Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus , targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose 50 (TCID 50 )/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID 50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ∼10 7 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ∼10 6.5 TCID 50 /ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID 50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.
Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi.
Rift Valley fever virus (RVFV) is a zoonotic arbovirus in the family Phenuiviridae. In Africa, outbreaks of Rift Valley fever (RVF) usually occur following periods of heavy rainfall that prompt population surges in mosquito vectors. RVF causes significant mortality in ruminants, especially in ovine fetuses and neonates (up to 100%), while human infections cause mild febrile illness to hemorrhagic fever and death. Macrophages play a critical role as phagocytes in the innate immune system and produce cytokines that stimulate the adaptive immune system. As in other arboviral diseases, macrophages and dendritic cells are thought to be early infection targets of RVFV, enabling the virus to suppress the immune response. To further investigate their role in RVF early immune response and cytokine induction, bovine peripheral blood mononuclear cell-derived macrophages were infected with RVFV MP-12 (an attenuated vaccine strain) in the presence and absence of Culex tarsalis saliva. We hypothesized that in macrophages treated with Culex saliva and RVFV will have increased transcription of Th2-associated cytokines compared to those infected with virus only due to immunomodulatory proteins present in mosquito saliva. The macrophage lineage of the cells and RVFV infection were confirmed by dual label immunofluorescence for IBA-1 (macrophage marker) and RVFV nucleoprotein. Total RNA was extracted from 3 biological replicates of macrophages treated respectively with saliva only, MP-12 only, MP-12 and saliva at 0, 8 and 24 hours post-infection (hpi) as well as untreated controls. RNA was reverse-transcribed and the resulting cDNA analyzed by qPCR of four housekeeping genes (ActinB, H3F3, PPIA, YWHAZ), 6 early response genes (TLR3, TLR7, TLR8, RIG1, MDA5, IFNa) and 9 cytokine genes (IL4, IL6, IL10, IL12b, IL18, IFNg, TNFa, IL1a, IL1b). Relative gene expression (RGE) was determined by the method described by Vandesompele which uses multiple reference genes for normalization and does not assume equal PCR efficiencies for the reference genes and genes of interest. RGEs for each target were log-transformed and analyzed with two-way ANOVAs and Tukey's post-hoc test with p-value correction for multiple tests to evaluate statistical differences between treatments and time points. There was no statistical increase in transcription of Th2-associated cytokines when bovine macrophages were infected with MP-12 in the presence of saliva at any of the time points when compared to virus only. In contrast, significant differences in gene expression were present when infected samples were compared to uninfected controls. At 8 hpi, transcription was significantly different than controls for all virus infected samples for TLR3, RIG1, MDA and IFNa as well as IL6, IL10, IL12b, IL18, TNFa, IL1a and IL1b. This trend continued at 24 hpi with the exception of IL12b. While Culex saliva may not enhance suppression of the early immune response in MP-12 infection, it may still have a role in virulent RVFV strain infections.
Following a summer of severe drought and abnormally high temperatures, a major outbreak of EHDV occurred during 2012 in the USA. Although EHDV-1, -2 and -6 were isolated, EHDV-2 was the predominant virus serotype detected during the outbreak. In addition to large losses of white-tailed deer, the Midwest and northern Plains saw a significant amount of clinical disease in cattle. Phylogenetic analyses and sequence comparisons of newly sequenced whole genomes of 2012 EHDV-2 cattle isolates demonstrated that eight of ten EHDV-2 genomic segments show no genetic changes that separate the cattle outbreak sequences from other EHDV-2 isolates. Two segments, VP2 and VP6, did show several unique genetic changes specific to the 2012 cattle outbreak isolates, although the impact of the genetic changes on viral fitness is unknown. The placement of isolates from 2007 and 2011 as sister group to the outbreak isolates, and the similarity between cattle and deer isolates, point to environmental variables as having a greater influence on the severity of the 2012 EHDV outbreak than viral genetic changes.
Rift Valley fever virus, a zoonotic arbovirus, poses major health threats to livestock and humans if introduced into the United States. White-tailed deer, which are abundant throughout the country, might be sentinel animals for arboviruses. We determined the susceptibility of these deer to this virus and provide evidence for a potentially major epidemiologic role.
To test the hypothesis that RNA interference (RNAi) imposes diversifying selection on RNA virus genomes, we quantified West Nile virus (WNV) quasispecies diversity after passage in Drosophila cells in which RNAi was left intact, depleted, or stimulated against WNV. As predicted, WNV diversity was significantly lower in RNAi-depleted cells and significantly greater in RNAi-stimulated cells relative to that in controls. These findings reveal that an innate immune defense can shape viral population structure.
