PEDV and PDCoV Pathogenesis: The Interplay Between Host Innate Immune Responses and Porcine Enteric Coronaviruses

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host’s first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication

Conserved Determinants of Enhanced CCR5 Binding in the Human Immunodeficiency Virus Subtype D Envelope Third Variable Loop

Human immunodeficiency virus 1 subtype D (HIV-1D) contributes to a significant portion of the HIV-1 disease burden in eastern and central Africa, and is associated with more rapid disease progression. Its viral envelope sequences, particularly in the third variable region (V3), are highly divergent from other major subtypes yet have rarely been studied to date. We evaluated the V3 and select bridging sheet residues of the HIV-1D 94UG114 envelope by alanine-scanning mutagenesis to determine the residues involved in CCR5 usage conservation in the face of sequence variability. We found most single alanine mutations capable of abolishing CCR5 binding, suggesting binding contacts that are highly sensitive to mutation. Despite drastic binding defects across the board, most mutants mediated fusion at or near wild-type levels, demonstrating an ability to accommodate changes in CCR5 affinity while maintaining the ability to complete entry. Three of the alanine mutations did not abolish CCR5 binding but rather resulted in enhanced CCR5 binding. The positions of these residues were found to be conserved between strains of two subtypes, revealing similar V3 elements that suggest a conservation of constraints in V3 loop conformation

Induction of immune responses following immunization with VSV-ΔP expressing H1 and H7 HA.

<p>BALB/c mice (5 mice/group) were intravenously injected with 1×10⁷ pfu of VSVs in 100 μl at days 0 and 21. At day 28, sera were harvested to determine for H1N1-specific and H7N9-specific IgG levels at a titer of 5,120. Values are averages of two independent experiments with error bars showing SD.</p

P gene deletion attenuated replication of recombinant virus.

<p>(A) BHK-21 and BHK-P cells were infected with VSV-ΔP at an MOI of 1 and observed for cytopathic effects (CPE). Infected cells were then subjected to flow cytometry to quantify the percentage of mCherry-expressing cells. The pictures are representative of triplicate samples. (B) BHK-P cells were infected with VSV or VSV-ΔP at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing standard deviation (SD). (C) Viruses were serially diluted for plaque titration, and plaques were stained with neutral red for visualization. Representative images of VSV and VSV-ΔP were selected for plaque size comparison.</p

Decreased lethality in mice after intracranial injection with VSV-ΔP.

<p>ICR mice (5 mice/group) were lightly anesthetized with ether and then intracranially injected with either PBS or 1×10⁴ pfu of VSVs. (A) Body weight was measured daily and (B) survival was plotted using the Kaplan-Meier survival curve. Values are averages of five mice with error bars showing SD and are representative of two independent experiments. NS, not significant; *, p<0.5; **, p<0.05.</p

Expression of foreign antigens on the viral surface.

<p>(A) BHK-P cells were infected with VSVs at an MOI of 0.1. Supernatants were harvested at the indicated time points and were assessed using a MUNANA-based assay, (B) HA assay and (C) HAI assay. Supernatant and cell lysates were subjected to Western blot analysis using a β-actin monoclonal antibody as the primary antibody. Values are averages of triplicate wells with error bars showing SD. (D) 1×10⁷ purified VSVs were lysed and subjected to Western blot analysis using an HA (H1N1) polyclonal antibody and serum from VSV-immunized mice as the primary antibody. (E) To study the incorporation of the H7 HA gene in VSVs, RNA were extracted from purified VSVs and subjected to RT-PCR using primers specific for the N/M fragment or H7 HA genes. (F) BHK-P cells were infected with VSVs at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing SD. A.U., arbitrary units.</p

Construction of recombinant VSVs with P gene deletion.

<p>(A) The top schematic shows the VSV genome layout and the naturally occurring restriction sites used for cloning. To construct VSV-ΔP, the VSV genome was digested with <i>Eco</i>RV, religated in the absence of the P gene fragment and the mCherry gene was then inserted between the G and L genes. The mCherry gene was replaced by HN, H1 or H7 HA to generate VSV-ΔP-HN, VSV-ΔP-HA1 and VSV-ΔP-HA7, respectively. (B) Supporting cells (BHK-P) were constructed by transducing BHK-21 cells with lentivirus bearing the P gene, and the selected clone expressing the emerald fluorescent protein was examined by (C) bright field and (D) fluorescence imaging. (E) BHK-P cells were infected with VSV or VSV-ΔP, and supernatants were harvested for viral genome extraction and RT-PCR. RBZ, hepatitis virus delta ribozyme; T7, T7 RNA polymerase leader; T7 ter, T7 terminator; LTR, long terminal repeat; ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; SFFV, spleen focus-forming virus (promoter); WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔU3, U3 deletion.</p

Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.

<p>Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.</p