Investigating the Infection and Persistence of Sindbis Virus in Host Neurons

Sindbis virus, an Alphavirus in the Togaviridae family, is an enveloped, single-stranded positive-sense RNA virus. Found mostly in parts of Africa, Australia, Egypt, Philippines, and Northern Europe – it is known to cause Ockelbo or Pogosta disease [1]. This disease is characterized by the sudden onset of fever, headache, and arthralgia; followed by arthritis, rash, fatigue, and muscle pain. The symptoms are gone within 14 days, though cases have shown joint pain to last from 12 months to 2 and a half years [4]. Common to several other viruses, Sindbis is transmitted from birds (its reservoir) to humans via an arthropod vector, the mosquito [5]. The transmission and symptoms of Sindbis virus are well documented. Once inside a human host, however, much less is known. When Sindbis enters the body, its target is the nervous system. The mechanism of not only neuroinvasion, but neurovirulence and persistence is unknown. Both the virus and the host play important roles in the progression of a neurological, viral infection [2,6]. The aim of this study is to investigate the infection and persistence of Sindbis virus in an environment that replicates the neurological system of a rodent host using iCHIP (in-vitro Chip-Based Human Investigational Platform). The multi-electrode array (MEA) on the iCHIP is used to detect signaling between the seeded neurons and to map the effect of the virus on them [3]. After detection, samples are taken at intervals and tested to observe persistence of both live virus and viral RNA. We hypothesize that the samples will show evidence of viable virus for the first couple weeks of sampling via a TCID50 assay, but then observe drop in viral population while the levels of viral RNA remain constant

The Effect of Environmental Selection Pressure on the Rate of Recombination to an Advantageous Receptor Mutation in Bovine Coronavirus

Bovine Coronavirus (BCoV) is an important analogue in understanding the effectiveness of zoonotic, single-stranded, positive sense RNA viruses. Many of the most recent viral outbreaks have been attributed to RNA viruses that have one, or more, animal reservoirs [1]. BCoV is such a great candidate for studying these types of viruses because they are from the family Coronaviridae, which also contains the viruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The goal of this study was to observe changes in genetic makeup of the virus’ outer membrane Spike protein via recombination between two BCoV strains. The Nebraska strain and the Mebus strain were co-infected into a human cell line (HRT-18) in a 1 to 100 ratio and their rate of infection recorded. The Nebraska strain contains a 12 nt insert in its Spike protein which has been hypothesized to allow for trypsin-independent cell entry [2]. Like SARS, BCoV has been found to require proteolytic cleavage by host trypsin in order for it to infect its host. To test the ability of the coronavirus strains to recombine and transfer this insert through template swapping, some cell lines were infected with the virus strains and incubated in media containing trypsin and trypsin-free media. RNA extraction of the virus present in the supernatant from the infected cells and subsequent RT-PCR and TaqMan PCR was used to determine the level of successfully infecting virus of each strain. The study concluded that, even at small levels, the presence of the Nebraska strain allowed recombination to occur and therefore boost the speed of infection and replication of the Mebus strain. Specific primers also indicated that the Mebus strain acquired the insert through template swapping. This results points out the importance of understanding the quasispecies of emerging viruses

Ancient Pathogen Dna in Archaeological Samples Detected With a Microbial Detection Array

Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time

Listeria monocytogenes serotype identification by PCR

Serotyping is a universally accepted subtyping method for Listeria monocytogenes. Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a "gold standard" for comparing isolates analyzed in different labs and with different techniques. Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system. Serotyping PCR primers were designed from variable regions of the L. monocytogenes genome. Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122 L. monocytogenes strains into five serotype groups [1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c]. Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively

Mixed-Genome Microarrays Reveal Multiple Serotype and Lineage-Specific Differences among Strains of Listeria monocytogenes

Epidemiological studies and analysis of putative virulence genes have shown that Listeria monocytogenes has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of L. monocytogenes . We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that L. monocytogenes has a strongly clonal population structure ( I A = 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of L. monocytogenes . Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes

Serotyping of Listeria monocytogenes by Enzyme-Linked Immunosorbent Assay and Identification of Mixed-Serotype Cultures by Colony Immunoblotting

Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining