Mosquito surveillance studies to identify mosquito-borne flaviviruses have identified West Nile Virus (WNV) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.

Valosin-containing protein (VCP) is classified as a member of the type II AAA(+) ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. (C) 2016 Elsevier B.V. All rights reserved

Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV.

Mosquito-borne alphaviruses are disseminated globally and cause febrile illness in humans and animals. Since the prevalence and diversity of alphaviruses has not been previously investigated in Zambia, reverse transcription PCR was employed as a broad-spectrum approach for the detection of alphaviruses in mosquitoes. From 552 mosquito pools, a novel alphavirus, tentatively named Mwinilunga alphavirus (MWAV), was discovered from a single Culex quinquefasciatus mosquito pool. The full genome of MWAV was subsequently determined, and pairwise comparisons demonstrated that MWAV represented a new alphavirus species. Phylogenetic analyses and a linear discriminant analysis based on the dinucleotide ratios in various virus sequences indicated that MWAV is related to a mosquito-specific alphavirus distinct from other known mosquito-borne alphaviruses due to its inability to replicate in vertebrate cell lines. Further analyses of these novel alphaviruses will help to facilitate a greater understanding of the molecular determinants of host range restriction and the evolutionary relationships of alphaviruses.

West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.

West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.

The Smacoviridae has recently been classified as a family of small circular single-stranded DNA viruses. An increasing number of smacovirus genomes have been identified exclusively in faecal matter of various vertebrate species and from insect body parts. However, the genetic diversity and host range of smacoviruses remains to be fully elucidated. Herein, we report the genetic characterization of eleven c ircular r eplication-associated protein ( Rep ) e ncoding s ingle- s tranded (CRESS) DNA viruses detected in the faeces of Zambian non-human primates. Based on pairwise genome-wide and amino acid identities with reference smacovirus species, ten of the identified CRESS DNA viruses are assigned to the genera Porprismacovirus and Huchismacovirus of the family Smacoviridae , which bidirectionally encode two major open reading frames (ORFs): Rep and capsid protein ( CP ) characteristic of a type IV genome organization. The remaining unclassified CRESS DNA virus was related to smacoviruses but possessed a genome harbouring a unidirectionally oriented CP and Rep , assigned as a type V genome organization. Moreover, phylogenetic and recombination analyses provided evidence for recombination events encompassing the 3′-end of the Rep ORF in the unclassified CRESS DNA virus. Our findings increase the knowledge of the known genetic diversity of smacoviruses and highlight African non-human primates as carrier animals.

Background and Aim: The Sunda pangolin (Manis javanica) is on the International Union for Conservation of Nature Red List of Threatened Species (critically endangered) due to high levels of illegal trafficking for its products. Thailand is one of the habitats of this species, and it has become the main hub for its illegal trafficking. Rehabilitating these captive pangolins and reintroducing them back to the wild are challenging due to the limited knowledge on their diet, management, and diseases. Hemoparasites, including Babesia spp., can cause important protozoal infections in both domestic and wild animals, resulting in the failure of rehabilitation and conservation programs. However, Babesia spp. has not been reported in pangolins. The aim of the study was to determine the prevalence of Babesia spp. in the Sunda pangolin of Thailand. Materials and Methods: A total of 128 confiscated Sunda pangolins from across different regions in Thailand were investigated. These pangolins had been admitted to a regional Wildlife Quarantine Center for rehabilitation before release in the forest. Routine physical examinations were conducted on the animals. We collected blood samples from each pangolin for hematological analysis and to detect Babesia spp. using polymerase chain reaction (PCR) targeting the partial 18s rRNA gene. Results: Babesia-specific PCR detected 53 animals (41.4%) that were positive for Babesia spp. Blood smears were obtained from the positive samples and investigated under a light microscope to observe for trophozoites of Babesia spp. Examination of 40 PCR-positive and -negative samples found no significant differences between the hematological parameters of Babesia-positive and Babesia-negative samples. Eight PCR-positive samples were randomly selected and their DNA was sequenced. Seven and one of sequences match uncharacterized Babesia spp. with 100% and 99.2% similarity, respectively. Phylogenetic analysis demonstrated that our samples form a unique monophyletic clade along with other Babesia spp. detected in the wild. This clade is clearly separated from other Babesia spp. from small carnivores, ruminants, and rats. Conclusion: Our results provide evidence of infection of Sunda pangolins in Thailand by Babesia spp. These pangolins originated from different regions and had not lived together before blood collection. Thus, we suggest that the uncharacterized Babesia spp. found in this study constitute a new group of pangolin-specific Babesia spp. The prevalence of the uncharacterized Babesia spp. was not correlated to pangolin health. Further studies are required to characterize the genomes and phenotypes, including the morphology and pathogenicity of these protozoa. Such information will be helpful for the conservation and health management of the Sunda pangolin.