Kiran Sarvepalli V27 Develops Protocol to Detect Pathogens in FFPE Tissue Samples in Summer Research Program

Every summer, first- and second-year students at Cummings School of Veterinary Medicine at Tufts University participate in the Student Summer Research Training Program. Students pair up with faculty members to create a project that augments the research happening in Cummings School laboratories. Under a professor’s mentorship, each student conducts their research throughout the summer and generates a poster of their work to present at the National Veterinary Scholars Symposium (NVSS) and at Cummings School’s Veterinary Research Day, held this year on September, 6. Kiran Sarvepalli, V27’s research took an especially interesting turn this summer and led him to devise an effective protocol for two laboratories on campus to analyze banked tissue samples.

Kiran set his sights on a career in veterinary medicine in high school when he took his first biology course. Growing up in Fairfax, Virginia, he always liked catching reptiles and amphibians. While earning his Bachelor of Science in Biology from Carnegie Mellon University, he conducted research in computational biology, predicting how a molecule would fragment in a mass spectrometer and identifying the molecular structure of organic compounds.

He was drawn to Cummings School by Tufts Wildlife Clinic and the strong exotic wildlife program. The Summer Research Program appealed to Kiran as a way to incorporate his interest in wildlife with research. A few months into his first semester, he reached out to Dr. Amanda Martinot (she/her), E.A. Stevens Associate Professor in the Department of Infectious Disease & Global Health and co-director of Comparative Pathology and Genomics Shared Resource in the Department of Comparative Pathobiology, where she holds a joint appointment as a board-certified veterinary anatomic pathologist. The primary research focus of Dr. Martinot’s Lab centers on infectious diseases, such as tuberculosis and SARS-CoV-2, and the development of live-attenuated vaccines for tuberculosis. Dr. Martinot also supports other research groups by developing and validating animal models by evaluating tissue pathology.

“The Summer Research Program is incredible,” says Dr. Martinot. “I’m always so impressed with the caliber of students. It’s a great way to connect with students potentially interested in research and excited about the possibility of pursuing research as part of their career in veterinary medicine.”

Dr. Martinot describes the process for students to become a part of the Summer Research Program, “We talk to students about what they’re interested in, what they’d like to get out of the experience, what the lab is doing, and how we can craft a project that’s exciting for them.”

With Kiran’s interests in wildlife and conservation medicine, Dr. Martinot looked beyond what was happening in her lab to projects she collaborates on with other labs, including her work with Dr. Marieke Rosenbaum, (she/her) assistant professor of Veterinary Public Health in the Department of Infectious Disease & Global Health and assistant professor in the Department of Public Health and Community Medicine at Tufts School of Medicine. She analyzes samples from wild-caught monkeys in Peru to study infectious diseases in trafficked primates.

Dr. Rosenbaum has worked with students for years to assist with her research. “The Summer Research Program is a huge opportunity to move forward smaller parts of a research project and helpful to round out the research and fill in the gaps. Students quickly become competent—doing their own trouble-shooting, bringing different ideas, and becoming proficient in that topic area. It’s fun and advances the research that we’re doing.”

Working with two mentors across two labs was a fit for Kiran’s summer research project: “Detection of Herpes Simplex Virus One (HSV-1) in FFPE Samples from Neotropical Primates.”

“The transfer of pathogens from animals to humans isn’t considered as often as the opposite, from humans to animals,” explains Kiran. “But those infections can be just as devastating. HSV-1 is pretty ubiquitous in human populations, but when it’s transferred to primates, it can be fatal. We’re concerned with how it’s transmitted and how primates can get infected. My project is on the pathology of what happens when a primate is infected with HSV-1.”

Kiran originally set out to use RNAscope in situ hybridization to detect HSV-1 in primates’ tissue samples. Checking in with him in mid-summer, he reported that doing so proved to be a bigger challenge than anticipated and he changed direction on his original goal and hypothesis.

Kiran analyzed formalin-fixed paraffin-embedded (FFPE) tissue samples from the Peruvian monkeys. While the FFPE process is essential to deactivate any pathogens to prevent the spread of potential diseases, it also compromises the integrity of DNA and RNA. He initially found the DNA extracted from the FFPE tissues of poor quality, making downstream polymerase chain reaction (PCR) difficult and inconsistent, so this aspect of the project took much longer than expected.

“Every day I’m in the lab, hands-on, doing research that makes you think in ways maybe you hadn’t thought before,” says Kiran. “If the PCR doesn’t work, what factors can we change next time around? It can be frustrating, but it is also very satisfying when the protocol works. The best part of research is getting the result.”

Kiran decided to take his research in a new direction—how to optimize techniques using PCR to detect HSV-1 in FFPE-embedded tissues. “There’s a lot of information in these FFPE bio-banked tissue samples and they can last almost indefinitely, but we need to extract the DNA to understand the pathology. My part is to optimize the protocols that will be useful for Dr. Martinot and Dr. Rosenbaum down the road.”

Dr. Rosenbaum explains his work, “Kiran is helping to develop ways to better detect and demonstrate that HSV-1 is infecting primates and ending up in their tissues. Some animals don’t develop the disease and others die from it. Kiran is developing assays and probes to look at tissues and how HSV is behaving in primates.”

Kiran generated a poster of his work and presented it at the National Veterinary Scholars Symposium (NVSS) in St. Paul, Minnesota in early August with his fellow Summer Research Program students. He liked meeting veterinary students from across the country and around the world and seeing their research. He was struck by the diversity of research happening—from translational human medicine to conservation work to data analysis of antibiotic resistance.

At the end of the summer, Kiran reported that when he initially ran the RNAscope in situ hybridization, he was unable to see a certain signal of the RNA and DNA and assumed the protocol had not worked. He switched to a more robust microscope and could then see the results properly, though the technique still needed optimization, which became his focus for the rest of the program.

“One thing I accomplished is establishing the PCR protocol to detect the herpes virus in FFPE tissues,” says Kiran. “It’s important to help people in labs to screen tissues for the herpes virus. It’s useful to have, so I’m happy about that.”

This protocol brings tremendous value to both Dr. Martinot’s and Dr. Rosenbaum’s laboratories.

“Kiran developed a method to extract DNA to analyze tissues,” says Dr. Martinot. “He did a great job developing a protocol that’s very useful for a lot of reasons. Anyone who has access to these tissue block archives can use this method to extract DNA from the fixed tissues. He got the protocol to work, extracted DNA, and developed an in-situ hybridization protocol to show that tissues with active virus-producing RNA also had pathology, and we can appreciate it through a microscope. It’s about connecting the dots.”

Dr. Rosenbaum explains how Kiran’s work contributes to her research, “It allows us to better understand how the virus behaves in those monkeys and the pathophysiology of the herpes virus in primates. When we find herpes in monkeys, we need to be able to prove that it’s HSV-1. The more we can characterize tissues and how they are infected, the more concrete evidence we have that it’s the cause of infections. Kiran’s work helps to do that.”

In addition to presenting their work at NVSS and Cummings School’s Veterinary Research Day, students in the Summer Research Program also attend weekly seminars with biomedical researchers—from lab animal veterinarians to government researchers, from the corporate sector to academia—to learn about careers in veterinary research and new research approaches and techniques.

“Overall, it was a very interesting summer and gave me a lot of experience that traditional vets don’t always have, as well as perspective on what you can do as a veterinarian,” says Kiran. “I’m not sure yet what I want to do, but it opens doors and understanding of what’s possible.”

Kiran hopes to continue his research in the lab, improving the protocol and analyzing tissue samples suspected of having HSV-1.

“It’s a small amount of time to generate data in six weeks; it’s a high bar for students to come out with something,” says Dr. Martinot. “They have to be resilient and patient. Kiran had those struggles, workshopped it, pivoted, and adjusted his techniques, and at the end of the day, came out with great results, the best possible outcome. I’m hoping to keep Kiran connected to the lab. I’m so impressed with the students that come through here; I never want any of them to leave.”