CHIKV Case Study

In the article, we learn the Cache Valley virus is spreading quickly through mosquitos. In this unit we learned that viruses with RNA in their nucleic acid reproduce a lot faster than viruses that have DNA in their nucleic acid, therefore the Cache Valley virus probably has RNA in its nucleic acid. Throughout the unit we learned viruses can spread through many types of mediums such

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

The predicted antigenic regions were chimerically expressed in a bacterial system. Briefly, two sets of PCR primers were designed according to gD gene sequence available in GenBank (accession number: NC_001847) to amplify gene fragments encoding aa 20-160 (nucleotide 118953～119375, Δ gD1) and aa 257-344 (nucleotide 119664～119927, Δ gD2), respectively. The primers sequences for amplification of Δ gD1(P1 and P2) and Δ gD2 (P3 and P4) were listed in Table. 1.

They were run at 1X 94ºC for 3 minutes, 30X at 94ºC for 30 seconds; 50ºC for 30 seconds; 72ºC for 45 second and 1X at 72ºC for 5 minutes. The PCR reactions took about 1 hour and 30 minutes to complete. The PCR products, were then purified by removing the leftover primers, nucleotides and salts. 250 µl of Buffer BB were added to Tube B and the mixture was pipetted into a spin column. The mixture was centrifuged for 30 seconds at room temperature. Then 2 cycles were completed at 30 seconds each with 200 µl of Buffer WB to remove any impurities. Then 25µl of Buffer EB were added to the tube to release the pure DNA and the mixture was centrifuged for 30 seconds. As the PCR reaction was running, a microscope slide was prepared from the live bacterial culture to observe the individual cells of the unknown bacteria and determine its

The pathogen - West Nile virus belongs to the family Flaviviridae, genus Flavivirus and is part of an antigenic complex of Japanese encephalitis viruses. According to the classification of human-pathogenic microorganisms WNV belongs to II group of pathogenicity. Pathogen - flavivirus has an icosahedral shape, the approximate size is 40-50nm. Flavivirus has a single-stranded RNA, has a protein coat an enveloped capsid. Based on the differences of the nucleotide and amino acid sequences currently distinguish at least five genotypes WNV. Well-preserved frozen and dried state. Die at a temperature above 56 C after 30 min.

Retroviruses are a part of a large and diverse family of enveloped RNA viruses. They are defined by common taxonomic denominators that include composition, structure and replicative properties. “The virions are 80–100 nm in diameter, and their outer lipid envelope incorporates and displays the viral glycoprotein’s .The shape and location of the internal protein core are characteristic for various genera of the family. “ (Coffin, John M) The virion RNA is approximately 7–12 kb in size. It is linear, single-stranded, nonsegmented, and of positive polarity. A distinctive feature of this family is its replicative method which involves reverse transcription of the virion RNA into linear double

While dengue-like fever was the main sequelae thought to be caused by the virus, the recent outbreaks have shown a notable increase in neurological sequelae. The neurological commitment of the virus goes far beyond what was previously thought—with its potential link to autoimmune CNS infections (Guillain-Barré, osteomyelitis), neurodegenerative diseases, and microcephaly/subsequent development of mental retardation when infected in the fetus. This complex gamut of neuropathological responses has hence, and rightly so, taken to the forefront of research studies on the virus. The amount of evidence linking the virus and neurological disorders is largely anecdotal thus far, and to establish a definitive causative link, research needs to shift back to basic science.

Routine confirmation of coronavirus infection based on detection of unique sequences of viral RNA by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing. Any testing for the presence of this virus must be performed in appropriately equipped laboratories by staff trained in the pertinent technical and safety procedures. A number of RT-PCR assays that are specific for the coronavirus develop and publish. Currently described tests include an assay targeting upstream of the E protein gene (upE)1 and assays targeting the open reading frame 1b (ORF 1b) gene1 and the open reading frame 1a (ORF 1a) gene2. The assay for the upE target is considered highly sensitive, with the ORF 1a assay considered of equal sensitivity. The ORF 1b assay is considered less sensitive than the ORF 1a assay but may be more specific (12).

Viral DNA was monitored by collecting lavage or swab samples periodically. Active infection was detected at all the mucosal sites: the penis (P), the anus (A) and the oral cavity (O) at week seven post infection (Fig. 2). The infection persisted in all the sites in these animals.

Samples were subjected to PCR, samples that tested positive for HBoV were then tested for HBoV IgG antibodies using ELISA. 22 of the 100 samples tested positive for HBoV using PCR amplification. 18 of the of the 22 samples that tested positive tested positive HBoV IgM antibodies. In a study by Schildgen et al. (2013), random tumor samples were PCR amplified to screened for HBoV DNA. Samples that tested positive were subjected to fluorescence in situ hybridization to detect where the HBoV was present in the samples. The analysis revealed that HBoV DNA was present in 11 of the 60 lung samples and 9 of the 44 colorectal samples contained HBoV DNA. Research by L. Zhou et al. (2014), explains that HBoV is not always the primary virus infected an individual with a respiratory tract infection. In many cases, there is an other respiratory tract virus present. The presence of HBoV was detected by PCR amplifying 1229 samples collected from individuals with respiratory tract infections. Samples were also analyzed 7 respiratory tract virus using cultures and 15 respiratory viruses using PCR amplification. HBoV was identified in 127 of the samples, 66 of the 127 samples were infected by only HBoV. The other samples contained HBoV and another virus present in the

Researchers found the Zika virus is a member of the Flaviridae family and is transmitted to humans by, mosquitoes. Although these research results are promising, it is difficult to determine whether this experiment really did ZIKV virus is a members of the Flaviridiae family or different family. Was the experiment of the ZIKV conduct appropriately to all animal? And although this research promising, results for the most flaviuvirus family. Interestingly, the virus was also detected in testicles of infected mice. We have no idea whether these result are applicable to another types of virus. So this article is miss leading and this conclusion does not necessary apply to all virus.

Zika virus epidemic in the Americas is linked to increased prevalence in microcephaly in infants infected with the Zika virus (ZIKV) in utero. The purpose of the study was to develop a vaccine to for women of childbearing age and their sexual partners to prevent infection and Viremia due to ZIKV. The authors created a DNA vaccine that can produce pre-membrane and envelope proteins that offer immune protection in mice and primates. This study showed that DNA vaccine is an effective way to protect against ZIKV.

2 µl of HaeIII was added directly to the other PCR reaction tube. This enzyme cut the DNA at GGCC recognition sequences. The tube was mixed and then put into a hot water bath set at 37°C for 1 hour; this was the optimal temperature for the enzyme’s activity.

800μL of sterile water and 200μL of the DNA isolate were pipetted into a 1mL UV-transparent curvette and mixed well. 1mL of sterile water was added to a second 1mL curvette. On the spectrophotometer, a baseline correction was done and the absorbance spectrum of the DNA isolate was recorded. A PCR master mix was made. 5μL of DNA isolate was pipetted into two new labelled PCR tubes. 5μL of Milli-Q water were pipetted into a third labelled PCR tube. 20μL of the created master mix was pipetted into one PCR tube and 20μL of the given master mix was pipetted into the other PCR tube. PCR tubes were placed in a thermocycler, and then frozen. Tube identification was noted. Electrophoresis Gel apparatus was set up using 1% molten agarose. 3μL of each of the three PCR tubes were pipetted into 3 labelled microcentrifuge tubes. 6μ of sterile water and 1μL of 10x loading dye was added to each. Gel was loaded with samples and a DNA ladder and gel was run. An image was captured and interpreted. Sample with the largest quantity of DNA, was prepared for sequencing. 10μL of

It is estimated that urban areas of the world with high burden of dengue are expanding and the number of dengue cases are also growing exponentially (Division, 2002). In 2001, 69 countries reported dengue activity in WHO regions of South-East Asia, Western Pacific and Americas with more than one million reported cases in WHO Americas region in 2002. Also poor surveillance and non-reporting of cases from WHO African and Eastern Mediterranean Regions fails to reflects the true disease burden. All fur dengue virus serotypes are found in Asia, Africa and Americas regions with decrease in number of fatal cases since 2000.