Note: Further information on many of these FAQs and other questions can be found in the LITERATURE section of the website. Please consult the citations for more detail, and contact Center Director Peter L. Strick, PhD for any remaining concerns.
How do I request virus?
Does the Center offer training in the use of neurotropic viruses as tracers?
Can I conduct part or all of my experiment at the Center?
What is the advantage of using these viruses compared with conventional tracers?
Are there any special issues to keep in mind when planning to use herpesvirus?
Any special issues when planning to use rabies virus?
PRV: Can you provide some background about your stocks of pseudorabies virus?
HSV: Can you provide some background about your stocks of HSV-H129 recombinants?
Rabies virus: Can you provide some background about your stocks of N2c strains?
How do we handle and store the virus upon arrival?
Does the Center create new reagents?
How should we acknowledge the Center in our publications?
How do I request virus?
Here is the three-step process for ordering virus from the Center.
1) Choose the virus that is the best fit for your experiment. See RESOURCES for lists of available viruses and details about their characteristics. See LITERATURE for detailed protocols and technical specifications.
For guidance in the design of studies with rabies virus and/or nonhuman primates, contact Center Director Peter L. Strick, PhD to set up a ZOOM meeting or to talk by phone. For guidance in the design of studies using herpesviruses, contact Co-Investigator Georgina Cano, PhD to set up a ZOOM meeting or to talk by phone.
2) Submit approvals from your institution. We need documentation that investigators have the proper training, facilities, institutional biohazard and animal approvals to conduct BSL2 level experiments with herpesvirus and/or rabies virus. Note that to use rabies virus, your institution must also certify that lab personnel are fully vaccinated. Investigators who plan to use a herpesvirus may find the following links helpful for answering questions from safety committees:
Common IACUC and IBC questions about PRV
Herpesvirus Safety
Send approval letters from: 1) your Institutional Biosafety Committee; 2) your Institutional Animal Care and Use Committee; and 3) your institution’s Environmental Health &Safety Committee to Compliance Officer Tim McPartland.
3) Place your order. Once we have full documentation of approvals, you can request a virus.
For PRV or HSV, contact Herpesvirus Core Director Oliver Huang, PhD.
For rabies virus, contact Rabies Virus Core Director Matthias Schnell, PhD.
Does the Center offer training in the use of neurotropic viruses as tracers?
Yes, we provide hands-on training to investigators who visit our facilities at the University of Pittsburgh. Training includes all aspects of virus tracing, including preparation of protocols, acquisition of reagents, biohazard and vaccination procedures, sterile surgical techniques during virus injections, handling infected animals, immunohistochemical procedures to reveal infected neurons, and data analysis.
To arrange for training related to experiments with rabies virus and/or virus experiments with nonhuman primates, contact Center Director Peter L. Strick, PhD. For training related to experiments with herpesviruses in rodents, contact Co-Investigator Georgina Cano, PhD.
Can I conduct part or all of my experiment at the Center?
Yes. We provide access to our Biosafety Level 2 facility at the University of Pittsburgh for investigators who do not have the necessary facilities for viral tracing experiments at their home institutions. Our facility for doing research on animals infected with rabies virus is almost unparalleled in the United States. The resources in our facility make it possible to map cortical and subcortical areas and define specific sites for virus injections. The facility also contains a virus isolation suite that allows different animal species infected with rabies virus to be housed in adjacent but fully isolated space. The operating and recording rooms, the isolation suite and the space for aldehyde preservation of tissue using vascular perfusion are all contained in BSL2+ space. In addition, Pitt has specialized facilities, animal-care staff and housing for virus studies with nonhuman primates.
To arrange to use Center resources for independent pilot projects or collaborations with Center investigators, contact Center Director Peter L. Strick, PhD to set up a ZOOM meeting or to talk by phone.
What’s the advantage of using neurotropic viruses compared with other tracers?
Most conventional tracers used to define brain connections demonstrate where a neuron either receives its input or sends its output. With a few notable exceptions, these tracers do not pass across synapses. Thus, conventional tracers are limited to establishing the presence of connections between two brain sites. This limitation can be a significant barrier to unraveling brain circuitry because single neurons are components of complex networks of interconnected neurons.
This barrier has been overcome with the development of neurotropic viruses as transneuronal tracers. Specific strains of herpesvirus and rabies virus are transported transneuronally in a direction-specific and time-dependent manner. Transneuronal transport of neurotropic viruses occurs in every neural pathway examined, and makes it possible to trace multiple links in a chain of synaptically connected neurons. Thus, this technique has been used to unravel connections within sensory, motor, cognitive and affective circuits of the central nervous system.
Are there any special issues to keep in mind when planning to use herpesvirus?
Pseudorabies virus (PRV) is a swine herpesvirus (Herpesvirus suis), unrelated to rabies virus. The wild-type strain of PRV, Becker, is transported bidirectionally and is highly virulent. For these reasons, it is not suitable for transynaptic tracing, and it is mainly used to study immune responses and viral infectiveness in the nervous system. The attenuated strain of PRV, Bartha, is transported retrogradely and is widely used for transneuronal tracing in rodents (rats, mice and hamsters), ferrets and rabbits. PRV appears to infect some laboratory primates, but not others. Its usefulness as a transneuronal tracer following injections into the central nervous system or into the periphery in nonhuman primates has not been fully explored. When used as a transneuronal tracer, PRV is considered a BSL2 hazard. PRV is not known to cause human infections in the laboratory. However, caution when using PRV is appropriate because there are reports of rare human infection in farm workers.
We provide several recombinants of the H129 strain of herpes simplex virus Type 1 (HSV1) for transneuronal tracing. The H129 strain is transported anterogradely and can be used as a transneuronal tracer in rodents and New World monkeys (e.g., cebus), but it does not infect Old World monkeys (e.g., macaques). When used as a transneuronal tracer, H129 is considered a BSL2 hazard.
For guidance in designing studies with herpesviruses in rodents, contact Co-Investigator Georgina Cano, PhD to set up a ZOOM meeting or to talk by phone. For guidance in the use of herpesviruses in nonhuman primates contact Center Director Peter L. Strick, PhD to set up a ZOOM meeting or to talk by phone.
Are there any special issues to keep in mind when planning to use rabies virus?
Rabies virus is an RNA virus that is transported exclusively in the retrograde direction (the direction that is opposite to the flow of nerve impulses). Rabies virus, when used as a tracer, is considered a BioLevel 2+ hazard. To work with replication-competent rabies virus it is essential that you become familiar with and follow all the requirements for vaccination and titer monitoring. In the case of the single-cycle or replication-deficient rabies viruses (delta-G), your biosafety committee might not require vaccination (most do not).
There are several experimental variables to keep in mind when using rabies virus as a tracer. At the most basic level, the effectiveness of rabies virus as a transneuronal tracer depends on the virus strain, its concentration and the amount of virus injected at a site. Rabies virus is taken up at synaptic terminals. The virus does not spread very far from an injection site. Thus, virus injections must be placed near synaptic sites for the virus to be effectively taken up and transported.
The characteristics of virus uptake and transport and the effectiveness of infection of neuronal and non-neuronal processes are highly strain dependent. For example, some strains of rabies virus will take one day or more to move transneuronally. Other strains will move transneuronally at a rate of one synapse every 8-12 hours. Transport from virus injections into sites within the central nervous system are uniformly successful. However, virus injections into peripheral sites (muscle, adrenal medulla, stomach, etc.) can vary in their rate of success. Transneuronal transport following injections of rabies virus into eye and neck muscles is routinely successful. On the other hand, transneuronal transport of rabies virus following injections into intrinsic hand muscles of monkeys is only successful in about 30% of the cases.
In double-labeling experiments with two differently labeled strains of rabies, the timing of virus injections is a critical factor. If one strain of rabies arrives in a neuron before another strain, the first strain may exclude entry of the second strain, leading to false negatives for studies of branching.
For guidance in designing studies with rabies virus and/or nonhuman primates, contact Center Director Peter L. Strick, PhD to set up a ZOOM meeting or to talk by phone.
PRV: Can you provide some background about your stocks of pseudorabies virus?
PRV is the most widely used virus for polysynaptic neural circuit analysis in rodents. Its popularity emerges from the early identification of the attenuated Bartha strain that is only transported in the retrograde direction, the subsequent development of recombinants of Bartha that express unique reporters constitutively or conditionally, and the fact that it does not infect humans.
The Herpesvirus Core has generated several PRV-Bartha recombinants that express fluorescent reporters constitutively, conditionally, or as components of fusion proteins transcribed by the viral genome. Recombinants PRV-152 and PRV-614, which express EGFP and RFP respectively, are among the most highly requested Center reagents. These recombinants express cytoplasmic reporters that robustly label the somata and proximal dendrites of infected neurons. More recently, we generated a third PRV recombinant isogenic with PRV-152 and PRV-614, named PRV-290, which expresses a strong blue fluorescent protein called mTuquoise2.
The Core also provides several recombinants from the wild-type PRV-Becker strain, which are transported bidirectionally and are highly virulent. We have also created four PRV-Becker mutants that are only transported retrogradely and are less virulent.
For a list of available viruses, see the PSEUDORABIES VIRUS TABLE.
HSV: Can you provide some background about your stocks of HSV-H129 recombinants?
Although several recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits, only one strain -- the H129 strain of herpes simplex virus Type 1 -- selectively spreads in the anterograde direction. This virus was isolated from the brain of an individual who suffered from viral encephalitis and was first shown by Strick's lab to be transported preferentially in the anterograde direction following injection into the motor cortex of cebus monkeys. In the same study, Strick and colleagues demonstrated that the anterograde phenotype of H129 spread contrasted with the selective retrograde spread of the McIntyre-B strain of HSV1. These observations have been confirmed in multiple subsequent studies. Fluorescently labeled H129 recombinants are now available from the Herpesvirus Core (HSV-414, HSV-424, HSV-768, HSV-772, HSV-774, HSV-373).
For a list of available viruses, see the HERPES SIMPLEX VIRUS TABLE.
Rabies virus: Can you provide some background about your stocks of N2c strains?
The Rabies Virus Core provides well-characterized replication-competent and replication- deficient rabies viruses, particularly based on the recombinant N2c strain. We have introduced different marker genes into N2c, using the two single restriction enzyme sites within the transcription unit. We have a panel of N2c-based marker viruses and can easily increase the numbers as the need arises. We have received requests to express other genes in the N2c vector, such as channelrhodopsin. We also supply other novel rabies virus recombinants as well as a mouse monoclonal antibody (31G10) directed against rabies virus phosphoprotein. Overall, we provide 16 recombinant rabies viruses with different markers, envelopes and titers.
For a list of available viruses, see the RABIES VIRUS TABLE.
How do we handle and store the virus upon arrival?
Viruses are shipped in dry ice and need to be stored immediately at -70°C or colder.
Instructions specific to herpesvirus: Once you are ready to use, proceed to aliquot the virus keeping the vial in wet ice (as well as the aliquots). Divide the virus in 100-200 ul aliquots (never less than 50 ul) since smaller volumes may produce freezing artifacts that reduce the viral titer. Do not freeze in snap-cap tubes because the ultra-cold, very dry environment in the freezer will “freeze-dry” the sample and inactivate the virus. Avoid creation of large surface areas exposed to air during frozen storage (e.g. placing small volumes in tubes with a wide internal diameter). Never freeze aliquots at −20°C since the titer will drop 4-5 log units due to ice formation. The viruses are inactivated within 12 weeks at temperatures from -18°C to -25°C due to membrane rupture caused by ice formation. Virus stocks can be stored frozen in aliquots at −70°C for extended periods of time without consequences, as long as the temperature remains at −70°C or colder.
Once thawed, aliquots should not be refrozen, as this will reduce the viral titer. Multiple cycles of freezing and thawing will inactivate the virus. It is often desirable to use a single aliquot of virus to inject more than one animal during the same sitting. In this case, the virus should be kept cold by storing the vial in wet ice during the period throughout the multiple surgeries. We have determined that the titer of PRV-Bartha or its derivatives will be stable over a period of 3-6 hrs when stored in this manner.
IMPORTANT: Please follow the instructions above to avoid inactivating your virus. DO NOT store viral stocks in a -20C freezer!
Does the Center create new reagents?
Yes, part of the Center’s mission is to be responsive to the needs of the field at large, as well as to individual requests for novel reagents and advances in technology. For instance, the Herpesvirus Core has worked with:
1) Tony Zador at Cold Spring Harbor Laboratories to construct a PRV recombinant (PRV IE180 null) that can be used to deliver genes to neurons for long-term expression;
2) Jeff Friedman at Rockefeller University to develop a Cre-conditional PRV recombinant (this virus is not available through the Center); and
3) Stuart Mazzone at the University of Queensland, Australia, to develop HSV-H129 fluorescent recombinants for anterograde transsynaptic tracing of the rodent respiratory system.
Similarly, the Rabies Virus Core generated a panel of new N2c-based rabies virus vectors that are more sophisticated than rabies virus vectors, which are based on a vaccine strain of the virus. We now also provide a replication-deficient rabies virus vector at the unprecedented titer of 10^9.
NOTE: Custom reagents are available by special order only. These orders require consultation with the appropriate Core Director and the Center Director.
How should we acknowledge the Center in our publications?
Please cite the Center for Neuroanatomy with Neurotropic Viruses with our grant number,P40 OD010996. NIH requires us to track all publications that include results with reagents that we have provided. Please also let us hear directly from you about new and interesting findings you have made with the benefit of Center resources.
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