Suckling Mouse Brain Research Articles

Discovery and biological confirmation of a highly divergent Tacaribe virus in metatranscriptomic data from neotropical bats.

Clade B New World arenaviruses (NWA) include rodent-borne lethal hemorrhagic fever viruses, whereas Tacaribe virus (TCRV) stands out because of its detection in bats and its presumably low zoonotic potential. However, the bat association of TCRV was put into question by lethal experimental neotropical fruit bat infections and rare TCRV detection in bats. Scarce genomic data include near-identical viruses from Caribbean bats and ticks from the US sampled 50 years later. The prototype TCRV isolate used for experimental risk assessments has an extensive passage history in suckling mouse brains. Exploring the true genetic diversity, geographic distribution, and host range of bat-borne NWA is pivotal to assess their zoonotic potential and transmission cycles. We analyzed metatranscriptomic data for evidence of NWA identifying a highly divergent TCRV in bats and confirmed virus detection in original biological materials, supporting the association of TCRV with neotropical bats and warranting investigation of strain-associated TCRV pathogenicity.

Inflammatory damage caused by Echovirus 30 in the suckling mouse brain and HMC3 cells

Echovirus 30 (E30), a member of the species B Enterovirus family, is a primary pathogen responsible for aseptic meningitis and encephalitis. E30 is associated with severe nervous system diseases and is a primary cause of child illness, disability, and even mortality. However, the mechanisms underlying E30-induced brain injury remain poorly understood. In this study, we used a neonatal mouse model of E30 to investigate the possible mechanisms of brain injury. E30 infection triggered the activation of microglia in the mouse brain and efficiently replicated within HMC3 cells. Subsequent transcriptomic analysis revealed inflammatory activation of microglia in response to E30 infection. We also detected a significant upregulation of polo-like kinase 1 (PLK1) and found that its inhibition could limit E30 infection in a sucking mouse model. Collectively, E30 infection led to brain injury in a neonatal mouse model, which may be related to excessive inflammatory responses. Our findings highlight the intricate interplay between E30 infection and neurological damage, providing crucial insights that could guide the development of interventions and strategies to address the severe clinical manifestations associated with this pathogen.

Tanreqing injection inhibits dengue virus encephalitis by suppressing the activation of NLRP3 inflammasome

BackgroundEncephalitis caused by dengue virus (DENV) is considered a manifestation of severe dengue. Tanreqing injection (TRQ) is a well-known Chinese patented medicine, which has been used to treat brain-related disorders by inhibiting inflammation. Nevertheless, the effects of TRQ on DENV encephalitis have not been studied. The aim of this study was to evaluate the effects of TRQ on DENV encephalitis and to explore its potential mechanisms.MethodsThe cytotoxicity of TRQ was examined by MTT assay, and the anti-DENV activities of TRQ in BHK-21 baby hamster kidney fibroblast were evaluated through CCK-8 and plaque assays. The expression levels of NO, IL1B/IL-1β, TNFα and IL6 were measured by qRT‒PCR and ELISA in the BV2 murine microglial cell line. The inhibitory effects of TRQ on NLRP3 inflammasome activation in BV2 cells were examined by Western blotting, qRT‒PCR and ELISA. The effects of TRQ on HT22 mouse hippocampal neuronal cells were examined by CCK-8 assay, morphology observation and flow cytometry. Moreover, a DENV-infected ICR suckling mouse model was developed to investigate the protective role of TRQ in vivo.ResultsTRQ decreased the release of NO, IL6, TNFα and IL1B from BV2 cells and inhibited the activation of NLRP3. The presence of the NLRP3 agonist nigericin reversed the anti-inflammatory activities of TRQ. Furthermore, TRQ inhibited the death of HT22 cells by decreasing IL1B in DENV-infected BV2 cells. In addition, TRQ significantly attenuated weight loss, reduced clinical scores and extended the survival in DENV-infected ICR suckling mice. Critically, TRQ ameliorated pathological changes in ICR suckling mice brain by inhibiting microglia and NLRP3 activation and decreasing the production of inflammatory factors and the number of dead neurons.ConclusionTRQ exerts potent inhibitory effects on dengue encephalitis in vitro and in vivo by reducing DENV-2-induced microglial activation and subsequently decreasing the inflammatory response, thereby protecting neurons. These findings demonstrate the potential of TRQ in the treatment of dengue encephalitis.

Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs

Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.

Isolation of a Novel Bat Rhabdovirus with Evidence of Human Exposure in China.

ABSTRACTBats are well-recognized reservoirs of zoonotic viruses. Several spillover events from bats to humans have been reported, causing severe epidemic or endemic diseases including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome-CoV (MERS-CoV), henipaviruses, and filoviruses. In this study, a novel rhabdovirus species, provisionally named Rhinolophus rhabdovirus DPuer (DPRV), was identified from the horseshoe bat (Rhinolophus affinis) in Yunnan province, China, using next-generation sequencing. DPRV shedding in the spleen, liver, lung, and intestinal contents of wild bats with high viral loads was detected by real-time quantitative PCR, indicating that DPRV has tropism for multiple host tissues. Furthermore, DPRV can replicate in vitro in multiple mammalian cell lines, including BHK-21, A549, and MA104 cells, with the highest efficiency in hamster kidney cell line BHK-21, suggesting infectivity of DPRV in these cell line-derived hosts. Ultrastructure analysis revealed a characteristic bullet-shaped morphology and tightly clustered distribution of DPRV particles in the intracellular space. DPRV replicated efficiently in suckling mouse brains and caused death of suckling mice; death rates increased with passaging of DPRV in suckling mice. Moreover, 421 serum samples were collected from individuals who lived near the bat collection site and had fever symptoms within 1 year. DPRV-specific antibodies were detected in 20 (4.75%) human serum samples by indirect immunofluorescence assay. Furthermore, 10 (2.38%) serum samples were DPRV positive according to plaque reduction neutralization assay, which revealed potential transmission of DPRV from bats to humans and highlighted the potential public health risk. Potential vector association with DPRV was not found with negative viral RNA in bloodsucking arthropods.

Coxsackievirus B5 virus-like particle vaccine exhibits greater immunogenicity and immunoprotection than its inactivated counterpart in mice

Coxsackievirus B group 5 (CVB5) represents one of the major pathogens that cause diseases such as hand, foot and mouth disease (HFMD) and aseptic meningitis et al. Currently, no specific drugs and vaccines are available, and a safe and effective CVB5 vaccine is of great value for control of the diseases. In this study, CVB5 P1 precursor and 3CD protease were co-expressed in Sf9 cells by using a baculovirus expression system. The P1 was processed by 3CD and self-assembled into CVB5 virus-like particles (VLPs). VP1 and VP3 capsid proteins of CVB5 could be detected by SDS-PAGE and Western blotting. Transmission electron microscopy revealed that the CVB5 VLPs were spherical particles with a diameter of about 30 nm, mimicking wild-type CVB5 virus. Our study showed that the total IgG and neutralizing antibodies induced by CVB5 VLPs were higher than those induced by inactivated vaccine. More importantly, the CVB5 VLPs conferred full protection to the CVB5-challenged suckling mice via passive immunity while protection efficiency of the inactivated vaccine was only 80%. The CVB5 VLPs vaccine could protect the limb muscles, brain, and heart tissues of suckling mice from CVB5-induced damage. These results demonstrated that the CVB5 VLPs vaccine possessed stronger immunogenicity and provided more robust immunoprotection than the inactivated CVB5 vaccine, suggesting that the CVB5 VLPs promise to be a CVB5 vaccine candidate in future.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/-mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766’s virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.

Change in the Single Amino Acid Site 83 in Rabies Virus Glycoprotein Enhances the BBB Permeability and Reduces Viral Pathogenicity.

Lab-attenuated rabies virus (RABV) is a highly cellular adaptation and less pathogenic than wild-type RABV. However, the molecular mechanisms that regulate the cellular adaptation and pathogenicity remain obscure. In this work, we isolated a wild-type RABV (CNIM1701) from a rabid bovine in northern China. The original CNIM1701 was lethal in adult mice and restricted replication in cell cultures. After 20 serial passages in the brains of suckling mice, the virus was renamed CNIM1701-P20, which was safe in adult mice and replicated well in cell cultures. In addition, sequence comparison analysis of the original CNIM1701 and CNIM1701-P20 identified 2 amino acid substitutions on G protein (Lys83 → Arg83 and Pro367 → Ser 367) related to pathogenesis and cellular adaptation. Using site-directed mutagenesis to exchange Lys83 with Arg83 and Pro367 with Ser 367 in the G protein of the RABV SAD strain, the pathogenicity of rSAD-K83R was significantly decreased. Our data indicate that the decreased pathogenicity of rSAD-K83R is due to increasing the expression of RABV-G, which also induced a higher level of apoptosis in infected cells. Furthermore, the K83 mutation induced high expression of MMP-2 and MMP-9 on DCs and promoted blood–brain barrier (BBB) permeability. These results demonstrate that the pathogenesis of RABV is partially dependent on G expression and BBB permeability, which may help in the design and development of highly safe, live-RABV vaccines.

Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Evaluation of environment safety of a Japanese encephalitis live attenuated vaccine

JE vaccination is the most effective and economical method of preventing JE. A live attenuated JE vaccine has been widely used in many countries since 1989, playing an important role in controlling JE outbreaks. However, whether the large-scale use of the live attenuated JE vaccine will lead to the dissemination of the vaccine virus in the environment and whether reversion of the neuroattenuation of the virus will occur during the transmission process remain major concerns for some researchers. To evaluate the transmission of a live attenuated JEV vaccine in mosquitoes and hosts, JE SA14-14-2 attenuated vaccine virus was intrathoracically (i.t.) inoculated into Culex tritaeniorhynchus, a native vector. Subsequently, virus harvested from inoculated mosquitoes was inoculated into pigs, a mammalian reservoir. The virus was isolated from the pigs and passaged once again in Culex tritaeniorhynchus. The genome sequences and virulence of the passaged viruses were then investigated. While a few nucleotide substitutions occurred during passaging, there was no change in the encoded amino acids. After intracerebral (i.c.) inoculation of mice with the vaccine, no pathological effects were observed. In addition, virus virulence remained low after inoculation of suckling mouse brains. These results indicate that vaccination of individuals with the live vaccine will not result in transmission of the live SA14-14-2 vaccine virus through mosquito biting and virus amplified in pigs.

Ultrastructural Features of Human Liver Specimens from Patients Who Died of Dengue Hemorrhagic Fever.

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA–indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.

Proteomics analysis of suckling mouse brain infected with attenuated rabies virus strain SRV9.

Rabies virus is an enveloped negative-stranded RNA virus belonging to the family Rhabdoviridae. It can be successfully controlled by vaccination however, there are still tens of thousands of deaths each year caused by rabies virus due to its mutations and complexity. A better understanding of the interaction between the rabies virus and the host might help solve this problem. Therefore, in this study, we used two-dimensional electrophoresis to investigate the protein expression of rabies virus-infected mice. This can help us to understand the impact of rabies virus on host protein expression during infection. For our experiment, two-dimensional electrophoresis was used to analyze the differential proteomics of the brain of 10- and 20-day-old suckling mice infected with attenuated rabies virus strain SRV9. The results showed that the expression levels of 10 protein spots had been up- or down-regulated at least 2-fold. Using MALDI-TOF-MS, we identified 8 differentially expressed proteins. We have identified proteins, namely hnRNP L, DPYSL3, NECAPs, and transaldolase that might be closely related to the susceptibility of SRV9 in suckling mice. Keywords: rabies virus; attenuated strain; suckling mouse; two-dimensional electrophoresis; proteomics.

Effective inhibition of different Japanese encephalitis virus genotypes by RNA interference targeting two conserved viral gene sequences in vitro and in vivo.

Japanese encephalitis is a zoonotic, mosquito-borne, infectious disease caused by Japanese encephalitis virus (JEV), which is prevalent in China. At present, there are no specific drugs or therapies for JEV infection, which can only be treated symptomatically. Lentivirus-mediated RNA interference (RNAi) is a highly efficient method to silence target genes. In this study, two lentiviral shRNA, LV-C and LV-NS5, targeting the conserved viral gene sequences were used to inhibit different JEV genotypes strains in BHK21 cells and mice. The results showed that LV-C significantly inhibited JEV genotype I and genotype III strains in cells and mice. Quantitative RT-PCR analysis showed that JEV mRNA were reduced by 83.2-90.9% in cells by LV-C and that flow cytometry analysis confirmed the inhibitory activity of LV-C. The viral titers were reduced by about 1000-fold in cells and the brains of suckling mice by LV-C, and the pretreatment of LV-C protected 60-80% of mice against JEV-induced lethality. The inhibitory activities of LV-NS5 in cells and mice were weaker than those of LV-C. These results indicate that RNAi targeting of the two conserved viral gene sequences had significantly suppressed the replication of different JEV genotypes strains in vitro and in vivo, highlighting the feasibility of RNAi targeting of conserved viral gene sequences for controlling JEV infection.

E90 subunit vaccine protects mice from Zika virus infection and microcephaly

Zika virus (ZIKV) became a global threat due to its unprecedented outbreak and its association with congenital malformations such as microcephaly in developing fetuses and neonates. There are currently no effective vaccines or drugs available for the prevention or treatment of ZIKV infection. Although multiple vaccine platforms have been established, their effectiveness in preventing congenital microcephaly has not been addressed. Herein, we tested a subunit vaccine containing the 450 amino acids at the N-terminus of the ZIKV envelope protein (E90) in mouse models for either in utero or neonatal ZIKV infection. In one model, embryos of vaccinated dams were challenged with a contemporary ZIKV strain at embryonic day 13.5. The other model infects neonatal mice from vaccinated dams by direct injection of ZIKV into the developing brains. The vaccine led to a substantial reduction of ZIKV-infected cells measured in the brains of fetal or suckling mice, and successfully prevented the onset of microcephaly compared to unvaccinated controls. Furthermore, E90 could protect mice from ZIKV infection even at 140 days post-immunization. This work directly demonstrates that immunization of pregnant mice protects the developing brains of offspring both in utero and in the neonatal period from subsequent ZIKV infection and microcephaly. It also supports the further development of the E90 subunit vaccine towards clinical trials.

Familial cluster of exposure to a confirmed rabid dog in travelers to Algeria.

A 10 person-family originating from Algeria traveled in rural Algeria for the purpose of visiting friends and relatives without seeking pre-travel advice, did not received pre-travel rabies immunization, and were exposed to a confirmed rabid dog including 8 within less than 4 days of arrival. Three received suckling mouse brain rabies vaccine although WHO strongly recommends that its production and administration be discontinued and seven received insufficient doses of equine rabies immune globulin abroad. Rabies treatment was completed on returning to France. This reports underline the fact that travelers visiting friends and relatives in dog rabies endemic country are at high risk of rabies exposure and unaware of such a risk in most instances. Rabies risk warning should be reinforced and rabies pre-exposure vaccination should be considered in all individuals traveling to North Africa (and to sub-Saharan Africa) whatever the duration of stay.

The neuroattenuation of three clone viruses isolated from the attenuated CTN rabies virus

<p>Three attenuated rabies virus strains(CTN181-3，CTN181-16 and CTN181-19), were selected by plaque cloning from CTN-181 rabies attenuated virus，which was infected guinea pigs submandibular glands.After passaging those viruses in suckling mice brain for five consecutive passages or in submandibular gland of guinea pigs for one passage, non-virulence reversion or increase were detected.The neuroattenuation stability make the three newly selected CTN attenuated strains the promising live vaccine candidates for wild animal prophylactic vaccination and even postexposure prophylaxis of rabies for humans.</p>

Isolation of Zika Virus Imported from Tonga into Australia.

Introduction: The globally emergent Zika virus (ZIKV) is a threat to Australia, given the number of imported cases from epidemic regions and the presence of competent mosquito vectors. We report the isolation of ZIKV from a female traveler who recently returned from Tonga to Brisbane, Queensland, Australia in 2016.Methods: A specific TaqMan real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) assay was used to detect ZIKV in serum and urine samples. Conventional cell culture techniques and suckling mice were employed in an attempt to isolate ZIKV from serum and urine.Results: A ZIKV isolate (TS17-2016) was recovered from the serum sample after one passage in suckling mouse brains and harvested 11 days post inoculation. Phylogenetic analysis of complete envelope (E) gene sequences demonstrated TS17-2016 shared 99.9% nucleotide identity with other contemporary sequences from Tonga 2016, Brazil 2015 and French Polynesia 2013 within the Asian lineage.Discussion: This is the first known report of successful isolation of ZIKV from a human clinical sample in Australia and the first from a traveler from Tonga. This study highlights the potential difficulties in isolating ZIKV from acute clinical samples using conventional cell culture techniques, particularly in non-endemic countries like Australia where access to samples of sufficient viral load is limited. The successful isolation of TS17-2016 will be essential for continued investigations of ZIKV transmission and pathogenicity and will enable the advancement of new preventative control measures extremely relevant to the Australian and Pacific region.

First isolation and identification of Zika virus in China

Objective To establish a method for the isolation of Zika virus and to gather experiences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples positive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nucleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The sequences of NS5 gene were analyzed by using MEGA6.0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle threshold (Ct) values of 24.4 and 25.3, respectively. The second generation of Zika virus was identified in suckling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus (GDZ16002 strain) belonged to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage. Key words: Zika virus; Viral isolation; Arbovirus; Suckling mice