Kwonil Jung

Porcine Reproductive and Respiratory Syndrome Virus–Induced Immunosuppression Exacerbates the Inflammatory Response to Porcine Respiratory Coronavirus in Pigs

We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co-infections, and provide more definitive approaches for treatment.

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs.

To verify whether porcine deltacoronavirus infection induces disease, we inoculated gnotobiotic pigs with 2 virus strains (OH-FD22 and OH-FD100) identified by 2 specific reverse transcription PCRs. At 21–120 h postinoculation, pigs exhibited severe diarrhea, vomiting, fecal shedding of virus, and severe atrophic enteritis. These findings confirm that these 2 strains are enteropathogenic in pigs.

Prevalence of porcine circovirus type 2 in aborted fetuses and stillborn piglets

A retrospective study of natural cases of abortion, recorded between October 2000 and September 2002, was made to determine the prevalence of abortions associated with porcine circovirus type 2 (PCV-2). The virus was detected by PCR, immunohistochemistry and in situ hybridisation. A total of 46 (13.1 per cent) of 350 aborted fetuses and stillborn piglets were positive for PCV-2 by PCR, and the virus was detected in fetuses at all stages of gestation. Viral antigen was detected in macrophages from the aborted fetuses and stillborn piglets by immunohistochemistry, and viral DNA was detected by in situ hybridisation.

The Effects of Simvastatin or Interferon-α on Infectivity of Human Norovirus Using a Gnotobiotic Pig Model for the Study of Antivirals

The lack of an animal model for human norovirus (HuNoV) has hindered the development of therapeutic strategies. This study demonstrated that a commonly used cholesterol-lowering statin medication, simvastatin, which increases HuNoV replication in an in vitro replicon system, also enhances HuNoV infectivity in the gnotobiotic (Gn) pig model. In contrast, oral treatment with interferon (IFN)-α reduces HuNoV infectivity. Young piglets, all with A or H1 histo-blood group antigens on enterocytes, were treated orally with 8 mg/kg/day of simvastatin; 5 days later, the pigs were inoculated orally with a GII.4 HuNoV (HS194/2009/US strain) and then treated with simvastatin for 5 more days. Simvastatin induced significantly earlier onset and longer duration of HuNoV fecal shedding in treated pigs, frequently with higher fecal viral titers. Simvastatin impaired poly (I:C)-induced IFN-α expression in macrophages or dendritic cells, possibly due to lowered toll-like receptor (TLR) 3 expression; however, the mechanisms were not related to interferon regulatory factor 3 or nuclear factor kappa B signaling pathway. Thus, the enhanced, earlier infectivity of HuNoV in simvastatin-treated pigs coincided with the inhibitory effect of simvastatin on innate immunity. In contrast to the increased HuNoV shedding that simvastatin induced, viral shedding during the treatment period was reduced or curtailed in the HuNoV-inoculated pigs pre-treated/treated with human IFN-α. Our findings are the first to indicate that IFN-α has potential as antiviral therapy against HuNoV. Based on these intriguing and novel findings using the Gn pig model, we confirmed that HuNoV infectivity is altered by treatment with simvastatin or IFN-α. Collectively, these findings indicate that Gn pigs are a useful model to test immunomodulators or efficacy of antivirals against HuNoV.

Altered Pathogenesis of Porcine Respiratory Coronavirus in Pigs due to Immunosuppressive Effects of Dexamethasone: Implications for Corticosteroid Use in Treatment of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 × 107 PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2+, CD3+, CD4+, and CD8+ T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-γ)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-γ-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.

The effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets

Abstract The effects of transplacental porcine circovirus type 2 (PCV2) infection on porcine epidemic diarrhoea virus (PEDV)-induced enteritis were examined in neonatal piglets. Six pregnant sows were randomly allocated to an infected (n =3) or control group (n =3). Three pregnant sows were inoculated intranasally with 6mL of tissue culture fluid containing 1.2×105 tissue culture infective doses 50% (TCID50)/mL of PCV2 strain SNUVR000470 three weeks before the expected farrowing date. Three control pregnant sows were similarly exposed to uninfected cell culture supernatants. Thirty piglets from PCV2-infected sows were randomly assigned to two groups (A and B) of 15 piglets each. Another 30 piglets from noninfected sows were randomly assigned to two groups (C and D) of 15 piglets each. The piglets in groups A and C were dosed orally at three days of age with 2mL of virus stock (1×106.5 TCID50/mL) of the PEDV strain, SNUVR971496, at the third passage. The mean villous height and crypt depth (VH:CD) ratio in PEDV-infected piglets from PCV2-infected sows (group A) were significantly different from those of the PEDV-infected piglets from PCV2 negative sows (group C) at 36, 48, and 72h post-inoculation (hpi) (P <0.05). In PEDV-infected piglets from PCV2-infected sows (group A), significantly more PEDV nucleic acid was detected in the jejunal tissues (P <0.05) at 24hpi than in the same tissues of the PEDV-infected piglets from PCV2 negative sows (group C). Thereafter, at 36, 48, 60, and 70hpi significantly more PEDV nucleic acid (P <0.05) was detected in the jejunal tissues of the PEDV-infected piglets from PCV2 negative sows (group C) than those of the PEDV-infected piglets from the PCV2-infected sows (group A). It is concluded that the clinical course of PEDV disease was markedly affected by transplacental infection of PCV2.

Outbreak of salmonellosis in pigs with postweaning multisystemic wasting syndrome

POSTWEANING multisystemic wasting syndrome (PMWS) is an infectious viral disease cause by porcine circovirus type 2 (PCV-2) (Allan and Ellis 2000). It is characterised by progressive weight loss, respiratory signs and jaundice (Clark 1997, Chae 2004). The disease occurs in herds that are usually otherwise in good health. PMWS is endemic in many pigproducing countries and continues to be a major cause of wasting disease in pigs. In the Republic of Korea, PMWS is commonly associated with respiratory diseases (Kim and others 2003). However, secondary infections with bacterial pathogens, particularly Salmonella Typhimurium and Salmonella Choleraesuis, typically occur concurrently with PMWS in many herds. Most herds undergo severe devastating diarrhoea. This short communication describes an outbreak of salmonellosis in pigs in herds with PMWS in the Republic of Korea. Between January 2003 and April 2004, there were 37 cases of severe diarrhoea and retardation of growth from 24 pig farms. Twenty-two cases were from 2003 and 15 cases occurred in 2004. All 37 cases were weaned pigs, aged from 28 to 63 days. Morbidity was 10 to 20 per cent but mortality was 70 to 80 per cent in all of the herds affected. The diarrhoea was initially yellowish in colour but progressed to black; antibiotic therapy was ineffective in treatment of the diarrhoea in all cases. Postmortem examinations were performed on the 37 pigs and samples of lung, liver, kidney, small and large intestines, spleen, tonsil, and inguinal lymph nodes were collected in neutral buffered formalin. Small and large intestines from all of the pigs were examined for viral pathogens such as PCV-2 and classical swine fever (CSF) virus, and for bacterial pathogens such as Salmonella species, Brachyspira pilosicoli and Brachyspira hyodysenteriae. Homogenates of the small and large intestines were inoculated into cultures of PCV-free porcine kidney 15 cells, MARC-145 cells, swine testicular cells, primary porcine fallopian tube cells and Vero cells. In situ hybridisation and immunohistochemistry were performed for PCV-2 and CSF virus, respectively, as described by Kim and Chae (2001) and Choi and Chae (2003). Positive and negative control tissues of each virus were included for each in situ hybridisation and immunohistochemistry procedure (Kim and Chae 2001, Choi and Chae 2003). PCR was carried out as previously described for Lawsonia intracellularis, B pilosicoli, B hyodysenteriae and salmonellae in porcine intestinal specimens (Elder and others 1997, Kim and others 1998, Choi and others 2002). The most consistent and predominant histopathological feature seen was lymphoid depletion in the lymphoid follicle and the paracortical zone of the lymph nodes, with the lymphoid follicles and the paracortical zone being replaced by fibrovascular stroma and occasionally macrophages. Multinucleated giant cells were scattered between stromal components, and grape-like clusters of intensely basophilic intracytoplasmic inclusion bodies appeared in these areas (Fig 1). Depletion of the medullary cords and empty medullary sinuses were also observed. A strong hybridisation signal for PCV-2 was detected in the cytoplasm of macrophages in lymph nodes, spleen and Peyer’s patches (Fig 2) in all 37 pigs tested. In the colon, necrosis frequently extended to involve the lamina propria (Fig 3), submucosa, muscularis mucosa and lymphoid follicles. Necrotic tissues were separated from the healthy mucosa by a marginal zone of leucocytes, and the deeper tissues were heavily infiltrated by neutrophils. The lamina propria and submucosa contained numerous neutrophils and macrophages. Salmonellae were detected in the small and large intestines from all 37 cases by PCR. Three pigs were also positive for L intracellularis and two pigs were also positive for B pilosicoli. B hyodysenteriae was not detected in the small and large intestines by PCR. S Typhimurium was isolated from 23 pigs and S Choleraesuis was isolated from two pigs. No other bacterium was isolated from the small and large intestines. Cytopathic viruses were not detected after five passages.

Expression of Mx Protein and Interferon-α in Pigs Experimentally Infected with Swine Influenza Virus

Expression of Mx protein and interferon-α (IFN-α) was examined by immunohistochemistry in pigs experimentally infected with swine influenza virus. In infected pigs euthanatized at 1 day postinoculation (dpi), the lumen of bronchioles were filled with large numbers of mononuclear cells, small numbers of neutrophils, sloughing epithelial cells, and proteinaceous fluid. Lesions at 3 and 5 dpi were similar but less severe. Alveolar spaces were filled with neutrophils. By 7 and 10 dpi, microscopic lesions were resolved. The immunohistochemical signals for Mx protein and IFN-α antigen were confined to cells in areas that had hybridization signal for swine influenza virus. In situ hybridization and immunohistochemistry of serial sections of lung indicated that areas containing numerous swine influenza virus RNA-positive cells also have numerous Mx and IFN-α antigen-positive cells. Mean immunohistochemical scores for Mx protein-positive cells were correlated with mean immunohistochemical scores for IFN-α antigen-positive cells (rs = 0.8799, p < 0.05). These results indicated that Mx protein and IFN-α antigen were expressed in the lung from pigs experimentally infected with swine influenza virus, but their biological functions remain to be examined.

Expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 in lungs from pigs experimentally infected with Actinobacillus pleuropneumoniae.

The expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 was studied, by reverse transcription–polymerase chain reaction and by in situ hybridization with a non-radioactive digoxigenin-labelled cDNA probe, in formalin-fixed, paraffin-wax-embedded lung tissue from pigs experimentally infected withActinobacillus pleuropneumoniae. Forty-eight 7-week-old colostrum-deprived pigs were randomly allocated to infected (n = 24) or control (n = 24) groups. Three pigs from each group were euthanized at 3, 6, 9, 12, 24, 36, 48 and 60 h post inoculation (hpi). IL-10 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-10 increasing at 36 hpi. IL-12p35 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-12p35 increasing at 36 and 48 hpi and rapidly decreasing thereafter whereas IL-12p40 mRNA was constitutively expressed at low levels during the experiment. Hybridization signals for IL-10, IL-12p35 and IL-12p40 were always associated with inflammation, in particular with macrophages and neutrophils within alveolar spaces. Expression of these cytokines was minimal in non-lesional lung ofA. pleuropneumoniae-infected pigs and in normal lung from control pigs. In situ hybridization ofA. pleuropneumoniae and these cytokines in serial sections of lung tissues indicated close co-localization ofA. pleuropneumoniae and these cytokines in pleuropneumonia. The results suggest that the expression of IL-10 and IL-12 play a role in pathogenesis ofA. pleuropneumoniae infection.

Lack of evidence of porcine circovirus type 1 and type 2 infection in piglets with congenital tremors in Korea.

Chae (2003). Positive and negative control tissues for each virus were included in each in situ hybridisation procedure (Kim and Chae 2001, Choi and Chae 2003). On microscopic examination of the tissues, mild to moderate cerebellar and spinal hypomyelination was detected in 33 of the 36 piglets. Neither PCV-1 nor PCV-2 nucleic acid was detected in any brain or spinal cord samples from the 36 piglets by in situ hybridisation; hybridisation signals of PCV-1 were seen occasionally in liver tissue from one piglet and lymph node from another piglet. PCV-2 DNA was also detected occasionally in the liver and spleen in one piglet, and in the spleen and lymph node in two other piglets with congenital tremor. No piglet had hybridisation signals of both PCV-1 and PCV-2. The granulomatous inflammatory reaction that is typical of lesions associated with PCV-2 infection in pigs with PMWS was not observed in the liver or lymph node from the PCV-1or PCV-2-positive piglets. CSF virus was not detected in any tissue of any of the piglets by in situ hybridisation. All the tests for PCV-1, PCV-2 and other cytopathic viruses were negative by the virus isolation procedure. In the present study of 36 cases of congenital tremor in piglets in Korea, no evidence of PCV-1 or PCV-2 nucleic acid was found in the central nervous system by in situ hybridisation. PCV-1 and PCV-2 were detected in non-neuronal tissues, such as the liver, spleen and lymph node, in two and three piglets, respectively. However, the identification of PCV-2 may not be significant, since PCV-2 can be detected in the lymph nodes of normal pigs by PCR and in situ hybridisation (Chae 2004). The detection of PCV-1 and PCV-2 in day-old piglets was interpreted as a consequence of infection in utero. However, it could not be ruled out that the piglets had been infected with PCV-1 or PCV-2 postnatally. The absence of CSF virus, the involvement of both male and female piglets, the absence of British Saddleback parentage and the lack of prenatal exposure to organophosphates excludes congenital tremor types A1, A3, A4 and A5, respectively. Therefore, the present cases with cerebellar and spinal hypomyelination may be classified as congenital tremor type A2; the two cases with no such histological lesions can be classified as congenital tremor type B. These results support those of researchers in Europe who failed to detect PCV infection in the central nervous system or non-neuronal tissues of piglets with congenital tremor (Kennedy and others 2003). These results are contradictory to those of a previous study, which found that PCV isolates from cases of congenital tremor type A2 can cause congenital tremor when inoculated into susceptible pregnant sows (Hines and Lukert 1994). Furthermore, PCV-2 was detected mainly in the brain and spinal cord from piglets with congenital tremor by Stevenson and others (2001). The apparent lack of consistency regarding the presence or absence of PCV infection in piglets with congenital tremor is difficult to explain. It is possible that a geographical difference exists in the involvement of PCV in congenital tremor; PCV may be associated with congenital tremor in pigs in the USA, but not in European and Asian countries. Further studies are therefore needed to determine the role of PCV in congenital tremor type A2.