Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine

BACKGROUND: Sucrose density gradient centrifugation and cross-flow filtration methods have been developed and standardised for the safe and reproducible production of inactivated arbovirus antigens which are appropriate for use in diagnostic serological applications. METHODS: To optimise the maximum titre of growth during the propagation of arboviruses, the multiplicity of infection and choice of cell line were investigated using stocks of Ross River virus and Barmah Forest virus grown in both mosquito and mammalian cell lines. To standardise and improve the efficacy of the inactivation of arboviral suspensions, stocks of Ross River virus, Barmah Forest virus, Japanese encephalitis virus, Murray Valley encephalitis virus and Alfuy virus were chemically inactivated using binary ethylenimine at a final concentration of 3 mM. Aliquots were then taken at hourly intervals and crude inactivation rates were determined for each virus using a plaque assay. To ensure complete inactivation, the same aliquots were each passaged 3 times in Aedes albopictus C6/36 cells and the presence of viral growth was detected using an immunofluorescent assay. For larger quantities of viral suspensions, centrifugation on an isopycnic sucrose density gradient or cross-flow filtration was used to produce concentrated, pure antigens or partially concentrated, semi-purified antigens respectively. RESULTS: The results of the propagation experiments suggested that the maximum viral titres obtained for both Ross River virus and Barmah Forest virus were affected by the incubation period and choice of cell line, rather than the use of different multiplicity of infection values. Results of the binary ethylenimine inactivation trial suggested that standardised periods of 5 or 8 hours would be suitable to ensure effective and complete inactivation for a number of different arboviral antigens. CONCLUSION: Two methods used to prepare inactivated arbovirus antigens have been standardised to minimise production failure and expenditure and to provide reagents that conform to the highest quality and safety requirements of a diagnostic serology laboratory. The antigens are suitable for use in either enzyme linked immunosorbent assays or haemagglutination inhibition assays and the optimised protocols can be directly applied to produce antigens from new or emerging arboviral pathogens

Complete coding sequence of a case of chikungunya virus imported into Australia

A case of chikungunya virus infection was imported from India into Australia in late 2016. Infection was diagnosed by real-time reverse transcription-PCR and confirmed by culture isolation and genome sequencing. Phylogenetic analysis of the genome sequence indicated that the virus grouped with the east/central/south African genotype

Chikungunya Virus Transmission at Low Temperature by Aedes albopictus Mosquitoes.

Aedes albopictus is an important vector of chikungunya virus (CHIKV). In Australia, Ae. albopictus is currently only known to be present on the islands of the Torres Strait but, should it invade the mainland, it is projected to spread to temperate regions. The ability of Australian Ae. albopictus to transmit CHIKV at the lower temperatures typical of temperate areas has not been assessed. Ae. albopictus mosquitoes were orally challenged with a CHIKV strain from either Asian or East/Central/South African (ECSA) genotypes (107 pfu/mL), and maintained at a constant temperature of either 18 °C or 28 °C. At 3- and 7-days post-infection (dpi), CHIKV RNA copies were quantified in mosquito bodies, and wings and legs using real time polymerase chain reaction (qRT-PCR), while the detection of virus in saliva (a proxy for transmission) was performed by amplification in cell culture followed by observation of cytopathic effect in Vero cells. Of the ≥95% of Ae. albopictus that survived to 7 dpi, all mosquitoes became infected and showed body dissemination of CHIKV at both temperatures and time points. Both the Asian and ECSA CHIKV genotypes were potentially transmissible by Australian Ae. albopictus at 28 °C within 3 days of oral challenge. In contrast, at 18 °C none of the mosquitoes showed evidence of ability to transmit either genotype of CHIKV at 3 dpi. Further, at 18 °C only Ae. albopictus infected with the ECSA genotype showed evidence of virus in saliva at 7 dpi. Overall, infection with the ECSA CHIKV genotype produced higher virus loads in mosquitoes compared to infection with the Asian CHIKV genotype. Our results suggest that lower ambient temperatures may impede transmission of some CHIKV strains by Ae. albopictus at early time points post infection

Temperature alters gene expression in mosquitoes during arbovirus infection

ABSTRACTArthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya constitute a significant proportion of the global disease burden. The principal vector of these pathogens is the mosquito Aedes (Ae.) aegypti, and its ability to transmit virus to a human host is influenced by environmental factors such as temperature. However, exactly how ambient temperature influences virus replication within mosquitoes remains poorly elucidated, particularly at the molecular level. Here, we use chikungunya virus (CHIKV) as a model to understand how the host mosquito transcriptome responds to arbovirus infection under different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18 °C, 28 °C and 32 °C, and found higher temperature correlated with higher virus replication levels, particularly at early time points post-infection. Lower ambient temperatures resulted in reduced virus replication levels. Using RNAseq, we found that temperature significantly altered gene expression levels in mosquitoes, particularly components of the immune response. The highest number of significantly differentially expressed genes in response to CHIKV was observed at 28 °C, with a markedly more muted effect observed at either lower (18 °C) or higher (32 °C) temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2 in the RNAi pathway, was not substantially altered in response to CHIKV. Upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28 °C. Time post infection also led to substantially different gene expression profiles, and this effect varied depending upon the which temperature mosquitoes were exposed to. Taken together, our data indicate temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher ambient temperatures.</jats:p

Temperature modulates immune gene expression in mosquitoes during arbovirus infection.

The principal vector of dengue, Zika and chikungunya viruses is the mosquito Aedes aegypti, with its ability to transmit pathogens influenced by ambient temperature. We use chikungunya virus (CHIKV) to understand how the mosquito transcriptome responds to arbovirus infection at different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18, 28 and 32°C, and found that higher temperature correlated with higher virus levels, particularly at 3 days post infection, but lower temperature resulted in reduced virus levels. RNAseq analysis indicated significantly altered gene expression levels in CHIKV infection. The highest number of significantly differentially expressed genes was observed at 28°C, with a more muted effect at the other temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2, was not substantially altered in response to CHIKV. The upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28°C. Functional annotations suggested that genes in immune response and metabolic pathways related to energy supply and DNA replication were involved in temperature-dependent changes. Time post infection also led to substantially different gene expression profiles, and this varied with temperature. In conclusion, temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher temperatures

The Molecular Epidemiology and Evolution of Murray Valley Encephalitis Virus: Recent Emergence of Distinct Sub-lineages of the Dominant Genotype 1

© 2015 Williams et al. Background: Recent increased activity of the mosquito-borne Murray Valley encephalitis virus (MVEV) in Australia has renewed concerns regarding its potential to spread and cause disease. Methodology/Principal Findings: To better understand the genetic relationships between earlier and more recent circulating strains, patterns of virus movement, as well as the molecular basis of MVEV evolution, complete pre-membrane (prM) and Envelope (Env) genes were sequenced from sixty-six MVEV strains from different regions of the Australasian region, isolated over a sixty year period (1951–2011). Phylogenetic analyses indicated that, of the four recognized genotypes, only G1 and G2 are contemporary. G1 viruses were dominant over the sampling period and found across the known geographic range of MVEV. Two distinct sub-lineages of G1 were observed (1A and 1B). Although G1B strains have been isolated from across mainland Australia, Australian G1A strains have not been detected outside northwest Australia. Similarly, G2 is comprised of only Western Australian isolates from mosquitoes, suggesting G1B and G2 viruses have geographic or ecological restrictions. No evidence of recombination was found and a single amino acid substitution in the Env protein (S332G) was found to be under positive selection, while several others were found to be under directional evolution. Evolutionary analyses indicated that extant genotypes of MVEV began to diverge from a common ancestor approximately 200 years ago. G2 was the first genotype to diverge, followed by G3 and G4, and finally G1, from which subtypes G1A and G1B diverged between 1964 and 1994. Conclusions/Significance: The results of this study provides new insights into the genetic diversity and evolution of MVEV. The demonstration of co-circulation of all contemporary genetic lineages of MVEV in northwestern Australia, supports the contention that this region is the enzootic focus for this virus

Replication Limited, Trans-Complemented Ross River Virus

Conventionally, live virus propagation in either cell culture or animal systems has been employed for the production of Ross River virus (RRV) antigens for use in diagnostic applications. Due to the continual increase in safety and biological containment regulations imposed within modern virology laboratories, now only a few certified facilities remain within Australia where the growth and manipulation of live virus can be performed. Recently, rigorous binary ethylenimine (BEI) inactivation procedures have been included in the manufacture of RRV antigens to minimise viral safety risks, however, these methods pose other potential health hazards and may result in the loss of important antigenic epitopes. In the present study, an alternative method which could transpose the infectious propagation and time-consuming inactivation techniques currently used for RRV antigen production was sought. A unique replication system was developed permitting continued propagation of non-infectious, wild-type (wt) RRV particles. Firstly, a full-length RRV cDNA infectious clone (pCMV-RRV), which retained the genetic and biological properties of the RRV prototype T48 strain (originally isolated by Doherty R.L., 1959), was constructed under the control of the immediate early human cytomegalovirus (CMV) promoter. An essential gene, namely the structural envelope (E) E1 gene, was then deleted to facilitate the production of a mutant construct, pCMV-RRVΔE1. Normally, this lethal E1 gene deletion would disable infectious RNA transcription and prevent virus formation. However, further investigations revealed that RNA derived from pCMV-RRVΔE1 was capable of trans-complementation in the presence of E1 and therefore could be used to generate replication deficient, RRV particles. In order to perform the pCMV-RRVΔE1 trans-complementation experiments, a tetracycline inducible cell line (6K-E1 6B), constitutively expressing the RRV E1 glycoprotein was developed using human equine kidney (HEK) 293T-REX ™ cells (Invitrogen, California). The expression vector engineered to develop the transgenic cells included the 6K gene translocation signal sequence immediately upstream of the E1 gene sequence. Rescue of defective replicon RNA was achieved following transfection of pCMV-RRVΔE1 DNA into 6K-E1 6B cells and resulted in the formation of transcomplemented RRV (RRVcomp) particles. Examination of infected cell sections by electron microscopy (EM) revealed the presence of cytoplasmic viral RNA complexes known as spherules and virus-like structures which were morphologically similar to viral particles derived from the parent pCMV-RRV clone. Interestingly, budding of RRVcomp from the plasma membrane (PM) could not be demonstrated which inferred that particles may have budded via a defective or alternate mechanism. In addition, conspicuous cytoplasmic vacuoles (CVs) were present in many uninfected and RRVcomp infected 6K-E1 6B cells which suggested intracellular retention of the transgencially expressed E1. However, reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of nucleic acid recovered from RRVcomp provided evidence that the E1 gene sequence deletion had been maintained following ten passages. Furthermore, transmission could not be detected when RRVcomp was serially passaged in baby hamster kidney (BHK) cells which strongly suggested that replication of the recombinant was exclusively limited and therefore could only occur within the complementing 6K-E1 6B cells. Significantly, the nucleotide sequence analysis of the RRVcomp genome also revealed the presence of a single base insertion in the 3’ terminal end of the 6K gene sequence. This unprecedented mutation was shown to result from RRVcomp replication in vivo and was introduced into the replicon genome after passage 3. Given this evidence, it is highly likely that translation and processing of the RRVcomp structural C-PE2-6K polyprotein was adversely affected and it is doubtful that a functional 6K species was produced. Indeed, failure to synthesise an authentic 6K would probably have disrupted normal viral assembly processes and perhaps this could account for the budding defects observed by EM. The absence of 6K may also have led to inefficient membrane fusion events by reducing the stability of the RRVcomp trimer complex. Conceivably, this could partially explain why appreciable RRVcomp titres were not produced following growth curve experiments. However, despite these abnormal RRVcomp growth characteristics, it could be argued that the 6K mutation was an acquired adaptation which promoted host cell signalase cleavage of the PE2-6K precursor protein. Logically, this would be a favoured event and may have led to more efficient heterodimerization between RRVcomp derived E2 and complementing E1 in trans. Although originating from separate plasmid constructs, the production of both structural glycoproteins E1 and E2 from RRVcomp infected 6K-E1 6B cells was demonstrated by western blot analyses. A protein with a molecular weight (MW) of 49 kDa corresponding to the authentic E1 could be demonstrated from samples of cell lysates but not from culture supernatants which supported EM findings and confirmed that a large proportion of synthesised E1 was being retained within the transgenic cells. By comparison, the presence of an authentic E2 glycoprotein with a MW of 52 kDa could not be demonstrated or was at a level too low for detection. Alternatively, an aberrant E2 form, possibly corresponding to an uncleaved precursor species, was observed from samples of both cell lysates and culture supernatant. Resembling the aberrant E2 molecule (E2*) reported in previous Semliki Forest virus (SFV) studies, this secreted protein demonstrated altered sensitivity to endoglycosidase treatment compared to wt E2 and was indicative of abnormal glycoprotein processing. Clearly, these results together with the evidence of a 6K mutation, underscore the defective replication properties of RRVcomp in the trans-complementation system. Despite this, EM detection of intact replication complexes and virus-like structures, combined with evidence of exclusive transmission in 6K-E1 6B cells suggested that correctly packaged RRVcomp particles were being formed, albeit at reduced levels. In the final analysis of the study, an antigen produced from RRVcomp infected cultures was produced to investigate if this reagent was suitable for RRV diagnostic ELISA (enzyme linked immunosorbent assay) technologies. The complemented antigen was used in capture ELISA assays to analyse a panel of 60 patient sera for the presence of anti-RRV IgM and IgG antibodies. Patients were also tested in parallel assays using inactivated wt RRV antigen. The results of the separate RRVcomp antigen assays correlated highly with each of the wt antigen IgM and IgG assays respectively, indicating that replication limited, trans-complemented RRV may be useful for the development of safer, non-infectious diagnostic reagents

Article Commentary: Archival Collections are Important in the Study of the Biology, Diversity, and Evolution of Arboviruses

Historically, classifications of arboviruses were based on serological techniques. Hence, collections of arbovirus isolates have been central to this process by providing the antigenic reagents for these methods. However, with increasing concern about biosafety and security, the introduction of molecular biology techniques has led to greater emphasis on the storage of nucleic acid sequence data over the maintenance of archival material. In this commentary, we provide examples of where archival collections provide an important source of genetic material to assist in confirming the authenticity of reference strains and vaccine stocks, to clarify taxonomic relationships particularly when isolates of the same virus species have been collected across a wide expanse of time and space, for future phenotypic analysis, to determine the historical diversity of strains, and to understand the mechanisms leading to changes in genome structure and virus evolution