Laura C. Miller

Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

Abstract The pathogenesis of Type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in 10-week old swine in the United States was investigated. rJXwn06, rescued from an infectious clone of Chinese HP-PRRSV, replicated in swine with at least 100-fold increased kinetics over U.S. strain VR-2332. rJXwn06 caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to HP-PRRSV than to those infected with VR-2332. Novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of HP-PRRSV to rapidly transmit between animals. Furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in HP-PRRSV infected swine, and showed that contact swine differed in the degree of cytokine response.

Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains

Porcine reproductive and respiratory syndrome virus (PRRSV) is a ubiquitous and costly virus that exhibits substantial sequence and virulence disparity among diverse isolates. In this study, we compared the whole genomic sequence and virulence of 4 Type 2 PRRSV isolates. Among the 4 isolates, SDSU73, MN184, and NADC30 were all clearly more virulent than NADC31, and among the 3 more virulent isolates, there were subtle differences based on viral replication, lung lesions, lymphadenopathy, febrile response, decreased weight gains, and cytokine responses in the lung. Lesions consistent with bacterial bronchopneumonia were present to varying degrees in pigs infected with PRRSV, and bacteria typically associated with the porcine respiratory disease complex were isolated from the lung of these pigs. Genomic sequence evaluation indicates that SDSU73 is most similar to the nucleotide sequence of JA142, the parental strain of Ingelvac(®) PRRS ATP, while the nucleotide sequences of NADC30 and NADC31 are more similar to strain MN184. Both the NADC30 and NADC31 isolates of PRRSV, isolated in 2008, maintain the nonstructural protein 2 (nsp2) deletion seen in MN184 that was isolated in 2001, but NADC31 has two additional 15 and 36 nucleotide deletions, and these strains are 8-14% different on a nucleotide basis from the MN184 strain. These results indicate that newer U.S. Type 2 strains still exhibit variability in sequence and pathogenicity and although PRRSV strains appear to be reducing the size of the nsp2 over time, this does not necessarily mean that the strain is more virulent.

Role of Toll-Like Receptors in Activation of Porcine Alveolar Macrophages by Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Control of virus replication initially depends on rapid activation of the innate immune response. Toll-like receptor (TLR) ligands are potent inducers of innate immunity against viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense RNA virus, initiates infection in porcine alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of single-stranded RNA and double-stranded RNA (dsRNA) intermediates in eliciting host immunity, we sought to determine if TLRs, particularly those that respond to viral molecular patterns, are involved in PRRSV infection. Activation of TLR3 in PAMs with dsRNA increased gene expression for alpha interferon and suppressed PRRSV infectivity. In contrast, TLR4 activation by the treatment of PAMs with lipopolysaccharide did not influence PRRSV infectivity.

Role of the Cytoplasmic Domain of the β-Subunit of Integrin αvβ6 in Infection by Foot-and-Mouth Disease Virus

ABSTRACT Field isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, αvβ6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the αvβ6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the β6 cytoplasmic domain on infection. Our results show that although loss of the β6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in αvβ6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.

Evaluation of a Real-Time PCR Kit for Detecting Escherichia coli O157 in Bovine Fecal Samples

ABSTRACT A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g−1 in artificially inoculated bovine feces following enrichment.

Chinese and Vietnamese strains of HP-PRRSV cause different pathogenic outcomes in United States high health swine

An infectious clone of a highly pathogenic PRRSV strain from Vietnam (rSRV07) was prepared and was demonstrated to contain multiple amino acid differences throughout the genome when compared to Chinese highly pathogenic PRRSV strain rJXwn06. Virus rescued from the rSRV07 infectious clone was compared to rJXwn06 and US Type 2 prototype strain VR-2332 to examine the effects of virus genotype and phenotype on in vitro growth, and virus challenge dose on in vivo pathogenicity and host response. After swine inoculation at high- and low-doses of virus, rSRV07 was shown to replicate to an approximately 10-fold lower level in serum than rJXwn06, produced lower body temperatures than rJXwn06 and resulted in decreased mortality. Furthermore, a 9-plex cytokine panel revealed that the cytokine responses varied between different strains of PRRSV, as well as between tissues examined and by inoculum dose.

Utility of a Panviral Microarray for Detection of Swine Respiratory Viruses in Clinical Samples

ABSTRACT Several factors have recently converged, elevating the need for highly parallel diagnostic platforms that have the ability to detect many known, novel, and emerging pathogenic agents simultaneously. Panviral DNA microarrays represent the most robust approach for massively parallel viral surveillance and detection. The Virochip is a panviral DNA microarray that is capable of detecting all known viruses, as well as novel viruses related to known viral families, in a single assay and has been used to successfully identify known and novel viral agents in clinical human specimens. However, the usefulness and the sensitivity of the Virochip platform have not been tested on a set of clinical veterinary specimens with the high degree of genetic variance that is frequently observed with swine virus field isolates. In this report, we investigate the utility and sensitivity of the Virochip to positively detect swine viruses in both cell culture-derived samples and clinical swine samples. The Virochip successfully detected porcine reproductive and respiratory syndrome virus (PRRSV) in serum containing 6.10 × 102 viral copies per microliter and influenza A virus in lung lavage fluid containing 2.08 × 106 viral copies per microliter. The Virochip also successfully detected porcine circovirus type 2 (PCV2) in serum containing 2.50 × 108 viral copies per microliter and porcine respiratory coronavirus (PRCV) in turbinate tissue homogenate. Collectively, the data in this report demonstrate that the Virochip can successfully detect pathogenic viruses frequently found in swine in a variety of solid and liquid specimens, such as turbinate tissue homogenate and lung lavage fluid, as well as antemortem samples, such as serum.

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library.ResultsMajor changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold.ConclusionsThe magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

The Presence of Alpha Interferon at the Time of Infection Alters the Innate and Adaptive Immune Responses to Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating and costly diseases to the swine industry worldwide. Overall, the adaptive immune response to PRRS virus (PRRSV) is weak, which results in delayed elimination of virus from the host and inferior vaccine protection. PRRSV has been shown to induce a meager alpha interferon (IFN-α) response, and we hypothesized that elevated IFN-α levels early in infection would shorten the induction time and increase elements of the adaptive immune response. To test this, we measured both antibody and cell-mediated immunity in pigs after the administration of a nonreplicating human adenovirus type 5 vector expressing porcine IFN-α (Ad5–pIFN-α) at the time of PRRSV infection and compared the results to those for pigs infected with PRRSV alone. Viremia was delayed, and there was a decrease in viral load in the sera of pigs administered the Ad5–pIFN-α. Although seroconversion was slightly delayed in pigs receiving Ad5–pIFN-α, probably due to the early reduction in viral replication, little difference in the overall or neutralizing antibody response was seen. However, there was an increase in the number of virus-specific IFN-γ-secreting cells detected in the pigs receiving Ad5–pIFN-α, as well as an altered cytokine profile in the lung at 14 days postinfection, indicating that the presence of IFN-α at the time of infection can alter innate and adaptive immune responses to PRRSV.

Macrophage Polarization in Virus-Host Interactions

Macrophage involvement in viral infections and antiviral states is common. However, this involvement has not been well-studied in the paradigm of macrophage polarization, which typically has been categorized by the dichotomy of classical (M1) and alternative (M2) statuses. Recent studies have revealed the complexity of macrophage polarization in response to various cellular mediators and exogenous stimuli by adopting a multipolar view to revisit the differential process of macrophages, especially those re-polarized during viral infections. Here, through examination of viral infections targeting macrophages/monocytic cells, we focus on the direct involvement of macrophage polarization during viral infections. Type I and type III interferons (IFNs) are critical in regulation of viral pathogenesis and host antiviral infection; thus, we propose to incorporate IFN-mediated antiviral states into the framework of macrophage polarization. This view is supported by the multifunctional properties of type I IFNs, which potentially elicit and regulate both M1- and M2-polarization in addition to inducing the antiviral state, and by the discoveries of viral mechanisms to adapt and modulate macrophage polarization. Indeed, several recent studies have demonstrated effective prevention of viral diseases through manipulation of macrophage immune statuses.