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Highlights • PRRSV induces the formation of bona fide stress granules (SGs) at late time points post-infection. • PRRSV-induced SGs are closely associated with VRCs, yet not required for efficient viral replication. • Arrest of cellular translation coincides with SG formation in PRRSV-infected cells. Abstract Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions. [ABSTRACT FROM AUTHOR]

• PRRSV induces UPR through all 3 signaling branches, including PERK, IRE1a and ATF6. • Induction of UPR significantly suppresses PRRSV replication and RNA synthesis. • The PERK arm of the UPR modulates the PRRSV-induced cytokine responses. Replication of most RNA viruses is closely associated with the endoplasmic reticulum (ER) within permissive cells. As such, viruses often induce tremendous amounts of stress on cells during viral replication. To cope with the stress, cells initiate the unfolded protein response (UPR) to promote cellular survival. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen, was previously shown to induce cellular stress at late stages post-infection resulting in the formation of stress granules (SGs). Here in this study, we demonstrate that PRRSV also induces additional cellular response pathways, including the UPR. Confocal microscopy analysis demonstrated significant morphological changes in the ER of PRRSV-infected cells, indicative of pronounced ER stress. Further investigation revealed induction of all three branches of the UPR, including eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), serine-threonine protein kinase/endoribonuclease (IRE1), and cyclic AMP-dependent transcription factor (ATF6). Activation of these sensors resulted in significant transcriptional upregulation of downstream UPR effectors. Additionally, UPR activation was shown to be detrimental to PRRSV replication, as treatment of cells with chemical ER stress inducers potently suppressed viral replication and RNA synthesis. Further investigation into the molecular mechanisms of UPR suppression of PRRSV replication revealed that PERK exacerbates the PRRSV-induced cytokine response. Collectively, these results demonstrate that PRRSV infection induces UPR activation through all three branches, and that UPR signaling may play a role in PRRSV pathogenesis. The results of this study further our understanding of the underlying molecular mechanisms of PRRSV replication and host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Background: Genotype 3 hepatitis E virus (HEV) infection is generally associated with mild disease. However, recently eight genotype 3 HEV isolates were identified from patients with severe hepatitis. Importantly, three mutations (S605P, I978V and V1213A) in these genotype 3 isolates were found to be typical of genotype 4 HEV, which is sometime associated with more severe hepatitis. Therefore in this study we seek to determine if these unique mutations contribute to enhanced virus replication and thus potentially severe disease. Methods: In the lack of an efficient cell culture system to study the effect of mutations on HEV replication, we developed a genotype 3 HEV replicon with Renilla luciferase (Rluc) as reporter and subsequently used it to construct numerous mutants, including swMu-1 (V1213A), swMu-2 (Q1246H), swMu-3 (V1213A and Q1246H), swMu-4 (S605P and I978V), and swMu-5 (V1213A, S605P and I978V). RNA transcripts from mutant replicons were transfected into Huh7 S10–3 liver cells to measure the effect of mutations on HEV replication efficiency. Results: The results showed that the V1213A mutant had the highest reduction in HEV replication efficiency than other mutants. The V1213A and S605P + I978V mutations have a cumulative, if not synergistic, effect on HEV replication. The Q1246H mutant decreased HEV replication compared to the wild-type HEV Rluc replicon but replicated better than the V1213A mutant. The amino acid residue V1213 favors the replication of both genotypes 3 and 4 HEV strains, but not genotype 1 HEV. Conclusion: The results suggested that the V1213A mutation reduced HEV replication, but is likely not associated with the reported severe hepatitis caused by genotype 3 HEV isolates containing this mutation. [ABSTRACT FROM AUTHOR]

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge. [ABSTRACT FROM AUTHOR]

Highlights • Vaccination of sows with an inactivated MRV3 vaccine induced only a low level humoral response. • Maternal immunity acquired from vaccinated sows reduces MRV3 replication. • The U.S. MRV3 infects but only causes very mild diarrhea in pigs. Abstract A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs. [ABSTRACT FROM AUTHOR]

The ORF3 protein of hepatitis E virus (HEV) contains a “PSAP” amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting heterologous late domain motifs (PPPY, YPDL, and PSAA) for the HEV ORF3 late domain (PSAP), we demonstrated that deviation from the PSAP motif reduces virus release as measured by viral RNA in culture media. Virus release could not be restored by insertion of a heterologous late domain motif or by supplying wild-type ORF3 in trans , suggesting that the HEV PSAP motif is required for viral exit which cannot be bypassed by the use of alternative heterologous late domains. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) infection is a cause of hepatitis in humans worldwide and has been associated with a case-fatality rate of up to 30% in pregnant women. Recently, persistent and chronic HEV infections have been recognized as a serious clinical problem, especially in immunocompromised individuals. To date, there are no FDA-approved HEV-specific antiviral drugs. In this study, we evaluated antisense peptide-conjugated morpholino oligomers (PPMO) designed against HEV genomic sequences as potential HEV-specific antiviral compounds. Two genetically-distinct strains of human HEV, genotype 1 Sar55 and genotype 3 Kernow-C1, isolated from patients with acute and chronic hepatitis, respectively, were used to evaluate inhibition of viral replication by PPMO in liver cells. The anti-HEV PPMO produced a significant reduction in the levels of HEV RNA and capsid protein, indicating effective inhibition of HEV replication. PPMO HP1, which targets a highly conserved sequence in the start site region of ORF1, was also effective against the genotype 3 Kernow-C1 strain in stably-infected HepG2/C3A liver cells. The antiviral activity observed was specific, dose-responsive and potent, suggesting that further exploration of PPMO HP1 as a potential HEV-specific antiviral agent is warranted. [ABSTRACT FROM AUTHOR]