Fuxun Yu

Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

The dengue virus conceals double-stranded RNA in the intracellular membrane to escape from an interferon response.

The dengue virus (DENV) circulates between humans and mosquitoes and requires no other mammals or birds for its maintenance in nature. The virus is well-adapted to humans, as reflected by high-level viraemia in patients. To investigate its high adaptability, the DENV induction of host type-I interferon (IFN) was assessed in vitro in human-derived HeLa cells and compared with that induced by the Japanese encephalitis virus (JEV), a closely related arbovirus that generally exhibits low viraemia in humans. A sustained viral spread with a poor IFN induction was observed in the DENV-infected cells, whereas the JEV infection resulted in a self-limiting and abortive infection with a high IFN induction. There was no difference between DENV and JEV double-stranded RNA (dsRNA) as IFN inducers. Instead, the dsRNA was poorly exposed in the cytosol as late as 48 h post-infection (p.i.), despite the high level of DENV replication in the infected cells. In contrast, the JEV-derived dsRNA appeared in the cytosol as early as 24 h p.i. Our results provided evidence for the first time in DENV, that concealing dsRNA in the intracellular membrane diminishes the effect of the host defence mechanism, a strategy that differs from an active suppression of IFN activity.

Serologic Evidence of Nipah Virus Infection in Bats, Vietnam

To the Editor: Bats are potential reservoir for highly pathogenic viruses, such as Nipah virus (NiV) and Hendra virus, which can cross species barriers (1). However, only limited surveillance has been conducted to assess risk for infection by these deadly emerging viruses. We conducted a study in Vietnam from 2007 to 2008 to assess the prevalence of these pathogens in bats.

Serodiagnosis Using Recombinant Nipah Virus Nucleocapsid Protein Expressed in Escherichia coli

ABSTRACT Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for human and swine sera and an IgM capture ELISA for human sera were established using the recombinant NiV-N protein as an antigen. One hundred thirty-three suspected patient sera and 16 swine sera were used to evaluate the newly established ELISA systems in comparison with the CDC inactivated-virus-based ELISA systems. For the human sera, the NiV-N protein-based indirect IgG ELISA had a sensitivity of 98.6% and a specificity of 98.4%, and the NiV-N protein-based IgM capture ELISA had a sensitivity of 91.7% and a specificity of 91.8%, with reference to the CDC ELISA systems. The NiV-N-based IgM ELISA was found to be more sensitive than the inactivated-virus-based ELISA in that it captured eight additional cases. For the swine sera, the two test systems were in 100% concordance. Our data indicate that the Nipah virus nucleocapsid protein is a highly immunogenic protein in human and swine infections and a good target for serodiagnosis. Our NiV-N protein-based ELISA systems are useful, safe, and affordable tools for diagnosis of Nipah virus infection and are especially fit to be used in large-scale epidemiological investigations and to be applied in developing countries.

Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.

Isolation and Molecular Characterization of Banna Virus from Mosquitoes, Vietnam

We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were isolated from field-caught mosquitoes at various locations; Banna virus was previously isolated from encephalitis patients in Yunnan, China, in 1987. Together, these findings suggest widespread distribution of this virus throughout Southeast Asia.

Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein

SummaryThe complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn2+ was found to be 20 times more effective than Mg2+ in coordinating the catalytic reaction of RdRp, while Ca2+ inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.

Development of a rapid and comprehensive proteomics-based arboviruses detection system.

A rapid and comprehensive protocol, which combines simple purification and liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI/MS/MS), was developed for identification of arboviruses in infected culture fluid. Using this protocol, various arboviruses were detected including uncommon viruses that were described previously as Banna virus and Yunnan orbivirus. This approach is useful for the rapid screening of viral samples that cannot be identified by conventional gene amplification or immunological methods.

Recombinant Truncated Nucleocapsid Protein as Antigen in a Novel Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Severe Acute Respiratory Syndrome Coronavirus Infection

ABSTRACT We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.

The world first two cases of severe fever with thrombocytopenia syndrome: An epidemiological study in Nagasaki, Japan

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a novel phlebovirus belonging to the family Bunyaviridae, was reported in China for the first time in 2009. We observed two cases where the SFTSV was isolated for the first time in Nagasaki, Japan, in 2005. Two males in their 60s, a farmer and a hunter, respectively, living in Nagasaki developed SFTS during the same period. The patients developed similar clinical symptoms and signs, such as fever, loss of consciousness, and multiple organ dysfunction. The farmer died and the hunter survived. A retrospective diagnosis of SFTS was made in 2013, and genetic analysis revealed that the patients were infected with different SFTSV strains. Retrospective analysis of cytokine production in non-fatal case revealed interleukin (IL)-6, IL-8 and interferon-γ level of acute phase was low and could be potential prognostic factors. As there are no epidemiological studies of positive rate of SFTSV antibody in people living in endemic areas in Japan, a field study was performed. Volunteers at high risk for tick bites, such as hunters, farmers, and soldiers, were recruited in 6 regions, including the areas where the SFTS cases occurred. Three hundred and twenty six volunteers in Nagasaki prefecture were examined and none of these tested positive for the SFTSV antibody. Our data indicates that the risk for SFTSV infection is not high in Nagasaki prefecture. Further collection of blood samples from endemic areas is warranted for the prevention of SFTSV infection.