VIRAL GENETIC VARIATION: IMPLICATIONS FOR DISEASE DIAGNOSIS AND DETECTION OF SHRIMP PATHOGENS - PETER J. WALKER AND JEFF A. COWLEY

Co-operative Research Centre for Aquaculture, CSIRO Tropical Agriculture,

PMB3 Indooroopilly, Q 4068, Australia.

Introduction

During the past 10 years, the shrimp farming industry in the Asia-Pacific region and in the Americas has experienced the devastating impact of successive panzootics of viral disease (Chamberlain, 1999). In the ongoing effort to control and prevent these diseases, molecular methods are finding increasing application for differential diagnosis, epidemiological investigations and screening of covert infections in hatcheries and on farms. Methods such as the polymerase chain reaction (PCR), dot-blot hybridisation (DBH) and in situ hybridisation (ISH) have now been developed for a wide range of shrimp viruses and a number of significant bacterial pathogens (Lightner and Redman, 1998). Although technically complex and requiring specialised analytical equipment, these molecular methods have been adopted at a suprisingly rapid rate by diagnosticians, researchers and industry. This is primarily due to their exquisite sensitivity and specificity compared to standard histological procedures. The rapid uptake has also been driven by the lack of other methods that are used commonly in diagnosis of animal viral infections. Because of the absence of adaptive immunity in invertebrates (Fearon and Locksley, 1996), serology cannot be used to detect existing or prior infections with shrimp viruses. Virus propagation in vitro has had very limited application because of the absence of suitable cultured cell lines. Antibody-based methods for viral detection (eg. ELISA, indirect immunofluorescence or immunoperoxidase tests) appear to offer considerable potential but have yet to be explored adequately for most shrimp viruses.

Although molecular genetic methods can provide rapid and accurate information on the infection status of shrimp, there is a considerable risk of misdiagnosis if the various parameters that determine reaction specificity are not carefully monitored and controlled. In each of these methods, diagnostic specificity is determined by a hybridisation (annealing) reaction in which a DNA (or RNA) probe must bind to the target sequence in the infecting virus. The efficiency of this annealing reaction will be determined by various parameters such as temperature, ion concentration, the accessibility and integrity of the target, the size of the probe and the relative concentrations of target and probe. The test result will also be influenced by the accuracy of the match between the probe and target sequences. This is particularly a problem in PCR tests for which a single base mismatch can sometimes prevent primer efficient extension of the primer-template hybrid (Kwok et al., 1990; Sommer and Tautz, 1989).

This paper will consider the inherent genetic variability of viruses and the implications of sequence variation for pathogen detection and diagnosis. The potential to exploit variations in the genetic sequence of viral isolates to determine disease epidemiology and to track the movements of aquatic animal pathogens will also be considered.

Biomolecular basis of viral variation

Genetic variation is an essential feature of all living organisms. It provides the resource for natural selection and for the progressive adaptation of the population to a changing environment. Viruses face continuous environmental change as they pass from host to host. The most obvious and significant of these is the defensive or immunological response. Evasion of the host defences is a central feature of the survival strategy of all viruses. However, allelic variations in host genes or differences in their pattern of expression also present a changing environmental landscape that can determine susceptibility to infection or efficiency of replication (Gibbs et al. 1995; Morse, 1994).

Viral variation can be generated by a number of mechanisms. Major rearrangements in genome structure and organisation can occur by genetic recombination. Gene duplications, gene exchanges and gene adoptions also occur. However, the most common form of variation is mutation by nucleotide substitution. This occurs as a consequence of polymerase error in reading the template during replication. As viruses replicate rapidly and prodigiously, viral variation has significant implications for diagnosis and epidemiology (Morse, 1994).

Genetic variation in RNA and DNA viruses

From a genetic perspective, viruses can be classified according to whether the genome comprises RNA or DNA. RNA viruses are inherently hypervariable as RNA polymerases, which replicate the viral genome, lack proof reading and error editing functions that occur in cellular DNA polymerases (Steinhauer and Holland 1987; Steinhauer et al. 1992). The resulting rate of nucleotide misincorporation in RNA viruses (10^-3-10^-4) is at least 1000 times that of bacteria or eukaryotes, causing one or more base substitutions each time the viral genome replicates. Some mutations are lethal as they truncate or distort the resulting protein, rendering it non-functional. However, many mutations result in viable genomes that continue to replicate and contribute to the virus population. In this way, RNA viruses continually refine their genetic structure to accommodate the changing environment. Some RNA viruses may also undergo genetic rearrangements that allow exchange of corresponding genes or gene segments during mixed infections (Steinhauer and Holland, 1987). These recombination and reassortment events allow the most efficient and environmentally adapted combinations of genes to emerge from the available genetic pool, increasing the potential for viral survival.

RNA viruses known to infect farmed shrimp include Taura syndrome virus (TSV), yellow head virus (YHV), gill-associated virus (GAV), lymphoid organ virus (LOV) and rhabdovirus of penaeid shrimp (RPS). Each of these viruses is likely to replicate with a high mutation frequency. Some may also have a capacity for genetic recombination.

In DNA viruses, the mutation rate is usually far lower than in RNA viruses (Steinhauer and Holland, 1987). DNA polymerases, both cellular and viral, do employ proof reading and repair functions to reduce the intrinsic error rate. However, some small DNA viruses (eg. parvoviruses) appear to produce factors that suppress the repair function, generating an error rate similar to that of RNA viruses (Parrish et al., 1991). Even at the lower error frequency, the prodigious replication rate of DNA viruses generates mutations in the genome that can be observed over time. For example, during natural infections of palm beetles following experimental release, the Oryctes rhinoceros non-occluded baculovirus has been reported to mutate at the rate of approximately 0.05 % (or 100 nucleotides) per year. Over a monitoring period of 4 years, changes in genome structure such as insertions, point mutations and recombinations could easily be detected in molecular tests (Crawford and Zelany, 1990). DNA viruses have also been reported to produce sequence duplications and insert host DNA sequences into the viral genome.

DNA viruses known to infect farmed shrimp include white spot syndrome virus (WSSV), monodon baculovirus (MBV), baculoviral midgut necrosis virus (BMNV), infectious hypodermal and haematopoeitic necrosis virus (IHHNV), spawner mortality virus (SMV) and hepatopancreatic parvovirus (HPV). A recent study of WSSV DNA from sources in different geographic locations has suggested little sequence variation between isolates, except in some samples obtained from the USA (Lo et al., 1999). It is not yet known if crustacean parvo-like viruses (HPV, SMV and IHHNV) have a capacity to suppress the error repair function of DNA polymerases.

Genetic variation in viral detection and disease diagnosis

Observations of genetic variability in viruses proclaim the need for care in the use of molecular methods for disease diagnosis. Mutations in the nucleotide sequence can prevent binding of PCR primers to target sequences, cause primers to bind non-specifically to non-target sequences, or prevent PCR extension of the sequence from the primer site (Kwok et al., 1990; Sommer and Tautz, 1989). Sequence insertions or duplications can generate size variations in the PCR product. In each case, the result may appear falsely negative. At the protein level, mutations and other variations in sequence can affect the binding of diagnostic reagents such as monoclonal antibodies. Variations can also cause closely related strains to have significantly different biological properties such as pathogenicity, tissue tropism or host range. An understanding of these factors is important for accurate interpretation of data obtained for disease diagnosis, epidemiolgical investigation or screening for disease-free certification.

The YHV complex - a case study in viral variation

At least three RNA viruses with very similar morphology infect farmed P. monodon in the Asia-Pacific region. As the first of these to be reported was yellow head virus, the term 'YHV complex' has been adopted here to encompass this group of related agents. An understanding of the relationship between the viruses in the YHV complex is now emerging from molecular genetic studies that illustrate the importance of viral variation in disease diagnosis and epidemiology.

Yellow head virus (YHV) was first reported to be associated with mass mortalities of farmed P. monodon in Thailand in 1990 (Limsuwan, 1991). It now appears that YHV or related viruses may have been responsible for serious production losses in Taiwan, Indonesia, China, Malaysia and the Philippines since 1986 (Lightner, 1996). Yellow head disease affects juvenile to sub-adult prawns in which it usually causes a yellowish colouration of the cephalothorax and gills, and stimulates erratic swimming near the surface at the pond edge. YHV replicates in the cytoplasm of infected tissues that include lymphoid organ, haemocytes and gills. The virus infects a range of penaeid species but appears not to infect other crustaceans.

Gill-associated virus (GAV) has been the primary cause of a yellow head-like disease and associated mortalities that have affected the industry in Australia since 1994. The virus is indistinguishable from YHV by TEM, infects a similar range of tissues, and causes similar histopathology (Spann et al., 1997). In moribund prawns, the lymphoid organ displays extensive structural degeneration and cellular necrosis. In GAV infections, mortality is usually preceded by varying degrees of red colouration of the body and pink to yellow colouration of the gills. There has been no evidence of pale body colouration or yellowing of the cephalothorax as described for YHV.

Prior to the identification of GAV, a virus with similar morphology was observed to be common in healthy P. monodon in Australia (Spann et al., 1995). Lymphoid organ virus (LOV) causes the formation of distinct foci of hypertrophic cells (spheroids) in the lymphoid organ which otherwise remains structurally intact. ISH and TEM of lymphoid organ tissue indicate that LOV is contained only within spheroids. The virus has not been observed by TEM in other tissues but can be detected in haemolymph and gills by PCR tests. LOV infections appear to be non-pathogenic in uncompromised P. monodon.

The complete nucleotide sequence of the GAV genome has now been obtained (JA Cowley and PJ Walker, unpublished data). Analysis of the sequence has indicated that GAV is most closely related to RNA viruses in the family Coronaviridae. In order to assess the genetic relationship between GAV and YHV, sequence comparisons were conducted in 3 regions of the RNA replicase (ORF1b) gene that had been used for the development of PCR and ISH tests (Wongteerasupaya et al., 1997; Tang and Lightner, 1998; Cowley et al., submitted). The comparisons over a total of 1780 nucleotides (approximately 6.0 % of the total genome) indicate that GAV and YHV vary by 17.6 % in nucleotide sequence and 10.7 % in amino acid sequence. This degree of variation is typical of closely related RNA viruses that constitute distinct geographic topotypes (Cowley et al., 1999). As the sequence of viral polymerase genes usually is relatively conserved, more genetic variation between YHV and GAV might be expected in some other regions of the genome. Comparison of sequences amplified from the ORF1b gene of a large number of LOV isolates from healthy P. monodon has indicated that they vary from the prototype GAV nucleotide sequence by an average of £1.5 % (Cowley et al., 1999). This is within the range of variation expected within a single population of replicating RNA sequences and indicates that the GAV and LOV are pathogenic and non-pathogenic variants of the same virus.

The available nucleotide sequence data has been used to develop primary and nested RT-PCR tests to detect GAV in infected prawn tissue (Cowley et al., submitted). Each of these GAV RT-PCR tests will detect both GAV and LOV, which cannot presently be distinguished genetically. The GAV RT-PCR test will also amplify the expected product from a Thai isolate of YHV. This occurs because the nucleotide sequences of GAV and YHV in the regions targeted by the PCR primers are sufficiently related to allow primer hybridization under the conditions of the test. An RT-PCR test has also been described for detection of YHV (Wongteerasupaya et al., 1997). However, due to significant differences in sequence from YHV at one of the primer binding sites, this test will not detect either GAV or LOV (Cowley et al., 1999). A comparison of the sequences of YHV and GAV isolates in the primer binding sites for each of these PCR tests is shown in Fig.1, illustrating the poor correspondence in sequence at the site of primer 144R.

A first assessment of these tests might suggest that the GAV PCR is group-specific, detecting all 3 viruses, and that the YHV PCR discriminates between GAV and YHV. However, as the extent of variation among viruses associated with yellow head disease in Thailand and the Asian region is presently unknown, such a presumption is premature. It is also possible that other non-pathogenic LOV-like viruses are common in the region. We have observed that the prevalence of LOV in healthy P. monodon captured in northern Queensland is extremely high (Cowley et al., submitted) and it is likely that pathogenic YHV-complex viruses emerge from such a background of non-pathogenic infection. This clearly illustrates the need for care in the design and interpretation of PCR tests and the need for accreditation and standardisation of procedures. Ultimately, when adequate information on the nature and distribution of YHV complex viruses is available, sequence data could be used to devise a range of encompassing and discriminatory molecular tests.

Figure 1. Comparison of the YHV and GAV sequences at the primer binding sites for YHV PCR primers 10F and 144R Wongteerasupaya et al. (1997). Dots indicate the location of homologous nucleotides. GAV sequences are described in Cowley et al. (1999).

Molecular epidemiology and the movement of aquatic animal pathogens

Although presenting challenges for test design, variability in nucleotide sequence can be a very potent tool in understanding the epidemiology of disease. By applying nucleotide sequence analysis and other discriminating molecular techniques to the analysis of virus isolates, there is potential to trace the origin and movement of viruses on a local and regional basis. It may also be possible to discriminate between pathogenic and non-pathogenic strains that otherwise may be indistinguishable. Such molecular approaches to epidemiology are now commonly used in the study of viruses infecting terrestrial animals and humans.

In the case of YHV-complex viruses, sequence analysis of the 618 nucleotide product generated by the GAV PCR test has already demonstrated that LOV and GAV are variants in the same virus population of which individual isolates are genetically distinct (J.A. Cowley and P.J. Walker, unpublished data). It has also been possible to define the Australian viruses as a population that has evolved with a different lineage to that of YHV from Thailand. The accumulation of more comprehensive data from multiple domains of the genome and from YHV complex isolates obtained throughout the Asia-Pacific region will provide better understanding of these viruses, the origins of disease and the risk factors associated with farming practices.

The principles illustrated here for the YHV complex are equally applicable to other RNA viruses and may well apply to DNA viruses infecting aquatic animals. Through the use of modern PCR and sequencing technology and the development of bioinformatics systems, the capability for rapid accumulation and analysis of nucleotide data is now a reality. If standard analytical procedures and appropriate security protocols can be agreed, there is obvious potential for such an approach to be a powerful tool in managing disease and in defining a more rational basis for controlling the movement of aquatic animal pathogens.

Acknowledgements

Work reported in this paper was conducted as a co-operative project involving CSIRO Australia, and Mahidol University, NACA and the Aquatic Animal Health Research Institute in Thailand. This research has been supported by the Australian Centre for International Agricultural Research (ACIAR) and the National Centre for Genetic Engineering and Biotechnology (BIOTEC) in Thailand.

References

Chamberlain, G.W. (1999). Sustainability of world shrimp farming. In: Global Trends: Fisheries Management. EK Pikitch, DD Huppert and MP Sissenwine, Eds American Fisheries Society Symposium 20, Bethesda, MD.

Cowley, J.A., Dimmock, C.M., Spann, K.M. and Walker, P.J. Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR. Diseases of Aquatic Organisms (submitted).

Cowley, J.A., Dimmock, C.M., Wongteerasupaya, C., Boonsaeng, V., Panyim, S. and Walker, P.J. (1999). Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses. Diseases of Aquatic Organisms 36, 153-157.

Crawford, A.M. and Zelany, B. (1990). Evolution in Oryctes baculovirus: rate and types of genomic change. Virology 174, 294-298.

Fearon, D.T. and Locksley, R.M. (1996). The instructive role of innate immunity in the aquired immune response. Science 272, 50-54.

Gibbs, A.J., Calisher, C.H. and Garcia-Arenal, F. (Eds.) (1995). Molecular Basis of Viral Evolution. Cambridge University Press.

Kwok, S., Kellogg, D.E., McKinney, N., Spasic, D., Goda, L., Levenson, C. and Sninsky, J.J. (1990). Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies. Nucleic Acids Research 18, 999-1005.

Lightner, D.V. (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, LA, USA. 304 p.

Lightner, D.V. and Redman, R.M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture 16, 201-220.

Limsuwan, C. (1991). Handbook for cultivation of black tiger prawns. Tansetakit Co. Ltd, Bangkok.

Lo, C.-F., Hsu, H.-C., Tsai, M.-F., Ho, C.-H., Kou, G.-H. and Lightner, D.V. (1999). Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Diseases of Aquatic Organisms 35, 175-185.

Morse, S.S. (1994). Towards an evolutionary biology of viruses. In The Evolutionary Biology of Viruses. (S.S. Morse Ed.). Pp. 1-28. Raven Press, New York.

Parrish, C.R., Aquadro, C.F., Strassheim, M.L., Evermann, J.F., Sgro, J.-Y. and Mohammed, H.O. (1991). Rapid antigenic-type reeplacement and DNA sequence evolution of canine parvovirus. Journal of Virology 65, 6544-6552.

Sommer, R. and Tautz, D. (1989). Minimal homology for PCR primers. Nucleic Acids Research 17, 6749.

Spann, K.M., Vickers, J.E. and Lester, R.J.G. (1995). Lymphoid organ virus of Penaeus monodon from Australia. Diseases of Aquatic Organisms 23, 127-134.

Spann, K.M., Cowley, J.A., Walker, P.J. and Lester, R.J.G. (1997). A yellow-head-like virus from Penaeus monodon cultured in Australia. Diseases of Aquatic Organisms 31, 169-179.

Steinhauer, D.A., Domingo, E. and Holland, J.J. (1992). Lack of evidence fro proofreading mechanisms associated with RNA virus polymerase. Gene 122, 281-288.

Steinhauer, D.A. and Holland, J.J. (1987). Rapid evolution of RNA viruses. Annual Review of Microbiology 41, 409-433.

Tang, K.F-J. and Lightner, D.V. (1999). A yellow head virus gene probe: application to in situ hybridization and determination of its nucleotide sequence. Diseases of Aquatic Organisms 35, 165-173.

Wongteerasupaya, C., Tongcheua, W., Boonsaeng, V., Panyim, S., Tassanakajon, A., Withyachumnarnkul, B. and Flegel, T.W. (1997). Detection of yellow-head virus of Penaeus monodon by RT-PCR amplification. Diseases of Aquatic Organisms 31, 181-186.