Phylogenetic analysis and genotyping of Iranian infectious haematopoietic necrosis virus (IHNV) of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene

Abstract Background Infectious haematopoietic necrosis (IHN) is known as one of the most contagious systemic viral diseases in salmonids which can lead to significant mortality rates and negative impacts on the salmonid farming industry. Infectious haematopoietic necrosis virus (IHNV) was first detected in rainbow trout (Oncorhynchus mykiss) farms in Iran in 2003. Objectives We conducted the present study to determine the detection of IHN genotypes in rainbow trout (O. mykiss) in farms in the central parts of Iran, using molecular and phylogenetic techniques. Methods Samples were collected from fries exhibiting clinical signs such as darkening of the skin, abdominal swelling, and loss of appetite. Phylogenetic analysis was performed by the neighbour‐joining method, using MEGA 5.1 software. For phylogenetic analysis and genotyping of IHNV from central parts of Iran, the sequences of the glycoprotein gene were determined for two Iranian isolates (Jahad‐UT1 and Jahad‐UT2). Results Phylogenetic analysis revealed that the detected strains (Jahad‐UT1 and Jahad‐UT2 isolates) are closely related (97.23%–100%) to European isolates within genogroup ‘E’. Conclusions This finding indicates that Jahad‐UT1 and Jahad‐UT2 isolates have been widely transferred to Iran from European countries. Moreover, the nucleotide diversity of these Iranian isolates showed a close relationship with the North American and Asian isolates, although the Iranian isolates were collected from a smaller geographical area and within a shorter time period between 2014 and 2015.


Infectious haematopoietic necrosis (IHN) is one of the most conta-
gious systemic viral diseases in salmonids, which can lead to significant mortality rates and negative impacts on the salmonid farming industry (Amar, Kiron, Akutsu, Satoh, and Watanabe, 2012;Bootland and Leong, 2011;OIE, 2019;Ahmadivand et al., 2017). Infectious haematopoietic necrosis virus (IHNV), the known causative agent of the disease, is a single-stranded negative-sense RNA virus belonging to the genus Novirhabdovirus of the family Rhabdoviridae (Wolf, 1988;OIE, 2019).
This viral disease is usually associated with a range of clinical signs, including skin darkening, lethargy, exophthalmia, pale gills, distended abdomen and petechial haemorrhages leading to mass mortalities.
However, mortalities may also occur in the absence of significant clinical signs (Purcell et al., 2013). The natural host range of IHNV includes many species of wild and farmed salmon and trout . In the early 1950s, infectious haematopoietic necrosis virus was detected primarily in sockeye salmon (Oncorhynchus nerka) hatcheries in western North America (Rucker, Whipple, Parvin, and Evans, 1953). At that time, the virus spread to Asian and European countries via the transport of contaminated fish or eggs (Bovo, Giorgetti, Jørgensen, and Olesen, 1987;Rudakova, Kurath, and Bochkova, 2007). In Europe, the virus was reported in France and Italy in 1987 and later, in Germany in 1992 (Enzmann, Kurath, Fichtner, and Bergmann, 2005).
Diseased fish exhibit bleeding of skin lesions, exophthalmia, abdominal distention, pale gills, skin darkening and extensive necrosis of haematopoietic tissues such as liver, pancreas, and granular cells of the lamina propria of the intestine (Amend and Smith, 1975). An effective method to prevent IHNV is the vaccination of healthy fish, while there are no definitive treatment protocols (Wang et al., 2016). This study aimed to examine the sequence of Jahad-UT1 and Jahad-UT2 strains of IHNV and then to perform a comparative sequence analysis and a phylogenetic tree analysis between these strains and other reference strains available in GenBank.

Fish source and sampling
During the period from September 2014 to December 2015, an outbreak with mass mortality (70%-90%) occurred in rainbow trout (Oncorhynchus mykiss) farms in Tehran, Iran province ( Figure 1). The farms were operated with a flow-through system for fresh water at a temperature between 10 • C and 12 • C. Five moribund fish were selected from each farm and transferred to the virology laboratory.
Samples were collected from fries suspected of having IHN and exhibiting clinical signs such as darkening of the skin, abdominal swelling and loss of appetite. The samples were stored at −70 • C for further analysis. Pools of 10 whole fry samples were also used for molecular detection. The mixture was then incubated at 25 • C for 5 min, 42 • C for 60 min, 95 • C for 5 min and 4 • C for 1 min. The cDNA was stored at −20 • C until use.

RT-PCR and sequencing
The reverse transcription polymerase chain reaction (RT-PCR) for detecting IHNV was based on the OIE-recommended primers. The 1.5% agarose gel and visualised under UV light. Subsequently, the PCR products were analysed to determine the nucleotide sequences.

Phylogenetic analysis
Phylogenetic analysis was performed by the neighbour-joining method, using MEGA 5.1 software, and each tree was constructed with a consensus of 1000 bootstrap repeats (Tamura et al., 2011

RESULTS
Out of 10 pooled samples tested, 2 kidney tissues showed positive results in RT-PCR (20%). Both were sent for sequencing, and their sequences were submitted to GenBank under accession numbers KX901448.1 and KX901449.1. Glycoprotein gene comparisons revealed that the viruses detected in this study belonged to genogroup 'E' (Figure 2). In addition, two strains from this study had the highest nucleotide similarity with IHNV detected in Iran in 2014 (Table 1).
The global spread of novirhabdoviruses caused by aquaculture activities, notably the trade-in eyed eggs, has been proven by the genotyping of virus strains (Bovo et al., 2021;Kurath and Winton, 2008;Winton, 1991). With an annual demand for 300-400 million eyed eggs, of which 70% are imported from European nations, Iran is one of the world's top producers of freshwater rainbow trout (Ahmadivand et al., , 2017FAO, 2020). However, some of these imports are probably not under sufficient veterinary care because of the economic and political situation. Additionally, Iranian fish farms are closely connected and exchange eggs, fry and fish annually, creating several opportunities for the virus to spread throughout the industry (Ahmadivand, Palić, and Weidmann, 2021). Therefore, the results of the current study highlight the need to implement strict regulations on the trade of fish eggs to prevent the spread of the virus to other regions of the world where rainbow trout (Oncorhynchus mykiss) are farmed.

AUTHOR CONTRIBUTIONS
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Zahra Ziafati Kafi, Arash Ghalyanchilangeroudi, Donya Nikaein, Naser Sadri, Ahmad Erfanmanesh and Ala Enayati. The first draft of the manuscript was written by Amin Marandi and edited by Hooman Rahmati-Holasoo, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.