Koi Herpesvirus Isolates Research Articles

Development of a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for the detection of KHV.

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.

Genetic Diversity of Cyprinid Herpesvirus 3 from Different Geographical Locations during 1999-2019 in the United States of America.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important pathogen in common and koi carp Cyprinus carpio, varieties. Two main genotypes of KHV have been reported worldwide that are associated with Asian and European origins. In the USA, outbreaks of KHV diseases have been reported in different states since the early 1990s; however, the diversity of KHV is unknown. In the current study, 67 DNA samples that were extracted from clinical cases of koi tissues that were submitted for diagnosis during KHV outbreaks from 10 different states in the USA from 1999 to 2019 were used to investigate their genetic diversity. The thymidine kinase gene was amplified, sequenced, and used for phylogenetic analysis. Our results showed that the KHV isolates that were collected from the different states were clustered in the two known KHV genogroups, where 31 isolates belonged to the Asian genotype branch and 36 to the European genotype branch. The spatiotemporal analysis demonstrated fluctuation of KHV genotypes in the USA, as the main KHV genotype that was detected in koi in the USA from 1999 to 2013 was the European genotype, whereas the Asian KHV genotype appeared to emerge in the USA in 2008, increasing in incidence until 2019. The current study provides evidence on the genetic diversity of KHV in the USA. Future studies that evaluate the virulence of these genetically diverse isolates is warranted to obtain a better understanding of the epidemiology of this re-emerging pathogen. This may provide an improved awareness of the current status of KHV and help to control the disease in the koi population in the USA.

Analysis of Genotype and Phylogenetic of Koi Herpesvirus (KHV) on Carp (Cyprinus carpio) in Lombok Island of West Nusa Tenggara Province Based on Thymidine Kinase Sequence

Koi herpesvirus (KHV) is a virus that usually infects carp and koi causing mass mortality, as well as economic and social loss. The results of varied test are often found in its development. Allegedly, there are mutation or genetic variation of KHV in Indonesia. Based on these problems, this research aims to understand the genetic variation and kindship of KHV isolate in the carp ( Cyprinus carpio ) in Lombok Island of West Nusa Tenggara Province based on thymidine kinase sequence. Water quality of fish pond was checked. KHV clinical symptom was checked visually in each fish sample, then sacrifices the organ (gill) for PCR analysis and DNA sequencing. The research results show that clinical symptoms that appear are pale fish gill and bleeding on the surface of the body. The temperature in the research site shows an optimum range for KHV development. The results electrophoresis of carp suspected with KHV shows five positives out of a total six samples. Genetically, KHV isolates from Lombok Island of West Nusa Tenggara Province is identical with the GenBank isolate code KHV-J/U/I and Indo_0C05WNT2. Keywords: Carp, Koi herpesvirus, Phylogenetic, Thymidine kinase.

Examination of Infection Parameters for Replication of Israeli Isolate of Koi Herpesvirus in Common Carp Brain Cells

Research on koi herpesvirus (KHV) is mainly focused on fish trials for the investigation of virus entry, disease progress and immunological responses within the organism in order to develop new diagnostic and treatment strategies. However, examination of in vitro-replication of KHV might give useful insights supporting the understanding of this pathogen as well as contributing to the development of new methods for diagnostic purposes and/or vaccine development. Nevertheless, until today, only few studies have been published on this matter. Thus, this work concerns investigation of in vitro-replication of the Israeli isolate of KHV, KHV-I, in the widely used common carp brain cells (CCB) to identify the parameters leading to high-quality virus stock with high titers for further applications. Therefore, the propagation of the KHV-I at the influence of various cell densities at inoculation, virus load and time of harvest on the propagation of KHV-I was examined. Obtained results indicate that inoculation with relatively high cell densities is advantageous since the highest titers could be reached for 60 000 and 75 000 cells/cm2 already 3 days after inoculation. In contrast, the use of too high virus load for inoculation with too low cell density can result in much lower maximum or even decrease in virus titers. While viral DNA showed to be relatively stable after reaching the maximum, the infectivity of the virus can decrease shortly after the peak. Therefore, selection of the right combination of infection parameters is crucial for reaching the appointed goal. This work gives valuable insights into the replication of KHV-I in CCB cells and indicates the differences between various KHV isolates regarding in vitro-propagation.

First detection of koi herpesvirus from koi, Cyprinus carpioL. experiencing mass mortalities in Iran: clinical, histopathological and molecular study.

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV-infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post-mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I(++) II(+) allele of this Asian genotype.

IMMUNOGENICITY ASSESSMENT FOR DETERMINATION OF THE MOST POTENTIAL ISOLATES OF KOIHERPES VIRUS

Common carp and koi (Cyprinus carpio) are the main consumption fish commodity and ornamental fish in Indonesia. Diseases due to koiherpes virus (KHV) infection had caused a huge loss in both common carp and koi culture industries world-wide. This study was generated to select the most potential candidate from out of three koiherpes virus isolates based on their serological performances for vaccine development to control Koiherpes virus disease in koi. Virus collection, isolation, and propagation in cell culture have been conducted from common and koi samples originated from Banjarmasin, South Kalimantan; Takalar, South Sulawesi; Cirata Lake-Cianjur, West Java; Depok, West Java; Ciseeng, Bogor, West Java; and Purwokerto, Central Java. Determination of the causative agent of the disease was done by PCR (Polymerase Chain Reaction) assay and inoculation of filtrate homogenates from infected fish into cell culture. There are three koiherpes virus isolates (BJMN-1, BJMN-2, and SKBM) which are being successfully isolated, cultured and propagated in Koi Fin (KF-1) cell line. Calculation of tissue culture infection dose from each isolates were 104.55 TCID50/mL, 104.72 TCID50/mL, and 103.28 TCID50/mL. Immunoassay of the three isolates was conducted by Indirect-ELISA method. Polyclonal antibody was made by injecting each isolate to the experiment animals (rat/Rattus norwegicus strain Sprague Dawley). It was shown that all of three isolates have high level of immunogenicity, as seen from the absorbance values at 490 nm wavelength. BJMN-1 isolates have a higher potential for it’s ability to cross-react with the other isolates and provided the highest absorbance values. In future, all isolates can be used as KHV vaccine candidates to prevent KHV infection.

Phylogenetic Analysis of Thymidin Kinase Gene Koi HerpesVirus (KHV) in Some Freshwater Fish Aquaculture Centers in East Java

Koi Herpesvirus ( KHV ) is a virus that infects goldfish and koi that resulted in mass death and cause economic and social losses. The use of Polymerase Chain Reaction (PCR) technique to detect KHV was done in nila and gurame. Proof that KHV could infect nila and gurame KHV following genetic variation in each definitive host and spreading area have not been done. Based on these problems, this research was conducted to study the genetic variability and genetic relationships between KHV isolates in some freshwater fish as the definitive host koi, komet , koki, nila and gurame that exist in the area of East Java. The results showed that clinically the gills were pale and hemorrage the end of the tail fin. Electrophoresis results showed that all samples from KHV infected koi, koki, komet, nila and gurame were all positive but the sequencing results for tilapia and carp could not be further identified using the BLAST program. Genetically, koi fish, koki and komet isolates were identical to GenBank isolates code KHV-GZ11 and Indo_0K02SS.

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp, Cyprinus carpio L. using in situ hybridization.

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody-based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin-embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8h and following 10days post-infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1hpi. Respiratory epithelial cells were positive as early as 2hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.

CLONING, EXPRESSION AND PRELIMINARY ANTIGENICITY ANALYSIS OF STRUCTURAL PROTEINS OF A KOI HERPESVIRUS ISOLATE FROM KOI, CYPRINUS CARPIO IN TAIWAN

The aim of this study was to clone and express the ORF72 and ORF92 genes of koi herpesvirus (KHV) in a prokaryotic system and to examine the antigenicity of recombinant proteins. Phylogenetic analysis revealed that both ORF72 and ORF92 had 100% homology with KHV-J, and 99% homology with those from KHV-U and KHV-I in nucleotides. This suggests that the KHV isolate in Taiwan is more closely related to the Japanese strain (Asian genotype). In the antigenicity analysis, the crude recombinant ORF72 and ORF92 capsid proteins reacted with the positive sera of the survival fish after a KHV outbreak, indicating that these recombinant capsid proteins might mimic antigens of the wild type KHV to induce an immunological response in the infected host. Our results demonstrated potential for general applicability to serological tests and vaccine development.

Goldfish is Not a Susceptible Host of Koi Herpesvirus (KHV) Disease

We attempted to assess the risk that goldfish Carassius auratus potentially transmits KHV to naïve carp Cyprinus carpio. Goldfish including three varieties and koi carp were exposed to three KHV isolates from Japan, USA and Israel, and cohabited with recipient naïve ‘wild type’ common carp, a highly susceptible strain, from 2 to 24 days post viral exposure (dpe). All of the recipient carp cohabited with KHV-exposed koi carp died due to KHV infection, whereas no mortality was observed in the recipient carp cohabited with the exposed goldfish. In addition, no KHV was detected in organs of the goldfish at 24 dpe by PCR. These results indicate that goldfish is not a susceptible host.

Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae

The sequences of four complete genes were analysed in order to determine the relatedness of koi herpesvirus (KHV) to three fish viruses in the family Herpesviridae: carp pox herpesvirus (Cyprinid herpesvirus 1, CyHV-1), haematopoietic necrosis herpesvirus of goldfish (Cyprinid herpesvirus 2, CyHV-2) and channel catfish virus (Ictalurid herpesvirus 1, IcHV-1). The genes were predicted to encode a helicase, an intercapsomeric triplex protein, the DNA polymerase and the major capsid protein. The results showed that KHV is related closely to CyHV-1 and CyHV-2, and that the three cyprinid viruses are related, albeit more distantly, to IcHV-1. Twelve KHV isolates from four diverse geographical areas yielded identical sequences for a region of the DNA polymerase gene. These findings, with previously published morphological and biological data, indicate that KHV should join the group of related lower-vertebrate viruses in the family Herpesviridae under the formal designation Cyprinid herpesvirus 3 (CyHV-3).

Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.