Xianfu Wu

Complete genomes of Aravan, Khujand, Irkut and West Caucasian bat viruses, with special attention to the polymerase gene and non-coding regions

The purpose of this study was to generate complete genome sequences of Aravan (ARAV), Khujand (KHUV), Irkut (IRKV) and West Caucasian bat (WCBV) viruses, and to compare them with genomes of other lyssaviruses. We focused on RNA-dependent RNA-polymerase (L) and non-coding regions, because other genes of these viruses have been described previously. The L protein is organized into six conserved blocks (I-VI), previously detected in all Mononegavirales. Furthermore, lyssaviruses have two additional conserved regions, L1 and L2, located in the COOH part of the L. L1 may be responsible for methylation of viral mRNA cap structures, whereas the significance of L2 is unclear. Phylogenetic patterns based on the L are similar to those described for the nucleoprotein. The WCBV is the most divergent member of the genus. Besides phylogeny, it has a short trailer region (57 nucleotides versus 69-70 nucleotides in other lyssaviruses) and different intergenic region lengths, including an exceptionally long non-coding region of the glycoprotein (697 nucleotides) containing a potential open reading frame of 180 nucleotides. The absence of a flanking transcription initiation signal, as well as Northern and Western blot data, suggests that this region is not independently transcribed but is a part of G mRNA.

Reemerging Rabies and Lack of Systemic Surveillance in People’s Republic of China

Standardized protocols and diagnostic-based surveillance are imperative for detection and elimination.

Molecular Epidemiology of Rabies in Southern People’s Republic of China

Migration and transport of dogs may have caused recent epidemics of human rabies.

From brain passage to cell adaptation: the road of human rabies vaccine development

A major challenge for global rabies prevention and control is the lack of sufficient and affordable high quality vaccines. Such candidates should be pure, potent, safe, effective and economical to produce, with broad cross-reactivity against viral variants of public health and veterinary importance. The history of licensed human vaccines reviewed herein demonstrates clearly how the field has evolved to the current state of more passive development and postexposure management. Modern cell culture techniques provide adequate viral substrates for production of representative verified virus seeds. In contrast to outdated nervous tissue-based rabies vaccines, once a suitable substrate is identified, production of high titer virus results in a major qualitative and quantitative difference. Given the current scenario of only inactivated vaccines for humans, highly cell-adapted and stable, attenuated rabies viruses are ideal candidates for consideration to meet the need for seed viruses in the future.

Rabies virus pathogenesis in relationship to intervention with inactivated and attenuated rabies vaccines.

Despite progress in vaccine development in the past century the mechanisms behind immune responses elicited by rabies biologics or via natural infection remain largely unknown. In this study, we compared protection elicited by standard, early, or delayed prophylaxis with a reduced number of vaccine doses using inactivated and live-attenuated vaccines. Two-month-old Syrian hamsters, 4-week-old ICR mice or adult rhesus macaques were inoculated with canine rabies virus variants. Thereafter, prophylaxis was initiated 6h, 1, 2, 3, 4, 5, 6 or 7 days post-exposure (p.e.). One or several doses of inactivated (HDCV), or reverse genetically attenuated (live), or gamma-irradiated (inactivated)-ERAG333 vaccines were administered intramuscularly. The dynamics of virus spread were measured over time in the rodent models. Rabies virus reached the spinal cord at day 4 and brain at day 6 p.e. All hamsters succumbed in groups in which live ERAG333 was delayed until days 5 and 6 p.e. However, 78%, 44%, 56% and 22% of hamsters survived when one dose of live ERAG333 was administered 6h, 1, 2, 3, and 4 days p.e., respectively. Similarly, 67% survived when inactivated ERAG333 was administered at 24h p.e. All hamsters succumbed when standard prophylaxis (the Essen regimen) was delayed until days 3-6, but 67% and 33% of hamsters survived when PEP began 1 or 2 days p.e., respectively. Macaques were protected by one dose of attenuated ERAG333 at 24h p.e. The highly attenuated (live) and inactivated ERAG333 vaccines elicited potent protective immune responses, even when prophylaxis initiation was delayed. When 2-5 doses of commercial vaccine and HRIG were administered according to the Essen scheme, 89-100% of the animals survived. Reduced vaccine schedules provided efficacious intervention, regardless of the total number of vaccine doses administered.

Rabies in Ferret Badgers, Southeastern China

Ferret badger–associated human rabies cases emerged in China in 1994. We used a retrospective epidemiologic survey, virus isolation, laboratory diagnosis, and nucleotide sequencing to document its reemergence in 2002–2008. Whether the cause is spillover from infected dogs or recent host shift and new reservoir establishment requires further investigation.

Host–rabies virus protein–protein interactions as druggable antiviral targets

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.

Evolutionary dynamics of rabies viruses highlights the importance of China rabies transmission in Asia

Rabies in Asia is emerging as a serious public health issue. To explore the possible origin, phylogenetic relationships, and evolutionary dynamics of Asian Rabies viruses (RABV), we examined 200 complete nucleoprotein (N) gene sequences from RABV isolates in the region. Phylogeny supported the classification of Asian RABVs into five distinct clusters in lyssavirus genotype 1. Our geospatial and temporal analyses demonstrated that China appears to be the prime source of Asian RABVs. Understanding of rabies transmission and associated human activities, such as dog translocation, can help rabies control and elimination in Asia through collaborative efforts or programs.

Ferret badger rabies origin and its revisited importance as potential source of rabies transmission in Southeast China

BackgroundThe frequent occurrence of ferret badger-associated human rabies cases in southeast China highlights the lack of laboratory-based surveillance and urges revisiting the potential importance of this animal in rabies transmission. To determine if the ferret badgers actually contribute to human and dog rabies cases, and the possible origin of the ferret badger-associated rabies in the region, an active rabies survey was conducted to determine the frequency of rabies infection and seroprevalence in dogs and ferret badgers.MethodsA retrospective survey on rabies epidemics was performed in Zhejiang, Jiangxi and Anhui provinces in southeast China. The brain tissues from ferret badgers and dogs were assayed by fluorescent antibody test. Rabies virus was isolated and sequenced for phylogenetic analysis. The sera from ferret badgers and dogs were titrated using rabies virus neutralizing antibodies (VNA) test.ResultsThe ferret badgers presented a higher percentage of rabies seroconversion than dogs did in the endemic region, reaching a maximum of 95% in the collected samples. Nine ferret badger-associated rabies viruses were isolated, sequenced, and were phylogenetically clustered as a separate group. Nucleotide sequence revealed 99.4-99.8% homology within the ferret badger isolates, and 83-89% homology to the dog isolates in the nucleoprotein and glycoprotein genes in the same rabies endemic regions.ConclusionsOur data suggest ferret badger-associated rabies has likely formed as an independent enzootic originating from dogs during the long-term rabies infestation in southeast China. The eventual role of FB rabies in public health remains unclear. However, management of ferret badger bites, rabies awareness and control in the related regions should be an immediate need.

Transmission dynamics of rabies in China over the last 40 years: 1969–2009

BACKGROUND Rabies is a serious reemerging zoonosis in China. The molecular evolution and transmission patterns of rabies virus inferred from historical data can provide guidelines for better disease control and prevention in the future. OBJECTIVES To investigate the epidemiology and evolutionary dynamics of the rabies virus in China. STUDY DESIGN The molecular evolution of 132 viral glycoprotein gene sequences of Chinese rabies viruses collected in 17 provinces and 3 municipalities between 1969 and 2009 was analyzed. RESULTS Phylogenetic analysis revealed that Chinese rabies viruses are subdivided into 6 lineages (A-F) within Lyssavirus genotype 1. Lineage A represents the widely dispersed cosmopolitan lineage while lineage B is closely related to Arctic-like rabies viruses. The remaining lineages (C-F) are typical of those circulating across much of Southeast Asia. The evolutionary rate for Chinese rabies virus was 1.532 x 10(-4) substitutions per site per year, and the corresponding common ancestor was in about 1115. CONCLUSIONS The phylogeographic structure demonstrated Chinese rabies viruses have been transmitted intra-provincially and extra-provincially due to human-related activities.