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Hepatitis C Virus Homologs in Nature - Essay Example

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The paper explores the homolog Hepatitis C Virus in infected dogs termed as Canine Hepatitis C virus, and in healthy horses as well as bats. A whole array of techniques has been discussed in detail employed by the scientists in these discoveries such as nested PCR, RT- PCR as well as microarray techniques…
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Hepatitis C Virus Homologs in Nature
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? HCV homologs in Nature – A Comprehensive Insight Viruses are one of the microbes that have caused severe harm to human life and health. Out of the whole pool of viruses, is hepatitis C virus that has infected around 3% of the global population. The question of finding a HCV homolog in other animals is essential to probe deeper into its phylogenetic linkage in nature. This review highlights the homolog Hepatitis C Virus in infected dogs termed as Canine Hepatitis C virus, and in healthy horses as well as bats. A whole array of techniques has been discussed in detail employed by the scientists in these discoveries such as nested PCR, RT- PCR as well as microarray techniques. Recently, rodent models have also been reported using these techniques owing to the ease of handling and ethically appropriate. A comparison of the novel viruses reported by different scientists was also compared in order to assess the similarities and differences between these viruses. With such findings at our disposal, scientists can better understand disease mechanisms and thus conduct quality research in disease prevention in terms of vaccine development as well as cure. Introduction Viruses have been the ‘danger microbes’ since centuries. The world has suffered great losses in many pandemics such as 1918 flu pandemic targeting roughly around 50 -100 million people which makes 5% of the world population at that time. (Patterson, 1991) (Johnson, 2002). History has witnessed many such disease disasters such as AIDS pandemic in 1981 killing around 25 million people. (Mawar, 2005) With human life so vulnerable to viruses, it is of utmost importance to study the impact of novel viruses on human health. It is now believed that many animal viruses cause two-thirds of infectious diseases in humans. For example, the Nipah, severe acute respiratory syndrome (SARS) and Ebola viruses have been found abundantly in bats in a study conducted by researchers in Columbia University and Eco-health Alliance. During a four-year study from 2006 – 2010, hundreds of urine and fecal samples of bats were tested for viral sequences. Results showed the prevalence of nine virus families in the samples and discovery of 50 novel viruses (ten of which had similarity with Nipah virus, and was responsible for many outbreaks in South Asia). (Kupperschmidt, 2013) Hsieh et al discovered a novel strain of swine Hepatitis E virus (HEV) in pigs which was thought to be the causal agent in more than 10% non-A, non-B and non-C hepatitis patients in Taiwan. (Hseih, 1999) Thus, it becomes important to study the origin of novel viruses and the extent of danger they pose to human beings. The scope of this paper describes the connection between these viruses and human disease and the techniques used in identification of such viruses. Key Technologies Used Quan et al applied Unbiased High Throughput Sequencing technique for assessment of the serum specimens collected from 415 healthy bats that represented seven families, 26 genera and 33 species in taxonomic classification; these inhabited five countries - Guatemala, Democratic Republic of China, Nigeria, Cameroon and Kenya. (Quan, 2013) Unbiased High Throughput Screening (UHTS) is one of the best techniques known until now that enable the discovery of novel pathogens. Many novel pathogens have been identified using this technique. Palacios et al reported the discovery of novel arena virus that usually causes mild illness but numerous fatal infections have been identified in patients with solid-organ transplantation. (Palacios, 2008) UHTS provides the advantage that it is unbiased by nature and provides full opportunity to probe the entire tree of life. The protocol of novel pathogen discovery involves amplification and sequencing, raw sequences are assorted into non-redundant sequence sets. Sequences that are unique are assembled into contiguous sequences which are then compared against sequence databases using computational softwares. These softwares match these sequences at nucleotide and amino acid levels in 6 reading frames, which ensure maximum chance to explore the novelty of a sequence. (Lipkin, 2010) Kapoor et al conducted a dual approach in the quest for finding a homolog of Hepatitis C virus. This was a major contribution as until before no closely related animal related virus had been identified earlier. With an aim to characterize viruses infecting dogs, respiratory samples of such were collected and undergone Unbiased High-throughput Sequencing. This was followed by Bioinformatic analysis that involved the use of programs to assess the homology of these sequences. Results showed that these sequences were similar to flaviviruses. This was followed by genome mapping and phylogenetic analysis of the sequences that helped the researchers conclude the existence of a virus that is closely similar to Hepatitis C virus and was named as Canine hepacivirus. (Kapoor, 2011) Burbelo et al developed a screening serological assay for determination of the Canine Hepatitis C virus host, which holds the closest phylogenetic relationship with Hepatic C virus. The envelope protein E2 of HCV showed the greatest similarity with Canine Hepatitis Virus, though it also exhibits highly diverse portions in genome. This assay also serves to screen various animal classes for infection caused by viruses as well as genetic characterization of the viruses. The assay consisted of the following phases. Firstly, collection of serum samples were collected from a range of animal species that comprised of 14 rabbits, 84 cows, 81 deer, 103 horses and 80 dogs. A serum sample from a dog was taken for RT-PCR analysis followed in order to extract the template for NS3 serine protease/helicase coding sequence of CHV(Burbelo et al. 2012). This was followed by cloning of the CHV protein fragments, followed by DNA sequencing. Next, LIPS assay was then applied to animal sera samples. This consists of a lengthy protocol; a summarised version of it is described here. Initially samples were presented for dilution in examining buffer A that consisted of 50 mM Tris, pH 7.5, 100 mM NaCl, 5mM MgCl, 1% Triton X-100. Next, antigen-antibody reaction was measured by using HCV helicase antigen fragments as antigen and diluted sera as antibody. This mixture was then added to a filter plate containing ultralink protein A/G beads in phosphate-buffered saline solution, which was then incubated on a rotator shaker for an hour. Next, the beads were washed on a Biomek Workstation and LU were calculated in a luminometer. The LIPS data was then analyzed statistically. Results showed that horse serum test were up for anti-helicase antibodies and used a control for remaining sera samples (Burbelo et al. 2012).Quantitative PCR was then carried out after the overall RNA was changed to cDNA (Burbelo et al. 2012). This type of PCR enabled the researchers to determine the NPHV genome copy numbers in serum samples. (Burbelo, 2012) . This enabled the sequencing of fragments similar to flaviviruses and phylogenetic analysis of the sequences in order to determine the common ancestor. (Burbelo, 2012) Lastly, the LIPS data was then used for a more efficient testing of Canine Hepatitis virus in data samples. Here again, the CHV serine protease/ helicase sequence was used as a template. Results of the process showed that samples from horses showed greater similarity to Canine Hepatitis virus than Hepatitis C virus. (Burbelo, 2012) Are these viruses found across different species linked? Kapoor et al described the identification of the Canine Hepatitis Virus as they hold the credit behind its discovery. This contribution was used by Burbelo et al for the development of the serological assay system. Thus, the viruses discussed in both these pieces of research address same virus. No homology or phylogenetic linkage has been reported between hepatitis C viruses in bats (Kapoor, 2011) and Canine Hepatitis viruses in dogs. This question requires further study and analysis. However, as the Canine Hepatitis C virus hosts horses as well as dogs, an inference can be drawn that these viruses are linked phylogenetically. Further conclusion on these results cannot be drawn at the moment; this requires further study. Relevance of Reported Research to Human Health The afore-mentioned findings have great significance to human health as these viruses have led to destruction of human life in large numbers. Hepatitis C virus is reported to infect 2% of human population. (Wang, 2013) This virus is major cause of cirrhosis, hepatocellular carcinoma and chronic liver disease. (Quan, 2013) Evidence of Infection by These Viruses in Respective Hosts Quan et al reported that bats are one of the hosts of hepatitis C viruses. However, the bats were not reported to be affected by disease. The disease transmission route from bats to humans was analysed to be intake of bat bodies, food contaminated with bat feces, infection through in-between hosts or direct exposure to bat blood. (Quan, 2013) In order to test the samples of healthy bats for disease, Serological assay is one of the bets methods to assess the presence of virus infection in samples. This technique was proposed by Burbelo et al. This technique was applied across various animals to test for the presence of canine hepatitis virus; results of which showed that horses were another host to these viruses. (Burbelo, 2012) Kapoor et al analysed the sampled from infected dogs to test the presence of hepatitis C viruses, and a viral strain similar to Hepatitis C virus was found and reported in their study. Thus, these viruses caused disease in dogs. In this study, they also conducted detailed phylogenetic analysis thereby proving that this virus is genetically closest to Hepatitis C virus. (Kapoor, 2011) Advantages of Rodent Model of HCV Kapoor et al conducted an in-depth study to identify an homolog of Hepatitis C virus and Pegiviruses owing to the fact that these share similar structure and genomic organizations, high genetic diversity in terms of inter-host and intra-host as well as persistent nature of infection. (Kapoor A. S., 2013) Drexler et al conducted another study to test the hypothesis that rodent viruses are similar and connected to hepatitis C viruses. And using Quantitative Rt- PCR, Histopathology, RNA in-situ hybridization, they suggested a rodent model for further research. (Drexler, 2013) Though, the efficiency of this model is questionable, this model has many advantages which are as follows. Firstly,owing to the fact monkeys and apes cannot be used as models in terms of ethics and accessibility and horses as well as dogs cannot be used as animal models because of their large body size, a rodent model provides better ease of handling and is ethically acceptable. Secondly, the rodent viruses have a core gene similar to all hepaciviruses as well as Pegiviruses. These viruses have also been reported to have secondary structures containing elements both from hepaciviruses and pegiviruses. Thirdly, a protein called F protein was found conserved in rodent viruses and hepaciviruses that is thought to be involved in conferring host resistance. Fourthly, considering the phylogenetic linkage analysis, both strains exhibit high connectedness. The hepacivirus sequences as well as novel rodent viruses showed high polyprotein phylogeny. Last of all, Rodent viruses resemble Hepatitis C viruses in mode of infection. Limitation of Rodent Model Considering the advantages mentioned above, a rodent model for HCV proves to be a noteworthy and effective model for research. Owing to the fact that this research was reported just recently, the drawbacks of these models is yet a question and can only be answered with passage of time and extensive work. Targetting Cats for Experimentation Yes, further research can be conducted upon cats to assess the hypotheses tested on rodents. Owing to the taxonomic classification, cats seem to be good animal models. However, extensive experimentation is to be carried out to prove this hypothesis. Application of Reported Techniques by Lipkin Myriad applications have been reported in literature by the use of these techniques. Borna Disease virus was reported in 1985 to be a novel virus infecting patients with bipolar disorder. The strains of this virus were discovered in birds using microarray and pyrosequencing techniques. West Nile Virus has been reported to cause encephalitis in people residing in America. Following the outbreak, CDC Encephalitis project was set up to identify the causative agent. Thus, by employing RT-PCR assays, the virus was detected in swimming pools, which helped the research team to identify this virus responsible for disease.Enteroviruses led to a disease called amyotrophic lateral sclerosis; a disease that causes muscular atrophy and loss of motor neurons. Initially, the causative agent was not understood, but in 2000, Berger et al reported the virus to be responsible for this disease. They made use nested PCR technique as well as Rt-PCR technique for identification of the etiolating agent. This study was a great contribution as a drug named pleconaril was already reported to act against this group of viruses. Dandenog virus was identified using PCR as well as BioInformatic algorithms, when a few patients who underwent organ transplantation died after 3 to 4 weeks. Unbiased High Throughput Sequencing enabled accurate identification of the virus.Viral Genome Sequencing was used to analyse a high case fatality rate (CFR) in Argentina with 4.5%.In that scenario, PCR was used to test the samples with microbial agents. This enabled the researchers to come up with results that showed high incidence of Streptococcus pneumonia, thereby highlighting the danger posed to human health by this virus. Extensive research to understand it’s mode of infection is required. These array of techniques were also used to analyse the relationship between vaccines, autism and gastrointestinal disease when intestinal abnormalities were reported in autistic patients who were administered with Mumps-Measles-Rubella vaccine. According to some reports, the disease was attributed to this vaccine. However, this report led to many controversies and evoked the notion of vaccine safety. References Burbelo, P. D., Dubovi, E. J., Simmonds, P., Medina, J. L., Henriquez, J. A., Mishra, N., Wagner, J., ... Kapoor, A. (January 01, 2012). Serology-enabled discovery of genetically diverse hepaciviruses in a new host. Journal of Virology, 86, 11, 6171-8. Drexler, J. C. (2013). Evidence for Novel Hepaciviruses in Rodents. PLOS One, 1-17. Hseih, S. M. (1999). Identity of a Novel Swine Hepatitis E Virus in Taiwan Forming a Monophyletic Group with Taiwan Isolates of Human Hepatitis E Virus. J. Clin. Microbiol. Johnson, N. &. (2002). Updating the accounts: global mortality of the 1918–1920 "Spanish" influenza pandemic. Bull Hist Med., 76(1), 105-15. Kapoor, A. S. (2011). Characterization of a canine homolog of hepatitis C virus. PNAS, 11608-11613. Kapoor, A. S. (2013). Identi?cation of Rodent Homologs of Hepatitis C Virus and Pegiviruses. mBIO, E00216- 13. Kupperschmidt, K. (2013). Sizing Up the Viral Threat. AAAS. Lipkin, W. (2010). Microbe Hunting. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 363-377. Mawar, N. S. (2005). The third phase of HIV pandemic: social consequences of HIV/AIDS stigma & discrimination & future needs. Indian J. Med. Res., 122(6), 471- 484. Palacios, G. (2008). Identification of a New Arenavirus Using High-Throughput Sequencing. N Engl J Med, 988-989. Patterson, K. &. (1991). The geography and mortality of the 1918 influenza pandemic. Bull Hist Med, 65(1), 4-21. Quan, P. F. (2013). Bats are a major natural reservoir for hepaciviruses and pegiviruses. Proc Natl Acad Sci, 8194- 8199. Wang, T. &. (2013). Interactions Between Hepatitis C Virus and Mitochondria: Impact on Pathogenesus and Innate Immunity. Curr Pathobiol Rep, 179-187. Read More
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