproductive and respiratory syndrome virus: a potential target for heterologous gene expression. Virus Res. 128(1-2):106-14.

Progress 01/01/06 to 12/31/06

Outputs
We have developed the RT-PCR/hybridization assay for detecting porcine noroviruses or sapoviruses. The primer pairs for specific to porcine noroviruses and sapoviruses were designed, and optimum conditions were established. To solve problems with RT-PCR inhibitors in fecal RNA which frequently cause false-negative results, we developed a competitive internal control (IC) RNA. Microwell hybridization assays were also developed to confirm the specific RT-PCR products. We also generated cells expressing self-replicating Norwalk virus RNA (Norwalk virus replicon) following transfection of Norwalk virus RNA bearing an engineered neomycin resistance gene into cell lines of human (Huh-7) or hamster (BHK21) origin. We found that expression of replicon RNA was significantly reduced in the presence of interferon- in a dose-dependent manner in the Norwalk virus replicon-bearing cells, suggesting a role for innate immunity in the control of norovirus replication. This stable replicon system should lead to new insights into norovirus replication, virus-host interactions, and approaches for the treatment of norovirus disease.

Impacts
The replicon system is a platform to screen potential antiviral drugs against noroviruses.

Publications

• Wang, QH, Chang, KO, Han, MG, Sreevatsan, S, Saif, LJ. 2006. Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR. Mar;132(1-2):135-45.
• Chang, KO, Sosnovtsev, SO, Belliot, G, Green, KY. 2006. Stable expression of a Norwalk virus RNA replicon in a human hepatoma cell line. Virology. Sep;353(2):463-73.