Researchers study molecular mechanisms in early stages of IPN virus infection

National Veterinary InstituteDec 30 2010

Infectious pancreatic necrosis is a serious disease that leads to enormous losses in the salmon industry each year.  The virus that causes the disease, IPN, is very prevalent in farmed salmon and trout in Norway. The virus attacks a wide diversity of hosts and has been found to occur in many different fish species, in addition to shellfish and molluscs. Fish that survive an IPN infection often become bearers of the virus and these fish are a potential source of infection to other individuals.

Irene Ørpetveit's doctoral research, carried out at The National Veterinary Institute, studied molecular mechanisms in the early stages of an IPN virus infection. All viruses are dependent on host cells in order to reproduce. Knowledge about how a virus enters its host's cells can lead to a better understanding of the disease and host range in addition to contributing to the development of more efficient vaccines and medicines. Ørpetveit found that the IPN virus attaches itself specifically to molecules on the surface of a number of different cell types. This indicates that the IPN virus uses an uptake mechanism that is activated by an external signal, for example, when a virus attaches itself to surface molecules.

We know that the IPN virus enters cells via vesicles or membrane bladders, which are then detached from the cell membrane. Ørpetveit has studied these vesicles more closely and found that the IPN virus is not dependent on a low pH level while this uptake mechanism is in progress. This is an important step towards the identification of the uptake mechanism used by the virus. She has also studied IPN-virus infection in cells that are assumed to be non-receptive and the results of her study can provide new information both about uptake mechanisms and about host range.

During one stage of this doctoral research, a molecular biological method was developed - a so-called real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). In order to validate and enhance this method, and as part of a larger project, it was compared with virus isolation in cell cultures, which is the traditional method used for detecting the IPN virus. 

As part of this study, Ørpetveit examined the distribution of the IPN virus in various parts of the salmon's kidneys. The results of her study show that the head kidney and middle kidney contain approximately the same amount of the virus. This is good news for those who take samples for IPN tests in the field because taking samples from the head kidney is more time-consuming than from the middle kidney. At the same time, Ørpetveit's experiments showed that taking samples from the tail kidney should be avoided.

Ørpetveit tested the effect of different preservation and storage procedures on parallel head kidney samples. Samples for IPN virus tests are usually preserved either on a modified cell culture medium (transport medium) or in RNAlater®, which is a commercial, RNA-stabilising reagent. Ørpetveit's work reveals how some methods of conserving and storing kidney samples from fish carrying the virus can directly influence test outcomes by giving falsely negative results.

Based on the results of this study, it is recommended that samples that are stored by using a transport medium should be homogenized immediately on arrival at the laboratory. Samples must always be stored at a temperature of minus 80°C.

Kidney tissue submerged in RNAlater® can only be analysed by using molecular biological methods, such as real-time RT-PCR. Storage of kidney tissue in RNAlater® is no better than in a transport medium, and this indicates that it is only necessary to use the former when there is a high risk of transport to the laboratory being delayed.

Real-time RT-PCR is shown to be at least as sensitive as traditional virus isolation in cell cultures when it comes to detecting the IPN virus. Real-time RT-PCR is also much less expensive and time-consuming. In the long term, real-time RT-PCR will therefore be employed for diagnostic purposes and, for example, for studies of brood fish and fry that are too small for pathological examination.