Diagnosis of penaeus monodon-type baculovirus by PCR

Details Diagnosis of penaeus monodon-type baculovirus by PCR Diagnosis of penaeus monodon-type baculovirus by PCR

1999-07-13

2001-09-04

Salimi, Ali R. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S091100, C435S091200, C424S204100, C536S023720

Reexamination Certificate

active

06284455

ABSTRACT:

I. FIELD OF THE INVENTION
The present invention relates to a method for detecting occluded baculovirus, more specially, Penaeus monodon baculovirus (MBV), using polymerase chain reaction (PCR). Two pairs of primers are designed, the first pair of primers (i.e., SEQ ID NO:1 and SEQ ID NO:2) are occluded baculovirus (particularly nuclear polyhedrosis virus [NPV]) specific, which can detect baculovirus that produces polyhedra occlusion bodies. The second pair of primers (i.e., SEQ ID NO:3 and SEQ ID NO:4) are MBV specific, which can detect MBV exclusively. The present invention also relates to an enzyme-linked immunosorbent assay (ELISA) method for detecting MBV occlusion bodies.
II. BACKGROUND OF THE INVENTION
Since the technological development in artificial fertilization, mass seed production, and feed formulation for cultured crustaceans, cultured shrimp (especially penaeid shrimp) has become number one cultivated crustacean in Southeast Asia, especially in Taiwan. In recent years, there have been numerous reports regarding disease outbreaks which cause substantial losses in shrimp production in Taiwan and southeast Asia. Pathogens, such as bacteria, viruses, and fungi, along with environmental stressors such as temperature fluctuation, heavy rainfall, overfeeding, and industrial and agricultural pollutants, are considered to be the major contributors for the outbreaks. Among these, viral infections are particularly of concern, primarily because viral diseases cannot be cured by therapeutic reagents and an early and sensitive detection of the disease is the most effective means for containment.
To date, nearly twenty penaeid shrimp viral diseases have been discovered. Among them, six major viruses were found, in which four baculoviruses, namely Penaeus monodon baculovirus (MBV), baculoviral midgut necrosis virus (BMN), baculovirus penaei (BP), and white spot syndrome virus (WSSV), are the most important ones due to their wide distribution and causes of high mortality in penaeid shrimp.
Baculoviruses are a large group of viruses which infect only arthropods. There are three subgroups of baculovirus, namely nuclear polyhedrosis viruses (NPV), granulosis viruses (GV), and non-occluded baculoviruses. NPV and GV are occluded forms of baculoviruses. The virions (enveloped nucleocapsids) in NPV and GV are embedded in a crystalline protein matrix, which is referred to as an inclusion or occlusion body. The occlusion body is found responsible for spreading the infection between organisms. The characteristic feature of the NPVs is that many virions are embedded in each occlusion body. The NPV occlusion bodies are relatively large (up to 5 micrometers). Occlusion bodies of the GV viruses are smaller and contain a single virion each. The crystalline protein matrix of the occlusion bodies of both forms is primarily composed of a single 25,000 to 33,000 dalton polypeptide which is known as polyhedrin or granulin. Baculoviruses of the non-occluded subgroup do not produce a polyhedrin or granulin protein, and do not form occlusion bodies.
In nature, baculovirus infection is initiated when an arthropod ingests food contaminated with baculovirus particles, typically in the form of occlusion bodies for an NPV. The occlusion bodies dissociate under the alkaline conditions of the midgut, releasing individual virus particles which then invade epithelial cells lining the gut.
Within a host cell, the baculovirus migrates to the nucleus where replication takes place. Initially, certain specific viral proteins (such as DNA polymerase) are produced within the infected cell via the transcription and translation of so-called “early genes”. These proteins are required for the replication of the viral DNA. The infected cell later produces large amounts of “late viral gene products”, which include components of the nucleocapsid which surrounds the viral DNA during the formation of progeny virus particles. The progeny virus particles then migrate to the cell membrane where they acquire an envelope as they bud out from the surface of the cell. This non-occluded virus can then infect other cells.
Polyhedrin synthesis begins late in the post-infection period. At that time, there is a decrease in the number of budded virus particles, and progeny virus particles are then embedded in occlusion bodies. Occlusion body formation continues until the cell dies or lyses, thereby releasing the occlusion bodies which can then spread the infection to other host cells.
Among shrimp baculoviruses, MBV and BP are NPVs, which are characterized by the existence of occlusion bodies and polyhedrin. Insect baculoviruses, AcMNPV, BmSNPV, LdMNPV and OpMNPV, also belong to NPVs. Among insect baculoviruses, AcMNPV (Autographa californica nuclear polyhedrosis virus) has been extensively studied. ACMNPV has a genome of 130 kbps of doublestranded, circular DNA. It belongs to the family Baculoviridae, subfamily Eubaculo-virinae, genus Nuclear Polyhedrosis Virus (NPV), and the subgenus Multiple Nucleocapsid Virus, which are characterized by the formation of viral occlusion bodies or polyhedra in the nuclei of infected host cells. DNA sequencing within the genome of AcMNPV is well documented.
Recently, Kuo et al. (U.S. Pat. No. 5,824,535) has reported the identification, purification, and detection of WSSV, a baculovirus associated with white spot syndrome. However, WSSV is a non-occluded baculovirus, which can not be used to detect occluded baculovirus.
In 1993, Chang et al.,
J. Invertebr. Pathol
., 62:116-120, disclosed a PCR method using polyhedrin gene to detect MBV. However, polyhedrin exists in all NPVs, which includes not only occluded insect baculoviruses (such as ACMNPV, BmSNPV, LdMNPV, and OpMNPV), but also shrimp viruses such as BP and MBV. In addition, according to a report by Bjornson et al. (Bjornson et. al.,
J. General Virol
., 73:3177-3183), there is a 80% sequence homology in polyhedrin genes among NPVs. Therefore, using a pair of primers derived from the DNA sequences of insect polyhderin genes for PCR, as described by Chang et al. (supra), can not sensitively and exclusively detect MBV, and the industry is still in need of a highly specific and sensitive method for the detection of MBV infection as a means of containing the disease.
III. SUMMARY OF THE INVENTION
The first embodiment of the present invention provides a method for detecting occluded baculoviruses using polymerase chain reaction (PCR). Two pairs of primers are designed. The first pair of primers is originated from the conserved sequences of the DNA polymerase genes of nuclear polyhedrosis viruses (NPVs). This pair of primers can detect occluded baculoviruses using PCR. The preferable first pair of primers (Primer 1 and Primer 2) has the DNA sequences of SEQ ID NO:7 and SEQ ID NO:8. This pair of primers are designed from the conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdNNPV). The most preferable first pair of primers (P1 and P2) has the DNA sequences of SEQ ID NO:1 and SEQ ID NO:2. These two DNA sequences are derived from the DNA polymerase-like gene of MBV. When an MBV DNA template is used, both pairs of primers produce a 714-bp DNA PCR fragment (SEQ ID NO:5). When an AcMNPV DNA is used as template, both pairs of primers produce a 621-bp PCR fragment.
The second pair of primers (PA1 and PA2) (SEQ ID NO:3 and SEQ ID NO:4) is a pair of nested primers designed from the 714-bp PCR product produced by P1 and P2 using MBV DNA as template. Using this pair of nested primers, a 511-bp PCR product (SEQ ID NO:6) is produced when MBV DNA is used as template. PA1 and PA2 are specific for MBV detection because they do not produce any PCR products when DNA templates from other sources, such as a non-MBV NPV (e.g., AcMNPV), white spot syndrome virus (WSSV), or normal shrimp tissue homogenates, are used.
The PCR method comprises the following steps: (1) amplifying DNA in a sample comprising the first or second pair of primers described above by PCR

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