Virulence and Infectivity of UC, MD, and L Strains of Infectious Hematopoietic Necrosis Virus (IHNV) in Four Populations of Columbia River Basin Chinook Salmon.

Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon (Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB.

Molecular evolution and selection pressure analysis of infectious hematopoietic necrosis virus (IHNV) revealed the origin and phylogenetic relationship of Iranian isolates in recent epidemics in Iran.

Infectious hematopoietic necrosis virus (IHNV) is the causative agent for a lethal salmonid disease. In this study, we surveyed the IHNV's epidemiology, diversity and the origin of infection in Iran. Phylogenetic analysis revealed that Iranian isolates belonged to one of the two lineages of E genogroup. Subsequently, a combination of phylogenetic, antigenic and structural analysis was performed to investigate the evolution of E genogroup lineages. Site-specific analysis of the viral glycoprotein showed different co-evolving and positively selected sites in each lineage. Most of these sites were mapped to the predicted antigenic patches of the glycoprotein. Further characterization revealed E lineages can be differentiated, in part, by specific mutations at positions 91 and 130, which are located in the structurally flexible regions of the glycoprotein, suggesting a key adaptative role for these sites. These data may assist in better monitoring the emerging isolates in regions infected to IHNV from E genogroup.

Phylogeography and evolution of infectious hematopoietic necrosis virus in China.

Infectious hematopoietic necrosis virus (IHNV) is a well-known rhabdoviral pathogen of salmonid fish. In this study, a comprehensive analysis of 40 IHNV viruses isolated from thirteen fish farms in nine geographically dispersed Chinese provinces during 2012 to 2017 is presented. Identity of nucleotide and amino acid sequences among all the complete glycoprotein (G) genes from Chinese isolates was 98.0-100% and 96.7-100%, respectively. Coalescent phylogenetic analyses revealed that all the Chinese IHN virus characterized in this study were in a monophyletic clade that had a most recent common ancestor with the J Nagano (JN) subgroup within the J genogroup of IHNV. Within the Chinese IHNV clade isolates obtained over successive years from the same salmon fish farm clustered in strongly supported subclades, suggesting maintenance and diversification of virus over time within individual farms. There was also evidence for regional virus transmission within provinces, and some cases of longer distance transmission between distant provinces, such as Gansu and Yunnan. The data demonstrated that IHNV has evolved into a new subgroup in salmon farm environments in China, and IHNV isolates are undergoing molecular evolution within fish farms. We suggest that Chinese IHNV comprises a separate JC subgroup within the J genogroup of IHNV.

Phylogeny of the infectious hematopoietic necrosis virus in European aquaculture.

Infectious hematopoietic necrosis (IHN)-a highly lethal infectious salmonid disease-has caused substantial economic losses in the European production of rainbow trout (Oncorhynchus mykiss) since the late 1980s. The causal agent of IHN is the IHN virus (IHNV) introduced from overseas. However, until today, its phylogeographic spread in Europe remains poorly understood. We therefore sought to elucidate this unresolved topic by using the largest ever compiled dataset of European IHNV isolates (E isolates) (193 GenBank E isolates and 100 isolates from this study) for the complete glycoprotein (G) gene sequence. Our results clearly revealed that the active trout trade has left its traces in the E phylogeny. For example, the spread by trade of IHNV-infected trout was apparently the cause for the exposure of the E lineage to different local scenarios of selection and genetic drift, and therefore has led to the split of this lineage into various subordinated lineages. Accordingly, we also found evidence for E isolates being mixed Europe-wide by cross-border introduction events. Moreover, there were indications that this propagation of the E lineage within Europe corresponded with an extensive and rapid spread event, already during or shortly after its formation. Finally, in accordance with the high substitution rate of IHNV determined by previous studies, our dataset indicates that the mean period of occurrence of a single E haplotype is typically not longer than one calendar year.

Infectious hematopoietic necrosis virus (IHNV) outbreak in farmed rainbow trout in Iran: Viral isolation, pathological findings, molecular confirmation, and genetic analysis.

Infectious hematopoietic necrosis virus (IHNV) is the etiological agent of a contagious disease (IHN) mainly in salmonid fish. In the present study, we isolated and identified IHNV in trout fry from Iranian trout farms with unexplained high mortality in 2016. The affected fry showed cumulative mortality of 90% with the gross pathological signs including exophthalmia and hemorrhage of the eye, skin darkening, abdominal distension, ulceration of the snout, and the visceral pallor and yellowish fluid in the intestine. Histopathological examination revealed marked necrosis in the anterior kidney, liver and spleen with the intracytoplasmic inclusion bodies in the liver sections. Also, intranuclear inclusion body and marginated chromatin were observable in the hematopoietic cells of the kidney. The homogenates tissues of infected fry induced IHNV-positive cytopathic effects (CPE) in EPC cells and confirmed by RT-PCR reactions and sequencing. Phylogenetic analysis revealed the Iranian IHNV isolates belonged to the European (E) genogroup with 100% identity to some Italian isolates. This is the first report of IHNV infection in farmed trout fry in Iran describing the viral isolation, clinical symptoms, histopathological findings, molecular confirmation, and genetic analysis suggestion of the specific country of origin.

Genetic relatedness of infectious hematopoietic necrosis virus (IHNV) from cultured salmonids in Korea.

Infectious hematopoietic necrosis virus (IHNV; n = 18) was identified in the Korean national surveillance program between February 2013 and April 2015, suggesting that IHNV is a major viral pathogen in cultured salmonids. By phylogeny analysis, we found that the JRt-Nagano and JRt-Shizuoka groups could each be further subdivided into three distinct subtypes. The Korean strains were genetically similar to Japanese isolates, suggesting introduction from Japan. Interestingly, the amino acid sequences of the middle glycoprotein gene show that distinct Korean subtypes have circulated, indicating that the settled IHNVs might be evolved stably in cultured salmonid farm environments.

Analysis of the genome sequence of infectious hematopoietic necrosis virus HLJ-09 in China.

Infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of juvenile salmonid fish. Six genome target fragments of the complete genome sequence of IHNV HLJ-09 were amplified by RT-PCR, and the 3'-terminal and 5'-terminal region of the genomic RNA were amplified using the RACE method. The complete genome sequence of HLJ-09 comprises 11,132 nucleotides (nt) (Accession number JX649101) and is different from that of other IHNV strains published in GenBank. Homology comparison and phylogenetic analysis of six ORF sequences were carried out using HLJ-09 and other IHNV strains published in GenBank. From phylogenetic tree analysis, the N gene, M gene, and P gene had the closest genetic relationship to IHNV-PRT from Korea. Phylogenetic analysis for the full length of the G gene showed that the HLJ-09 strain exhibited very close homology to the ChYa07, RtNag96, RtUi02, and RtGu01 strains from Korea and Japan, indicating that the HLJ-09 strain belonged to the genotype JRt. Ultimately, the Chinese IHNV HLJ-09 strain may have originated in Korea and Japan.

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands.

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put-and-take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put-and-take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co-infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV-positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co-infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus-introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.

Phylogenetic relationships of Iranian infectious hematopoietic necrosis virus of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

Infectious hematopoietic necrosis virus (IHNV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes a highly lethal disease of salmon and trout. In Iran IHNV was first detected in 2001 on farms rearing rainbow trout (Oncorhynchus mykiss). To evaluate the genetic relationships of IHNV from northern and western Iran, the sequences of a 651-nt region of the glycoprotein gene were determined for two Iranian isolates. These sequences were analyzed to evaluate their genetic relatedness to worldwide isolates representing the five known genogroups of IHNV. Iranian isolates were most closely related to European isolates within the genogroup E rather than those of North American genogroups U, M and L, or the Asian genogroup J. It appears that Iranian IHNV was most likely introduced to Iran from a source in Europe by the movement of contaminated fish eggs.

Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus.

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

Differential susceptibility in steelhead trout populations to an emergent MD strain of infectious hematopoietic necrosis virus.

A significant emergence of trout-adapted MD subgroup infectious hematopoietic necrosis virus (IHNV) began in the coastal region of Washington State, USA, in 2007. This emergence event lasted until 2011 and caused both asymptomatic adult fish infection and symptomatic epidemic disease and mortality in juvenile fish. Incidence of virus during this emergence demonstrated a heterogeneous distribution among rivers of the coastal region, leaving fish populations of some rivers apparently untouched while others suffered significant and recurrent infection and mortality (Breyta et. al. 2013; Dis Aquat Org 104:179-195). In this study, we examined the possible contribution of variations in susceptibility of fish populations, age-related resistance, and virus virulence to the observed landscape heterogeneity. We found that the most significant variable was host susceptibility: by controlled experimental challenge studies steelhead trout populations with no history of IHNV infection were 1 to 3 orders of magnitude more sensitive than a fish population with a long history of IHNV infection. In addition, 2 fish populations from the same river, which descended relatively recently from a common ancestral population, demonstrated 1 to 2 orders of magnitude difference in susceptibility. Fish age-related development of resistance was most evident in the more susceptible of 2 related fish populations. Finally, the strain of virus involved in the 2007 coastal Washington emergence had high virulence but was within the range of other known M group viruses tested. These results suggest that one major driver of landscape heterogeneity in the 2007 coastal Washington IHNV emergence was variation in fish population susceptibility and that this trait may have a heritable component.

Determination of the complete genome sequence of infectious hematopoietic necrosis virus (IHNV) Ch20101008 and viral molecular evolution in China.

This study determined the complete genomic sequence of the infectious hematopoietic necrosis virus (IHNV) strain Ch20101008 isolated from farmed brook trout (Salvelinus fontinalis) that died from a disease caused by the virus in northeast China. The sequence was determined from 10 overlapping clones obtained through RT-PCR amplification. The whole genome length of Ch20101008 comprised 11,129 nucleotides (nt), and the overall organization was typical of that observed for all other IHNV strains. The phylogenetic analysis results of the 65 IHNV glycoprotein genes and 47 IHNV partial nucleoprotein genes presented five major genogroups (J, U, L, E and M). The J genogroup included the J Nagano and J Shizuoka subgroups. The IHNV Ch20101008 strain belonged to the J Nagano subgroup of the J genogroup and was significantly related to other Chinese IHNV strains. All Chinese IHNV isolates are monophyletic, with a recent common ancestor, except for the BjLL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in GenBank. The results indicated that 198 nt were substituted, 53 of which exhibited amino acid change in the Ch20101008 genome. An adenine nucleotide deletion was found at position 4959 of the 5' UTR of the L gene. In the G gene, specific nucleotides and amino acid variations of the Chinese IHNV strains were observed when compared with 23 isolates from other countries. Of the 15 nucleotide sites that changed, seven resulted in amino acid substitution. The data further demonstrated that the J genogroup IHNV was introduced to and evolved in salmon farm environments in China.

Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus.

Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution.

Dating the divergence of the infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an economically significant rhabdovirus that can cause severe disease to most salmonid fish. Phylogenetic analyses of worldwide IHNV isolates have defined five major genogroups. Herein, to further the understanding of the molecular epidemiology and evolution of IHNV, Bayesian coalescent analyses were conducted to the time-stamped coding sequences of the N, G and NV genes. The nucleotide substitution rates and the divergence times were assessed. Among the three genes, NV, the smallest one coding for a non-virion protein, was conferred the highest mean rate. Estimates for the G subsets based upon the five genogroups indicated that L and U evolved much slower than the others. Age calculations suggested that the first diversification event of the IHNV isolates analyzed might have happened before the notification of the disease during the early 1950s. Selection analyses suggested that the three genes were mainly subject to purifying selection. In addition, surveys of codon usage variation showed that the three genes had influences other than mutational bias.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Transcriptome analysis of rainbow trout infected with high and low virulence strains of infectious hematopoietic necrosis virus.

There are three main genetic lineages or genogroups of Infectious hematopoietic necrosis virus (IHNV) in N. America. Strains representing the M genogroup are more virulent in rainbow trout relative to the U genogroup. In this study, we used microarray analysis to evaluate potential mechanisms responsible for host-specific virulence in rainbow trout that were given intraperitoneal injections of buffer or a representative M or U type virus strain. Reverse transcriptase quantitative PCR (RT-qPCR) was used to assess viral load and gene expression of select immune genes. Viral load was significantly higher in trout infected with the M virus starting at 24h post-infection (p.i.) and continuing until 72 h p.i. Microarray analysis of the 48 h time point revealed 153 up-regulated and 248 down-regulated features in response to M virus infection but only 62 up-regulated and 49 down-regulated features following U virus infection. Translation and transcription features were among the most frequent down-regulated features in response to M virus infection and may be associated with the host cell shutoff phenomenon. A greater host cell shutoff response by the M virus may facilitate subversion of the host cell transcriptional machinery and enhance viral replication, suggesting the M virus may be better optimized to manipulate the rainbow trout transcriptional and translational machinery. Anti-viral associated features were the most commonly up-regulated features. A common set of features were up-regulated in both the M and U infection groups, but were induced to a higher magnitude in the M infection group. Gene expression of the anti-viral genes Mx-1 and Vig-1 was correlated but not entirely dependent on viral load in the anterior kidney. Slower replication of the U virus may allow the host more time to induce protective anti-viral immune mechanisms.

Resistance and protective immunity in Redfish Lake sockeye salmon exposed to M type infectious hematopoietic necrosis virus (IHNV).

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolates from the U and M phylogenetic subgroups is clearly evident in the Redfish Lake (RFL) strain of sockeye salmon Oncorhynchus nerka. In these fish, experimental immersion challenges with U isolates cause extremely high mortality and M isolates cause low or no mortality. When survivors of M virus immersion challenges were exposed to a secondary challenge with virulent U type virus they experienced high mortality, indicating that the primary M challenge did not elicit protective immunity. Delivery of a moderate dose (2 x 10(4) plaque-forming units [PFU]/fish) of virus by intraperitoneal injection challenge did not overcome RFL sockeye salmon resistance to M type IHNV. Injection challenge with a high dose (5 X 10(6) PFU/fish) of M type virus caused 10% mortality, and in this case survivors did develop protective immunity against a secondary U type virus challenge. Thus, although it is possible for M type IHNV to elicit cross-protective immunity in this disease model, it does not develop after immersion challenge despite entry, transient replication of M virus to low levels, stimulation of innate immune genes, and development of neutralizing antibodies in some fish.

Genetic signature of rapid IHHNV (infectious hypodermal and hematopoietic necrosis virus) expansion in wild Penaeus shrimp populations.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39x10(-4) substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.

Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic necrosis virus (IHNV).

The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems, partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype reached higher densities in both single and mixed infections. There was no effect of competition on the performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and virulence.

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout-derived cell line, RTG-2.

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.