Introduction for Hridita
Hi Hridita! Happy to have you in the lab. This website has some introductory information that you may need.
1 Project overview:
You will investigate mice infected with a gamma-herpesvirus called MHV68 for serum antibodies and auto-antibodies (using ELISA), serum inflammation (using ELISA), and neurological symptoms (observing/playing with the mice). You may end up testing for virus in the animals using cell culture and plaque assays, which would be done in a biosaftey cabnet.
The hypothesis, is that viral infection increases auto-antibodies against myelin basic protein (MBP) and inflammatory markers.
2 Protocols
Your project is going to use two different protocols. The main thing is going to be an ELISA for serum antibodies in animals infected with MHV68 (a relative of KHSV) and serum cytokines.
Second, you will test for lytic infection within infected animals. That will also take eukaryotic cell culture in the form of growing 3T3 fibroblasts in a sterile enviroment.
2.1 ELISA video and protocol:
The lab’s ELISA protocol is here, you will need access to the lab’s google Docs
This video goes over the basics:
Further reading:
- This paper has good general information on ELISAS. (Aydin et al. 2025)
After going through those videos, you should be able to answer:
Negative Controls: 1) PBS coated wells, which will determine background. 2) If you have serum from an uninfected/control source, that is also needed.
Positive Controls: 1) The known target, titrated in serial dilution. For your antibody titer experiments you will often have an infected animal from a wild type background that serves as a control. For your serum cytokine measurments, we have purified cytokine.
2.2 MHV68 lytic assay:
You will need to culture an adherent moue cell line called “3T3”. These are fibroblasts. The 3T3 cells in the tissue culture hood. Eventually, you will use them to test for viral reactivation. Together, you will build skills like: working in the cell culture hood, growing and passaging cells, and pipetting.
2.2.1 working in the hood
Before starting in the biosaftey cabinet (“cell culture hood”, you need to have all relevant safety training done.
Suggested reading:
This top 10 tips for cell culture blog post from Addgene (a company that sells plasmids) has good info
A general cell culture protocol for 3T3 cells is here, based upon this protocol form abcam
I wanted to find you a good checklist for working in the biosaftey cabnet. This checklist from the CDC is comprehensive, but many or most of the optional setps are not required for us.
2.2.2 MHV reactivation
This link contains a general plaque assay for mHV68, which we got from the Tibbets lab.
This link has a Video showing the complete MHV68 lytic virus assay: https://www-jove-com.proxy.lib.ohio-state.edu/v/3140/dissecting-host-virus-interaction-lytic-replication-model
3 Herpesvirus
In your project, you will be studying a herpesvirus called Murine Herpes Virus 68 (MHV68) This virus is a gammaherpesvirus, which is commonly used as a mouse model of human pathogens (reviewed here(Wang, Tibbetts, and Krug 2021)). The two human cancer-causing pathogens it is related to is Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV).
In your project, you will be studying the link between gamma-herpesvirus infection and multiple sclerosis (MS). While a good MS model, I noticed the mouse phenotype has elements similar to multicentric KHSV-associated Castleman’s Disease (HHV8-MCD).
So your experiments are designed to test for chronic inflammation observed during HHV8-MCD, also. 🔬🧪🧬
The following video is a general on herpesviruses. It is pretty good! Please watch it:
From that video you should be able to answer:
CMV - beta
EBV - gamma
KHSV - gamma
Okay! Now, how do they spread?
spread via fomites (lasts ~6hr), material to fetal, breast milk, daycares
spreads via salavary. By 18-21 there is > 97% seroprevelance worldwide. In kids, in high economic status areas, infection is delayed to young adulthood
unknown spread mechanism. Infects 2-5% of general US, 100% of Africans, near 100% of men who have sex with men and HIV+ men.
CMV, EBV, and KHSV are all associated with disease outside of cancers
From the options:
Congenital diseases of newborn and HIV, especially retinitis, and some long-term neurological consiquences
Infects 20% of B cells initially. An atypical CD8 cell response. Asymptomatic in children, but mononuclosis in adolescents. Ocasional neurologic symptoms
Typically no overt symptoms upon first infection. Infects B cells, and an atypical CD8 cell response occurs. Disease mostly in immunocompromised
CMV: Congenital diseases of newborn and HIV, especially retinitis, and some long-term neurological consiquences
EBV: Infects 20% of B cells initially. An atypical CD8 cell response. Asymptomatic in children, but mononuclosis in adolescents. Ocasional neurologic symptoms
KHSV: no overt symptoms upon first infection
You will be testing serum antibodies against MHV68. We are going to take a lession from EBV to learn about what the different antibody markers are.
IgG anti-EBNA peaks for EBV convalescence because it is a latent gene.
If EBV reactivates during convalescence, the patient makes IgM anti-EBNA.
Initial infection is against capsid antibodies, called VCA
Of the three herpesviruses we are talking about, all spread through direct fluid contact. However, only one spreads on fomites and can last outside the host for ~6 hours, and spreads via urine. This is important knowledge because the mouse equivalent does also.
answer: CMV (and thus murine CMV - mCMV - also spreads cage-to-cage via urine)
3.0.1 Additinoal reading and watching -
What is viral latency (video. ~30 min)
4 Multiple Sclerosis Info
4.1 What is MS?
Here is an introductory video from national multiple sclerosis society (NMSS). Please watch it! It is short and to the point
https://www.nationalmssociety.org/understanding-ms/what-is-ms
From the video you should understand that MS is:
A demyelnating disease of the central nervous system (relevant because mice have an anti-meylin basic protein T cell receptor)
We have immunomodulatory treatments for, but major chalanges are treating progressive patients, remeynelaing treatment, and understanding viral cause.
Important to our lab and your project, Ocrelizumab is the most successful treatment. It kills CD20+ B cells (infectious target of KHSV and MHV68) and is also used in immunotherapy for B cell cancers.
4.2 MS immunology
This video is a very in-depth on MS immunology.
https://hstalks-com.proxy.lib.ohio-state.edu/t/4347/the-immunology-of-multiple-sclerosis/?biosci
From the video you should be able to answer:
CD4
CD8 T cells (mostly)
B cells (also in brain, clonally expanded in the meninges)
Monocytes
Activated macrophages
The above answer differs greatly from the immune cells dominant in Experimental Autoimmune Encephalomitis (EAE), which are CD4 T cells, of Th1 or Th17 polarization. That is one reason why we do not call EAE mouse MS, instead we call it a model for MS.
Genetic link to CD4 T cells via MHC
Genetic link to defective Regulatory T cells via IL2RA polymorphisms. There is a funcitonal link to Foxp3 expression, but not frequency
Clinically Isolated Syndrome
Primary progressive
Relapsing Remitting
Secondary progressive
And the last question…
We have a broad range of immune thearpies. However, higher efficacy drugs that target T cells result in immunosupression unacceptable for long term treatment needed in a MS therapy.
We lack any meaningfull repearative treatments.
Disease modifying therapies delay immune-mediated damage. Eventually, patients will move to progressive disease. There is a need to target the underlying cause to stop further progression. That may be targeting the gamma-herpes/EBV cause.