Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2.

Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.

Superinfection Exclusion of Alphaherpesviruses Interferes with Virion Trafficking.

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection interferes with secondary viral infections within that same cell. Although SIE has been observed across many viruses, it has remained relatively understudied. A recently characterized glycoprotein D (gD)-independent SIE of alphaherpesviruses presents a novel mechanism of coinfection restriction for herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV). In this study, we evaluated the role of multiplicity of infection (MOI), receptor expression, and trafficking of virions to gain greater insight into potential mechanisms of alphaherpesvirus SIE. We observed that high-MOI secondary viral infections were able to overcome SIE in a manner that was independent of receptor availability. We next assessed virion localization during SIE through live microscopy of fluorescently labeled virions and capsid assemblies. Analysis of these fluorescent assemblies identified changes in the distribution of capsids during SIE. These results indicate that SIE during PRV infection inhibits viral entry or fusion while HSV-1 SIE inhibits infection through a postentry mechanism. Although the timing and phenotype of SIE are similar between alphaherpesviruses, the related viruses implement different mechanisms to restrict coinfection. IMPORTANCE Most viruses utilize a form of superinfection exclusion to conserve resources and control population dynamics. gD-dependent superinfection exclusion in alphaherpesviruses is well documented. However, the undercharacterized gD-independent SIE provides new insight into how alphaherpesviruses limit sequential infection. The observations described here demonstrate that gD-independent SIE differs between PRV and HSV-1. Comparing these differences provides new insights into the underlying mechanisms of SIE implemented by two related viruses.

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein.

Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.

The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes.

The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744-759, 747-774 and/or 756-771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371-1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499-600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.

Immunogenicity of a recombinant parapoxvirus expressing the spike protein of Porcine epidemic diarrhea virus.

The parapoxvirus Orf virus (ORFV), has long been recognized for its immunomodulatory properties in permissive and non-permissive animal species. Here, a new recombinant ORFV expressing the full-length spike (S) protein of Porcine epidemic diarrhea virus (PEDV) was generated and its immunogenicity and protective efficacy were evaluated in pigs. The PEDV S was inserted into the ORFV121 gene locus, an immunomodulatory gene that inhibits activation of the NF-κB signalling pathway and contributes to ORFV virulence in the natural host. The recombinant ORFV-PEDV-S virus efficiently and stably expressed the PEDV S protein in cell culture in vitro. Three intramuscular (IM) immunizations with the recombinant ORFV-PEDV-S in 3-week-old pigs elicited robust serum IgG, IgA and neutralizing antibody responses against PEDV. Additionally, IM immunization with the recombinant ORFV-PEDV-S virus protected pigs from clinical signs of porcine epidemic diarrhoea (PED) and reduced virus shedding in faeces upon challenge infection. These results demonstrate the suitability of ORFV121 gene locus as an insertion site for heterologous gene expression and delivery by ORFV-based viral vectors. Additionally, the results provide evidence of the potential of ORFV as a vaccine delivery vector for enteric viral diseases of swine. This study may have important implications for future development of ORFV-vectored vaccines for swine.

Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus.

BACKGROUND: A novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection. RESULTS: The nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes. CONCLUSION: These new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies.

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.