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Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates.

ABSTRACT: Detection of Classical swine fever virus (CSFV) nucleic acid in archival formalin fixed tissue samples and their use for phylogenetic analysis was investigated. Ten samples were examined for the presence of CSFV nucleic acid by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of 5'UTR and E2 gene. RT-PCR was found positive for 5'UTR fragment in eight samples while only one tissue samples showed amplification for E2 gene target fragment. For molecular epidemiology of the disease, 5'UTR PCR product of sample from Darbhanga (Bihar), was cloned and sequenced. The sequence was compared with the sequences available in database. The phylogenetic analysis reveals that the isolate belongs to subgroup 2.2 sharing 98.7% nucleotide identities with Vietnamese isolate (CaTh05-1, AB252170), indicating towards the possible origin of genogroup 2.2 CSFV isolates involved in the outbreak from Vietnam. From the study, it can be concluded that the tissue samples collected and stored in buffer formalin for years can be used to detect CSFV nucleic acid. Results are also suggestive of that the 5'UTR region of genome is more suitable target for RT-PCR based detection of CSFV in archival formalin fixed specimens. The study also indicates the potential of archival formalin fixed tissues for molecular epidemiology and genotyping of the CSF virus.

SUBMITTER: Singh VK

PROVIDER: S-EPMC5377871 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates.

Singh Vinod Kumar VK Rajak Kaushal Kishore KK

Virusdisease 20170213 1

Detection of Classical swine fever virus (CSFV) nucleic acid in archival formalin fixed tissue samples and their use for phylogenetic analysis was investigated. Ten samples were examined for the presence of CSFV nucleic acid by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of 5'UTR and <i>E2</i> gene. RT-PCR was found positive for 5'UTR fragment in eight samples while only one tissue samples showed amplification for <i>E2</i> gene target fragment. For molecular epidemiol ...[more]

PMID: 28466064

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Project description:There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.

Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

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Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates.

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Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates.

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