Histopathology using differential staining on eyes and nervous tissues is the easiest and simple. This can be followed by virus isolation of the suspected infected tissues on suitable fish cell lines.  

Histopathology using differential staining on eyes and nervous tissues is the easiest and simple. This can be followed by virus isolation of the suspected infected tissues on suitable fish cell lines.  

In addition to Prof Hassans' comment, molecular detection using PCR is a rapid and sensitive method to confirm the infection.

http://vdi.sagepub.com/content/20/1/38.full

Methods currently used for surveillance and diagnostic testing for Betanodavirus

Method / Targeted surveillance / Presumptive Diagnosis / Confirmatory diagnosis

Histology                      d                                       c                                       d

Histology followed by IHCT or IFAT:                                                                                                                        c                                       a                                       a

Transmission EM      d                                      c                                       b

Virus isolation            c                                       b                                       b

Virus isolation followed by IHCT or IFAT or RT-nPCR and sequencing :                                          b                                      b                                        a

RT-nPCR                        b                                      a                                        c

RT-qPCR                        a                                      a                                        b

RT-nPCR followed by sequencing :                                                                                                                              b                                      a                                       a

The designations used on the table, according to the OIE Manual indicate:

a = the recommended method for reasons of availability, utility, and diagnostic specificity and

b = standard method with good diagnostic sensitivity and specificity, but cost and availability limit its application;

c = the method has application in some situations but other factors severely limit its application;

d = the method is not recommended for this purpose.

These are somewhat subjective as suitability involves issues of reliability, sensitivity, specificity and utility.

(Reference: Australia and New Zealand Standard Diagnostic Procedure, Betanodavirus Infections of Finfish, NJG Moody and MStJ Crane, 2014)

Its a 3-D (3 directional) approach. Symptomatics, antibody-based (both previous exposure and causative) and gene-based (Real time PCR). Though there are many commercial kits available, they need to be validated for your set of samples. In my case only SJNNV amplification (real time and conventional PCR) only gave positive results while the same samples were negative with 2 of the commercial kits. I suggest, a close monitoring of the conditions of the live fish and screening by PCR using optimized primers or kits for regular monitoring. Given that many wild fish (natural collections from the open seas) samples are known to be harboring the NNV (nodavirus), its the disease-favored husbandry condition that triggers mortality and losses than the presence of virus per-se.

Dear Dr Azad,

Thanks a lot for your valued and practical response.

 I agree with you about limitation in using commercial kits to recognise of causative agents in suspected outbreaks to VNN.

We have the similar experience to detection of Betanodavirus by commercial kits so I strongly recommended to use some modified  RT-qPCR and then RT-nPCR followed by sequencing in each affected region.

De Jalil

Thanks for the reply. RNA-based detection kits are even more tricky. I see many practicality loop holes in them compared to the DNA-based ones. The protein-based ones are much more reliable simply because you have the end result of the RNA-to DNA -to amplification then to etc to peptide synthesis. That way, ADL (Sterling), as far as I know has MAbs against the NNV of diff species.  If there are polyclonals against the coat protein conserved region of Piscine Nodaviruses, it would be much better for screening and diagnostic purposes. 

Dear Dr Azad,

Thanks again.

I agree with you about the selection of the coat protein conserved region of Piscine Nodaviruses as a biomarker for detection of VNN but  I am sure that polyclonals Ab not appropriate for this target and we should design and produce MAbs against mentioned VNN coat protein.

Am i right?

Dear Dr.Zorie Zahra

Salalm Alaikom

First of all I wish you have recovered complete health. Re method I think we should have look at different types ( outbreaks vs carrier states). And even spcies. Classical lesions of  Vacuolization and necrosis of the brain, spinal cord and/or retina are the most common histopathological findings. However, in the case of experimentally challenged larval Atlantic Halibut, lesions were reported to become more severe and widespread being found in the intestine, liver, olfactory epithelium, yolk-sac epithelium, gills and pectoral fins, as well as in the retina and central nervous system at later stages of infection. On the other side  Fish that are subclinical carriers of NNV, including survivors of disease outbreaks, may show different histopathological lesions including cell degeneration and reactive changes such as hypercellularity, and proliferation or hypertrophy of glial cells. So histopathology should be interpreted according to type , spcie and other factors. However for presumptive diagnosis  IFAT, Cell Culture or molecular techniques could be used. For confirmatory purposes I am not sure but think cell culture is best method.For subclinical cases or carrier we may use cell culture or molecular techniques.