Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth's average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector-virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy.

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

Zika Virus and Host Interactions: From the Bench to the Bedside and Beyond.

Before the emergence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of the current COVID-19 (coronavirus disease 2019) pandemic [...].

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

7-Deaza-7-fluoro-2'-C-methyladenosine inhibits Zika virus infection and viral-induced neuroinflammation.

Zika virus (ZIKV) has gained a lot of attention in the past few years due to its rapid spread worldwide and its close association to severe neurological outcomes, such as microcephaly and Guillain-Barre syndrome. In this study, the in vitro and in vivo anti-ZIKV activity of 7-deaza-7-fluoro-2'-C-methyl-adenosine (DFMA) was evaluated. In vitro, using primary mouse neuronal cells and human neural stem cells infected by ZIKV, treatment with DFMA resulted in impaired viral replication and protection against virus-induced cell death. In vivo, when administrated prior to infection, DFMA prevented lethality and markedly reduced viral loads and neuroinflammation, including microgliosis and overall brain damage. Additionally, as an early therapeutic treatment, DFMA increased survival rates in mice. Collectively, these findings demonstrate that the nucleoside analog DFMA inhibits ZIKV infection and viral-induced neuroinflammation in vitro and in vivo without apparent untoward effects, suggesting it may be useful in individuals infected with ZIKV.

An RNA Thermometer Activity of the West Nile Virus Genomic 3'-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching.

The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

Quantitative Inventory of Mosquito Larvae (Diptera: Culicidae) and Physicochemical Analysis of Aquatic Habitats in the Region of Fez, Morocco.

Mosquitoes occupy a major health problem, as a potential vector for the transmission of many pathogens, the most important of which in Morocco are Plasmodium parasites and West Nile fever virus. Acquiring knowledge about the biotypology of these mosquitoes is an essential step in any control strategy. Thus, the objective of this study is to draw up an inventory of the culicidian fauna in the Fez region (North-central part of Morocco) and to determine the physicochemical parameters influencing the production of mosquito larvae of aquatic habitats in this area. Mosquito larvae of the extant biotopes were sampled every two months between November 2015 and December 2016, by means of the "dipping" method. The physicochemical parameters of the various larval biotopes were collected six times during the year of follow-up. Mosquito species were identified using morphological criteria. The results were analyzed using ecological indices and the generalized linear model (Poisson model) on R software. The identification of the 1,122 mosquito larvae collected from the various studied breeding areas revealed the presence of 11 species belonging to two subfamilies (Culicinae and Anophelinae) and five genera (Anopheles, Culex, Culiseta, Aedes, and Uranotaenia). Anopheles were observed in small numbers in permanent, stagnant, and shallow habitats. The larvae of An. maculipennis s. l. (major vector of malaria in Morocco) were collected. Culex larvae, especially Cx. pipiens (the main vector of WNV in Morocco), were collected in the majority of the larval habitats and on the different types of environments. Two other species of Culex, which plays a significant role in the transmission of WNV, were also collected, particularly, Cx. perexiguus and Cx. theileri. The results found, via this first study of its kind at the regional level, also made it possible to update the list of Culicidae species reported in the Fez region. Four new species have been recorded: Aedes flavescens, Uranotaenia unguiculata, Culiseta longiareolata, and Culex perexiguus. The results of this study will contribute to the provision of information for entomological surveillance and to better plan and guide vector control activities at local and national levels.

Les moustiques constituent un problème majeur de santé, en tant que vecteur potentiel de la transmission de nombreux agents pathogènes dont les plus importants au Maroc sont le Plasmodium et le virus de la fièvre du West Nile. Acquérir des connaissances sur la biotypologie de ces moustiques est une étape essentielle dans toute stratégie de lutte. Ainsi, l'objectif de ce travail est de dresser l'inventaire de la faune culicidienne dans la région de Fès (Centre Nord du Maroc) et de déterminer les paramètres physicochimiques influençant la production en larves de moustiques des habitats aquatiques dans cette zone. L'échantillonnage des larves a été réalisé tous les deux mois dans les différents biotopes existants, entre novembre 2015 et décembre 2016, à l'aide de la méthode de dipping. Les paramètres physicochimiques des différents biotopes larvaires ont été relevés six fois durant l'année du suivi. L'identification des caractères morphologiques des larves a été déterminée. Les résultats ont été analysés moyennant des indices écologiques et le modèle linéaire généralisé (GLM), suite à un modèle de Poisson sur le logiciel R. L'identification des 1 122 larves de culicidés récoltées au niveau des différents gîtes prospectés a révélé la présence de 11 espèces appartenant à deux sous-familles (Culicinae et Anophelinae) et cinq genres (Anopheles, Culex, Culiseta, Aedes et Uranotaenia). Les anophèles ont été observés en faible nombre dans des habitats permanents, à eaux stagnantes, et peu profondes. Les larves d'An. maculipennis s. l. (vecteur majeur du paludisme au Maroc) ont été récoltées. Les larves de Culex, en particulier de Cx. pipiens (principal vecteur du WNV au Maroc), ont été collectées dans la majorité des gîtes prospectés et dans les différents types de milieux. Deux autres espèces de Culex, dont le rôle dans la transmission du WNV est reconnu, ont été également collectées au cours de cette étude ; il s'agit de Cx. perexiguus et Cx. theileri. Les résultats trouvés via cette étude, première du genre au niveau de la région, ont permis également de mettre à jour la liste des espèces culicidiennes signalées au niveau de la région de Fès par l'ajout de quatre espèces : Aedes flavescens, Uranotaenia unguiculata Culiseta longiareolata et Culex perexiguus. Les résultats de cette étude contribueront à l'apport d'informations pour la surveillance entomologique et pour mieux planifier et orienter les activités de la lutte antivectorielle au niveau local et national.

Insect anionic septapeptides suppress DENV replication by activating antiviral cytokines and miRNAs in primary human monocytes.

Dengue viruses (DENVs) have threatened 2/3 of the world population for decades. Thus, combating DENV infection with either antiviral therapy or protective vaccination is an urgent goal. In the present study, we investigated the anti-DENV activity of insect cell-derived anionic septapeptides from C6/36 mosquito cell cultures persistently infected with DENV. These molecules were previously shown to protect C6/36 and Vero cells against DENV infection. We found that treatment with these septapeptides strongly and rapidly downregulated the multiplication of DENV-1 16007, DENV-3 16562, and DENV-4 1036 but not that of DENV-2 16681 in primary human monocytes. This inhibitory effect was likely mediated through various routes including the increased production of antiviral cytokines (IFN-I), activation of mononuclear cell migration, and upregulation of the expression of antiviral miRNAs (has-miR-30e*, has-miR-133a, and has-miR-223) and inflammation-related miRNAs (has-miR-146a and has-miR-147). In conclusion, anionic septapeptides exerted anti-DENV activity in human monocytes through the upregulation of innate immune responses and the activation of several previously reported antiviral and inflammation-related miRNAs.

Antimalarial drugs and their metabolites are potent Zika virus inhibitors.

Studies aimed at repurposing existing drugs revealed that some antimalarial compounds possess anti-Zika virus (anti-ZIKV) activity. Here, we further tested 14 additional antimalarial drugs and their metabolites or analogs for anti-ZIKV activity using a phenotypic screening approach. We identified four compounds with varying anti-ZIKV activity, including a metabolite of amodiaquine termed desethylamodiaquine (DAQ) and N-desethylchloroquine (DECQ), a metabolite of chloroquine, which both exhibited low micromolar effective concentrations against three different ZIKV strains. Two other compounds termed dihydroartemisinin (DHA) and quinidine (QD) exhibited only partial inhibition of ZIKV replication. Characterization of the inhibitory mechanisms of DAQ and DECQ showed that both drugs target the entry step as well as postentry events of the viral replication cycle. These hits represent attractive starting points for future optimization of new anti-ZIKV drug candidates derived from antimalarial drugs and their analogs.

Antiviral RNA Interference Activity in Cells of the Predatory Mosquito, Toxorhynchites amboinensis.

Arthropod vectors control the replication of arboviruses through their innate antiviral immune responses. In particular, the RNA interference (RNAi) pathways are of notable significance for the control of viral infections. Although much has been done to understand the role of RNAi in vector populations, little is known about its importance in non-vector mosquito species. In this study, we investigated the presence of an RNAi response in Toxorhynchites amboinensis, which is a non-blood feeding species proposed as a biological control agent against pest mosquitoes. Using a derived cell line (TRA-171), we demonstrate that these mosquitoes possess a functional RNAi response that is active against a mosquito-borne alphavirus, Semliki Forest virus. As observed in vector mosquito species, small RNAs are produced that target viral sequences. The size and characteristics of these small RNAs indicate that both the siRNA and piRNA pathways are induced in response to infection. Taken together, this data suggests that Tx. amboinensis are able to control viral infections in a similar way to natural arbovirus vector mosquito species. Understanding their ability to manage arboviral infections will be advantageous when assessing these and similar species as biological control agents.

Development of standard methods for Zika virus propagation, titration, and purification.

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.

Analysis of Pesticides and Toxic Heavy Metals Contained in Mosquito Coils.

In this study, 10 mosquito coils manufactured in China were obtained in Suriname, South America, where they are used extensively. The coils were analyzed for organics (allethrin, permethrin, and butylated hydroxytoluene) and heavy metals (Cr, Co, As, Cd, and Pb) by GC-MS and ICP-MS, respectively. Allethrin was the only target organic compound detected in all mosquito coils with concentrations ranging from ~1900 to ~4500 µg/g. The concentrations of heavy metals varied as follows (in µg/g): Cr: 2.9-9.4, Co: 0.1-1.2, Cu: 0.7-16.1, Se: 0.10-0.4, Ni: 2.1-5.8, As: 0.10-2.2, Cd: 0.10-0.2, and Pb: 1.1-3.6.

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.

Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

Sub-genomic RNA of defective interfering (D.I.) dengue viral particles is replicated in the same manner as full length genomes.

The predicted secondary structure of sub-genomic RNA in dengue virus defective interfering (D.I.) particles from patients, or generated in vitro, resembled that of the 3' and 5' regions of wild type dengue virus (DENV) genomes. While these structures in the sub-genomic RNA were found to be essential for its replication, their nucleotide sequences were not, so long as any new sequences maintained wild type RNA secondary structure. These observations suggested that these sub-genomic fragments of RNA from dengue viruses were replicated in the same manner as the full length genomes of their wild type, "helper", viruses and that they probably represent the smallest fragments of DENV RNA that can be replicated during a natural infection. While D.I. particles containing sub-genomic RNA are completely parasitic, the relationship between wild type and D.I. DENV may be symbiotic, with the D.I. particles enhancing the transmission of infectious DENV.

Mosquito cell lines: history, isolation, availability and application to assess the threat of arboviral transmission in the United Kingdom.

Mosquitoes are highly effective vectors for transmission of human and animal pathogens. Understanding the relationship between pathogen and vector is vital in developing strategies to predict and prevent transmission. Cell lines derived from appropriate mosquito hosts provide a relatively simple tool for investigating the interaction between the host and viruses transmitted by mosquitoes. This review provides a brief overview of the development of mosquito cell lines, methods of isolation, their availability and application for investigating insect-virus interactions.

Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses.

In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated 'Nhumirim virus'; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus's capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 10(6)-fold and 10(4)-fold reduction in peak titres, respectively.

A new species of mesonivirus from the Northern Territory, Australia.

Here we describe Casuarina virus (CASV), a new virus in the family Mesoniviridae. This is the first report of a mesonivirus in Australia, which extends the geographical range of this virus family to 3 continents. The virus was isolated in 2010 from Coquillettidia xanthogaster mosquitoes during surveillance in the suburbs of Darwin, the capital of the Northern Territory. Cryo-electron microscopy of the CASV virions revealed spherical particles of 65 nm in size with large club-shaped projections of approximately 15 nm in length. The new virus was most closely related to Alphamesonivirus 1, the only currently recognized species in the family. In 2013 a further 5 putative new mesonivirus species were described: Hana, Méno, Nsé, Moumo and Dak Nong viruses. The evolutionary distance between CASV and two of its closest relatives, Cavally and Hana viruses (Jones-Taylor-Thornton distance of 0.151 and 0.224, respectively), along with its isolation from a different genus of mosquitoes captured on a separate continent indicate that CASV is a new species.

Chloroquine inhibits dengue virus type 2 replication in Vero cells but not in C6/36 cells.

Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 µg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.

Non-permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus.

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi-square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10(5)  cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10(5)  cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10(4) and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.