MethodsTo clone HPV58, the pSP72 vector was utilized. The vector DNA was propagated and isolated using the Qiagen maxiprep kit and protocol. The isolated plasmid was then cut with the BglII restriction enzyme and CIP treated. HPV58 DNA and the pSP72 vector were ligated together and then trasnformed into competent bacteria cells. The CIP treated pSP72 vector alone was ligated and transformed as a negative control. The transformed competent cells were grown on an agar plate in the presence of the selective drug ampicillin overnight at 37C. Bacterial colonies were then chosen, grown in culture, and DNA was harvested using the Qiagen miniprep kit. The concentration of the resulting DNA was measured utilizing a nanodrop spectrophotometer. The DNA was then screened by digestion with BglII and gel electrophoresis to see if the ligation and transformation was successful.

Results Future DirectionsOnce successfully cloned, HPV58 DNA will be electroporated in primary foreskin and cervical cells to create immortalized cell lines that stably maintain the HPV58 genomes. These cell lines can then be grown in organotypic raft culture for virion production. Virus can then be used for further studies.

In Vitro System for Growing and Studying Infectivity of HPV 58 Terence Griffin, Jennifer Biryukov, and Craig Meyers The Pennsylvania State University College of Medicine

Department of Microbiology and Immunology

Figure 2: Colony growth post ligation and transformation of HPV58. After ligation and transformation, the negative control, with transformation of just the CIP treated vector showed no bacterial growth (left). The ligation of HPV58 and the pSP72 vector yielded growth of numerous bacterial colonies (right).

Figure 1: pSP72 plasmid DNA. The first is the DNA ladder, the second lane contains the pSP72 DNA after being cut with the BglII enzyme and the third lane contains the uncut pSP72 plasmid DNA.

IntroductionHigh-risk human papillomaviruses (HPVs) are one of the highest risk factors for cervical cancer, the second most common cancer in women. The high-risk HPV types are HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, and 66.1 Out of the high-risk types, 16 and 18 are the most prevalent worldwide. However, while uncommon in most of the world, there is a high prevalence of HPV 58 in Chinese women.2 The aim of this current study is to try and successfully grow native HPV58 in vitro in organotypic raft culture in order to study the virus life cycle and develop and test antivirals against HPV58, since little is known about the HPV58 type.3

ConclusionThe negative control plate did not grow colonies and numerous colonies grew on the plate with HPV58 insert. This result would generally indicate a successful cloning procedure with multiple colonies having the HPV58 genome. However, after screening 30+ colonies and re-doing the ligation and transformation, we were unable to find colonies that contained the HPV58 genome. More troubleshooting is necessary to determine why the HPV58 genome is not correctly being ligated into the pSP72 vector.

ReferencesRemmink, A. et al., The Presence of Persistent High-Risk HPV Genotypes in Dysplastic Cervical Lesions is Associated with Progressive Disease: Natural History up to 36 Months. Int. J. Cancer. 1995; 61:306-311

Chan, P. et al., High prevalence of human papillomavirus type 58 in Chinese women with cervical cancer and precancerous lesions. Journal of Medical Virology. 1999; 59:232-238

Wang, X. et al., Type-specific interaction between human papillomavirus type 58 E2 protein and E7 protein inhibits E7-mediated oncogenicity J. Gen. Virol. 2012; 93: 1563-1572