Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells. Received March 7, 2000 Accepted August 16, 2000     

The localization of persistent foot and mouth disease virus in the epithelial cells of the soft palate and pharynx

After contact with foot and mouth disease virus (FMDV), cattle may become persistently infected, regardless of their pre-existing immune status or whether they develop clinical disease. The cellular sites of FMDV persistence have not previously been determined. The use of in-situ hybridization in combination with tyramide signal amplification (TSA) provided the first direct evidence that FMDV RNA is localized within the epithelial cells of the soft palate and pharynx during persistent infection, indicating that these cells remain persistently infected after contact with FMDV. Copyright Harcourt Publishers Ltd.     

Consistent change in the B-C loop of VP2 observed in foot-and-mouth disease virus from persistently infected cattle: implications for association with persistence

The mechanisms of foot-and-mouth disease virus (FMDV) persistence are poorly understood. It is thought the existence of viral quasispecies that encompass sub-populations with varying survival competencies and antigenicities may play some role in the maintenance of virus in persistently infected animals. By analyzing nucleotide sequences encoding the viral VP2 protein in oesophageal-pharyngeal fluid (probang) samples from cattle at different stages of infection, the significance of any amino acid changes in relation to persistence was investigated. Twenty-two experimentally infected cattle (including six carriers) from three animal experiments with FMDV type O UKG34/2001 were studied. Comparison of VP2 sequences in these samples with the inoculum sequence revealed a consistent change in the B-C loop in FMDV from persistently infected cattle. Residue 2079 changed from Y to H in five carrier animals and residue 2080 changed from A to Q in one carrier from 14 days post-infection onward. In contrast, there were no changes evident in any of the non-carriers up to 28 days post-infection. The results indicate that a substitution change in the B-C loop of VP2 may be associated with persistent FMDV infection in cattle.     

Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein

Summary  Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E. coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection. Received June 17, 1996 Accepted September 11, 1996     

Mucosal disease induced in cattle persistently infected with bovine viral diarrhea virus by antigenically different cytopathic virus

Summary.  Four cattle persistently infected with non-cytopathic (NCP) bovine viral diarrhea (BVD) virus were challenged with cytopathic (CP) BVD virus that was antigenically different from the persistent virus. Two of the animals were injected with dexamethasone (DM) and then challenged. They developed mucosal disease on days 21 and 33 post-challenge. CP-BVD viruses were isolated from their lymph nodes but not from the sera. The isolates were antigenically different from the persistent virus and the nucleotide sequence of a 787 base region in the E2 gene was markedly different. One of the isolates was indistinguishable from the challenge virus by virus neutralization tests and the nucleotide sequence showed high homology with that of the challenge CP-BVD virus. The other two cattle, challenged with the CP-BVD virus without DM treatment, developed mucosal disease at 30 and 264 days post-inoculation. CP-BVD virus was isolated from the sera as well as the lymph nodes of the cattle and was antigenically and genetically similar to the persistent virus and different from the challenge CP-BVD virus. The present results indicate that cattle persistently infected with NCP-BVD virus can develop mucosal disease induced by antigenically different CP-BVD viruses when their cellular immunity is suppressed, although they are not immunotolerant to the virus. Accepted November 1, 2000 Received July 25, 2000     

Anti-CD8 treatment alters interleukin-4 but not interferon-γ mRNA levels in murine sensory ganglia during herpes simplex virus infection

Summary.  Clearance of herpes simplex virus (HSV) from sensory ganglia of infected mice is dependent on CD8⁺ cells but not on the death of infected neurons. The mechanism of action of CD8⁺ cells in HSV infected ganglia is therefore unknown. The determine which cytokines might be involved in the CD8⁺ cell dependent response to ganglionic HSV infection, IL-2, IL-4, IL-6, IL-10, and IFN-γ mRNA levels were measured in infected ganglia from immunocompetent and anti-CD8 treated mice. Anti-CD8 treatment increased the abundance of mRNA encoding IL-4, and, to a lesser extent, IL-2, and IL-6. Significantly, IFN-γ mRNA was not affected. Received May 17, 1999 Accepted June 29, 1999     

Domain disruptions of individual 3B proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle

Picornavirus RNA replication is initiated by a small viral protein primer, 3B (also known as VPg), that is covalently linked to the 5' terminus of the viral genome. In contrast to other picornaviruses that encode a single copy of 3B, foot-and-mouth disease virus (FMDV) encodes three copies of 3B. Viruses containing disrupted native sequence or deletion of one of their three 3B proteins were derived from a FMDV A24 Cruzeiro full-length cDNA infectious clone. Mutant viruses had growth characteristics similar to the parental virus in cells. RNA synthesis and protein cleavage processes were not significantly affected in these mutant viruses. Cattle infected by aerosol exposure with mutant viruses developed clinical disease similar to that caused by the parental A24 Cruzeiro. Therefore, severe domain disruption or deletion of individual 3B proteins in FMDV do not affect the virus' ability to replicate in vitro and cause clinical disease in cattle.     

Potential secondary pathogenic role for bovine herpesvirus 4

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbor persistent BoHV-4. We found that the addition of prostaglandin E2 (PGE2) to bovine macrophage cells persistently infected with BoHV-4 increases viral replication. Because opportunistic infection can increase PGE2 production, we propose a link between opportunistic infection, PGE2 production, and BoHV-4 replication.     

Pathogenic characteristics of the Korean 2002 isolate of foot-and-mouth disease virus serotype O in pigs and cattle

Experimental infection of susceptible cattle and pigs showed that the O/SKR/AS/2002 pig strain of foot-and-mouth disease virus (FMDV) causes an infection that is highly virulent and contagious in pigs but very limited in cattle. Pigs directly inoculated with, or exposed to swine infected with, strain O/SKR/AS/2002 showed typical clinical signs, including gross vesicular lesions in mouth and pedal sites. In addition, FMDV was isolated from, and FMDV genomic RNA was detected in, blood, serum, nasal swabs and oesophageal-pharyngeal (OP) fluid early in the course of infection. Antibodies against the non-structural protein (NSP) 3ABC were detected in both directly inoculated and contact pigs, indicating active virus replication. In contrast, the disease in cattle was atypical. After inoculation, lesions were confined to the infection site. A transient viraemia occurred 1 and 2 days after inoculation, and this was followed by the production of antibodies to NSP 3ABC, indicating subclinical infection. No clinical disease was seen, and no antibodies to NSP 3ABC were present in contact cattle. Additionally, no virus or viral nucleic acid was detected in blood, nasal swab and OP fluid samples from contact cattle. Thus, the virus appeared not to be transmitted from infected cattle to contact cattle. In its behaviour in pigs and cattle, strain O/SKR/AS/2002 resembled the porcinophilic FMDV strain of Cathay origin, O/TAW/97. However, the latter, unlike O/SKR/AS/2002, has reduced ability to grow in bovine-derived cells. The porcinophilic character of O/TAW/97 has been attributed to a deletion in the 3A coding region of the viral genome. However, O/SKR/AS/2002 has an intact 3A coding region.     

Studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) can be spread by a variety of mechanisms and the rate of spread, the incubation period and the severity of disease depend on a multitude of parameters, including the strain of virus, the dose received, the route of introduction, the animal species and the husbandry conditions. More knowledge with regard to these parameters is urgently needed to improve resource-efficient disease control. This report describes detailed studies of FMDV load, excretion and transmission in pigs infected with FMDV O UKG 2001, O TAW 1997 and C Noville virus and in cattle infected with the O UKG 2001 virus to facilitate use of a "FMDV load framework" for the assessment of transmission risks. Virus replicated rapidly in pigs and cattle exposed by direct contact. The mean incubation period was around 3-4 days for cattle-to-cattle and 1-3 days for pig-to-pig transmission, depending on the intensity of contact. The results confirmed that a strong relation exists between dose and length of incubation period. Clinical disease was severe in pigs but relatively mild in inoculated cattle; contact infection of cattle appeared to increase the severity of lesions. FMDV RNA was recovered in nasal and mouth swabs from inoculated animals soon after they developed a viraemia and probably reflected the early production and excretion of virus. FMDV RNA in nasal and mouth swabs from contact animals could be detected several days before they showed other signs of infection, indicating the possibility of detecting exposed animals during the incubation period. FMDV RNA could also be detected in swab samples after the viraemic phase. This may have represented background environmental virus that had been trapped in the respiratory tract and mouth. Alternatively, it may have indicated a somewhat slower clearance or half-life of viral RNA or an extended low level of FMDV replication at these sites. The pattern of FMDV RNA concentrations in pigs was closely similar to that in cattle, but the amounts of FMDV RNA were higher.     

Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

Summary Treatment of porcine kidney (PK-15) cells with either interferon-gamma (IFN-γ) or endosomal- lysosomal system acidification inhibitors increases replication of porcine circovirus type 2 (PCV2). In the present study, the effect of a combination of these treatments on the number of infected cells and virus yield was tested. The number of PCV2 (Stoon-1010)-infected PK-15 cells increased in cells treated with ammonium chloride (445 ± 39% increase), IFN-γ (446 ± 8%), ammonium chloride + IFN-γ (1721 ± 283%), chloroquine diphosphate (1037 ± 121%), chloroquine diphosphate + IFN-γ (2199 ± 255%), monensin (950 ± 178%) and monensin + IFN-γ (1948 ± 60%). Combined IFN-γ and endosomal-lysosomal system acidification inhibitors increased PCV2 yield by up to 50 times compared to untreated PK-15. This augmented virus replication in PK-15 cells may be helpful in the production of PCV2 vaccines. Correspondence: Hans J. Nauwynck, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium     

Cytokine production by human leukocytes with different expressions of natural antiviral immunity and the effect of antibodies against interferons and TNF-±

Introduction: Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines. Materials and Methods: Samples of blood were taken from healthy blood donors and from persons with frequent infections of the upper respiratory system. PMBCs were isolated by gradient centrifugation. Vesicular stomatitis virus (VSV) was used as the indicatory virus to infect PMBCs. The cytokines: IFN, TNF, and IL-6 were titrated by biological methods and IL-10 by ELISA. Results: Blood donors were divided for two groups: those with VSV-resistant and those with VSV-sensitive PMBCs and secretion of cytokines by them was compared. The resistant PMBCs produced more cytokines than the sensitive ones. A statistically significant difference, was found only in the case of the IFNs. To examine the contribution of IFNs and TNF in maintaining resistance, leukocytes from both groups were treated with specific anti-cytokine antibodies. The authors’ previous study showed that the elimination of spontaneous IFN-α, IFN-β, IFN-γ, and TNF-α from resistant leukocytes resulted in increased VSV replication This indicates the important role of cytokines. In VSV-sensitive PMBCs, anti-IFN-α showed the opposite effect (decreased virus replication). In the absence of spontaneous IFN-α, disturbances in cytokine production were observed. Conclusions: Complete resistance of PMBC to VSV infection is accompanied by higher cytokine release, The paradoxical effect of anti-IFN-α on virus replication in leukocytes sensitive to viral infection may be attributed to changes in the cytokine profile balance, i.e. high TNF production by VSV-infected leukocytes and a complete reduction of IL-6 production.     

Experimental infection of cattle and goats with a foot-and-mouth disease virus isolate from the 2010 epidemic in Japan

In this study, we carried out experimental infections in cattle and goats using a foot-and-mouth disease virus (FMDV) isolate from the 2010 epidemic in Japan to analyze clinical manifestations, virus-shedding patterns and antibody responses in the animals. We found that the FMDV O/JPN/2010 isolate is virulent in cattle and goats, produces clinical signs, is spread efficiently by direct contact within the same species, and is persistently infectious in cattle. Quantitative analysis of levels of viral RNA in the tissues of cattle and goats infected with the isolate showed that the pharyngeal region is an important major target of the FMDV O/JPN/2010. Time course data of viral loads, excretion and transmission of the FMDV O/JPN/2010 in this study are key in providing quantitative data essential for epidemiological investigation and risk analysis in relation to disease controls.     

Comparison of interferon-γ response between tuberculosis and non-tubercular Pneumonia

Objective and design: Macrophages aided by interferon-gamma (IFN-γ) are vital to controlling Mycobacterium tuberculosis (M. tuberculosis) infection. Although numerous studies have compared IFN-γ response between tubercular patients and healthy controls, no studies have investigated IFN-γ response in patients with pulmonary tuberculosis and non-tubercular pneumonia. The aim of this work was to examine the difference in IFN-γ response between patients with tuberculosis and non-tubercular pneumonia. Methods: IFN-γ production was detected based on the difference in supernatants between non-stimulated and stimulated peripheral blood mononuclear cells by phytohemagglutinin in 83 tubercular patients and 47 patients with pneumonia. Presence of a cavity on chest radiography and co-morbidities of pneumoconiosis, bronchiectasis, liver cirrhosis, renal failure on hemodialysis, diabetes mellitus (DM) and lung cancer were recorded for analysis. Results: Interferon-gamma response, DM and a cavity on chest radiography were independent factors for predicting active pulmonary tuberculosis. Interferon-gamma response was decreased in patients with pulmonary tuberculosis compared with that in patients with non-tubercular pneumonia. Notably, M. tuberculosis infection was the principal factor correlated with IFN-γ response. Conclusion: The IFN-γ response was principally affected by M. tuberculosis infection and not by other co-morbidities. Further study is required to identify the mechanism of decreased IFN-γ production. Received 19 May 2006; returned for revision 28 June 2006; accepted by G. Wallace 20 July 2006     

A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens

Changes in the NP gene of H5N1 highly pathogenic avian influenza (HPAI) viruses have previously been shown to affect viral replication, alter host gene expression levels and affect mean death times in infected chickens. Five amino acids at positions 22, 184, 400, 406, and 423 were different between the two recombinant viruses studied. In this study, we individually mutated the five amino acids that differed and determined that the difference in virus pathogenicity after NP gene exchange was a result of an alanine to lysine change at position 184 of the NP protein. Infection with viruses containing a lysine at NP 184 induced earlier mortality in chickens, increased virus titers and nitric oxide levels in tissues, and resulted in up-regulated host immune genes, such as α-interferon (IFN-α), γ-interferon (IFN-γ), orthomyxovirus resistance gene 1 (Mx1), and inducible nitric oxide synthase (iNOS). This study underlines the importance of the NP in avian influenza virus replication and pathogenicity.     

Establishment of cell lines persistently infected with foot-and-mouth disease virus

Cell lines persistently infected with foot-and-mouth disease virus (FMDV) have been established by growth of BHK-21 (c-13) or IBRS-2 (c-26) that survived standard cytolytic infections with FMDV. They maintain cytoplasmic FMDV RNA sequences, as shown by dot blot hybridization tests, using cloned FMDV cDNA as probes. Cell line C1-BHK-Rc1 was derived by infection of cloned BHK-21 c1 cells and plaque-purified FMDV C-S8 c1. Indirect immunofluorescence assays indicated the presence of FMDV antigens. It was resistant to superinfection by FMDV C-S8 c1, O-S7, or A5, but not by encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or Semliki forest virus (SFV). Infectious FMDV was detected in the culture medium only up to cell passage 65. The virus isolated from C1-BHK-Rc1 cells showed decreased plaque size and diminished yield in infections at 42 degrees. Multiple mutations in the intracellular FMDV RNA have been detected by T1 oligonucleotide fingerprinting of genomic RNA segments hybridized to FMDV cDNA fragments. At late cell passages, when no infectious FMDV is detected, cells continue to express viral antigens and FMDV RNAs with deletions of up to 3 kb have been identified by Northern blot analysis. We conclude that persistent infections of cell cultures with FMDV are readily established and that multiple genetic and phenotypic variations occur in the virus during persistence.