Generation of a Bactrian camel hepatitis E virus by a reverse genetics system.

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA-transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

Full genome-based characterization of G4P[6] rotavirus strains from diarrheic patients in Thailand: Evidence for independent porcine-to-human interspecies transmission events.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand.

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.

Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection.

The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.

Determination of Ferret Enteric Coronavirus Genome in Laboratory Ferrets.

Ferret enteric coronavirus (FRECV) RNA was detected in laboratory ferrets. Analysis of the complete genome sequence of 2 strains, FRCoV4370 and FRCoV063, revealed that FRECV shared 49.9%-68.9% nucleotide sequence identity with known coronaviruses. These results suggest that FRECV might be classified as a new species in the genus Alphacoronavirus.

Production of infectious dromedary camel hepatitis E virus by a reverse genetic system: Potential for zoonotic infection.

BACKGROUND & AIMS: The pathogenicity, epidemiology and replication mechanism of dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus (HEV), has been unclear. Here we used a reverse genetic system to produce DcHEV and examined the possibility of zoonotic infection. METHODS: Capped genomic RNA derived from a synthetic DcHEV cDNA was transfected into human hepatocarcinoma cells PLC/PRF/5. The DcHEV capsid protein and RNA were detected by an enzyme-linked immunosorbent assay (ELISA) or RT-qPCR. A neutralization test for DcHEV was carried out by using antisera against HEV-like particles. DcHEV was used to inoculate two cynomolgus monkeys to examine the potential for cross-species infection. RESULTS: The transfection of PLC/PRF/5 cells with capped DcHEV RNA resulted in the production of infectious DcHEV. The genome sequence analysis demonstrated that both nucleotide and amino acid changes accumulated during the passages in PLC/PRF/5 cells. The cynomolgus monkeys showed serological signs of infection when DcHEV was intravenously inoculated. DcHEV was neutralized by not only anti-DcHEV-LPs antibody, but also anti-genotype 1 (G1), G3 and G4 HEV-LPs antibodies. Moreover, the monkeys immunized with DcHEV escaped the G3 HEV challenge, indicating that the serotype of DcHEV is similar to those of other human HEVs. CONCLUSIONS: Infectious DcHEV was produced using a reverse genetic system and propagated in PLC/PRF/5 cells. The antigenicity and immunogenicity of DcHEV are similar to those of G1, G3 and G4 HEV. DcHEV was experimentally transmitted to primates, demonstrating the possibility of a zoonotic infection by DcHEV. LAY SUMMARY: Dromedary camel hepatitis E virus (DcHEV) was produced by a reverse genetic system and grows well in PLC/PRF/5 cells. Cynomolgus monkeys experimentally infected with DcHEV indicated serological signs of infection, suggesting that DcHEV has the potential to cause zoonotic HEV infection.

An Outbreak of Acute Hepatitis Caused by Genotype IB Hepatitis A Viruses Contaminating the Water Supply in Thailand.

BACKGROUND: In 2000, an outbreak of acute hepatitis A was reported in a province adjacent to Bangkok, Thailand. AIMS: To investigate the cause of the 2000 hepatitis A outbreaks in Thailand using molecular epidemiological analysis. METHODS: Serum and stool specimens were collected from patients who were clinically diagnosed with acute viral hepatitis. Water samples from drinking water and deep-drilled wells were also collected. These specimens were subjected to polymerase chain reaction (PCR) amplification and sequencing of the VP1/2A region of the hepatitis A virus (HAV) genome. The entire genome sequence of one of the fecal specimens was determined and phylogenetically analyzed with those of known HAV sequences. RESULTS AND CONCLUSIONS: Eleven of 24 fecal specimens collected from acute viral hepatitis patients were positive as determined by semi- nested reverse transcription PCR targeting the VP1/2A region of HAV. The nucleotide sequence of these samples had an identical genotype IB sequence, suggesting that the same causative agent was present. The complete nucleotide sequence derived from one of the samples indicated that the Thai genotype IB strain should be classified in a unique phylogenetic cluster. The analysis using an adjusted odds ratio showed that the consumption of groundwater was the most likely risk factor associated with the disease.

Neutralization activity of patient sera collected during the 2008-2009 Chikungunya outbreak in Thailand.

Chikungunya virus (CHIKV) infection typically causes fever, rash, myalgia, and arthralgia and sometimes results in recurrent joint pain or, in severe cases, neurological disorders or death. How CHIKV infection leads to prolonged or severe symptoms is still not well understood. In this study, we examined the neutralization (NT) titer of 98 serum samples collected from patients during the 2008-2009 chikungunya outbreak in Thailand. While all serum samples showed neutralizing activity, virus was detected in 58% of the serum samples. When we analyzed a possible association between virus and antibody titers and the presence of typical symptoms of CHIKV infection, fever and joint pain, there was no significant association except that the number of patients with fever was over three times more than the number of those without fever when CHIKV was detectable in serum. This study indicates that although neutralizing antibody is critical to eliminate CHIKV, it appears not to be the main factor associated with clinical symptoms in some cases, so that other aspects of immune responses, such as those involving proinflammatory mediators and adaptive immune cells, should be considered altogether.

Long-term viral shedding and viral genome mutation in norovirus infection.

The duration of viral shedding in the patients from two outbreaks and four sporadic cases of norovirus (NoV) infections was investigated. The longest period of viral shedding into feces was for 173 days in an inpatient from one case of outbreak. The VP1 sequence from two long-term viral shedding cases in the outbreak revealed four synonymous and one non-synonymous mutations in one inpatient at 26 days from the onset of illness, and nine synonymous and two non-synonymous mutations and a deletion, 10 synonymous mutations and a deletion in other inpatient at 29 days and 54 days from the onset of illness, respectively. Ten of the 11 amino acid positions detected in these two inpatients were in the outermost P2 domain of the viral capsid protein, and mutations at positions 295, 297, and 394 were shared in the inpatients. Mutations in the P2 domain were in epitopes A and D or near epitopes A, C, and E, suggesting that the long-term carrier state of norovirus infection contributes to the generation of escape mutants by host immunoselection.

Monkeys and Rats Are Not Susceptible to Ferret Hepatitis E Virus Infection.

Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in the Netherlands, Japan, and the US. To determine whether ferret HEV transmits to other animals, we inoculated laboratory rats (Wistar), nude rats (Long-Evans-rnu/rnu), and cynomolgus monkeys with ferret HEV (F4351) by intravenous injection. None of the animals demonstrated a positive sign for virus replication, indicating that rats and monkeys are not susceptible to ferret HEV.

Complete genome of hepatitis E virus from laboratory ferrets.

The complete genome of hepatitis E virus (HEV) from laboratory ferrets imported from the United States was identified. This virus shared only 82.4%-82.5% nt sequence identities with strains from the Netherlands, which indicated that the ferret HEV genome is genetically diverse. Some laboratory ferrets were contaminated with HEV.

Impact of amino acid substitutions in the V2 and C2 regions of human immunodeficiency virus type 1 CRF01_AE envelope glycoprotein gp120 on viral neutralization susceptibility to broadly neutralizing antibodies specific for the CD4 binding site.

BACKGROUND: The CD4 binding site (CD4bs) of envelope glycoprotein (Env) gp120 is a functionally conserved, important target of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Two neutralizing human monoclonal antibodies, IgG1 b12 (b12) and VRC01, are broadly reactive neutralizing antibodies which recognize conformational epitopes that overlap the CD4bs of Env gp120; however, many CRF01_AE viruses are resistant to neutralization mediated by these antibodies. We examined the mechanism underlying the b12 resistance of the viruses using CRF01_AE Env (AE-Env)-recombinant viruses in this study. RESULTS: Our results showed that an amino acid substitution at position 185 in the V2 region of gp120 played a crucial role in regulating the b12 susceptibility of AE-Env-recombinant viruses by cooperating with 2 previously reported potential N-linked glycosylation (PNLG) sites at positions 186 (N186) and 197 (N197) in the V2 and C2 regions of Env gp120. The amino acid residue at position 185 and 2 PNLG sites were responsible for the b12 resistance of 21 of 23 (>91%) AE-Env clones tested. Namely, the introduction of aspartic acid at position 185 (D185) conferred b12 susceptibility of 12 resistant AE-Env clones in the absence of N186 and/or N197, while the introduction of glycine at position 185 (G185) reduced the b12 susceptibility of 9 susceptible AE-Env clones in the absence of N186 and/or N197. In addition, these amino acid mutations altered the VRC01 susceptibility of many AE-Env clones. CONCLUSIONS: We propose that the V2 and C2 regions of AE-Env gp120 contain the major determinants of viral resistance to CD4bs antibodies. CRF01_AE is a major circulating recombinant form of HIV-1 prevalent in Southeast Asia. Our data may provide important information to understand the molecular mechanism regulating the neutralization susceptibility of CRF01_AE viruses to CD4bs antibodies.

Isolation of cross-reactive human monoclonal antibodies that prevent binding of human noroviruses to histo-blood group antigens.

In order to identify the repertoire of antibodies generated on natural infection of norovirus (NoV) in humans, and to characterize the human monoclonal antibodies against NoV, three phage-displayed antibody libraries originating from healthy person(s) were screened using purified virus-like particles (VLPs) of strain Narita 104 (r104, genogroup II, genotype 4) or strain Chiba 407 (rCV, genogroup I, genotype 4) as antigens. On screening with r104, 62 clones were isolated. Among these antibodies, two clones, 12A11 and 12B10, showed intra-genogroup cross-reactivity to genotypes 1, 3-7, 12, and 14, and genotypes 1, 4, 6, and 7 of genogroup II, respectively. In addition, antibodies belonging to the same group were isolated from two different libraries. On screening with rCV, five clones were isolated, two of which were cross-reactive. One, CV-2F5, reacted to genotypes 1-4, and 8 of genogroup I, and the other, CV-1A5, showed inter-genogroup cross-reactivity to all the VLPs employed in this study. The blocking activities of the monoclonal antibodies against the interaction of homotypic VLPs (VLPs used in the panning procedure) with histo-blood group antigens were also assessed as an alternative to neutralization assay. Although the blocking activity of 12A11 was partially limited 12B10 prevented the binding of r104 to histo-blood group antigens that had been reported to bind r104. The blocking activity of CV-2F5 against the attachment of rCV to suitable histo-blood group antigens was weak, but the blocking activity of CV-1A5 was well recognized. Thus, 12B10 and CV-1A5 were suggested to be cross-reactive monoclonal antibodies with neutralizing activity.

Characterization of full genome of rat hepatitis E virus strain from Vietnam.

We amplified the complete genome of the rat hepatitis E virus (HEV) Vietnam strain (V-105) and analyzed the nucleotide and amino acid sequences. The entire genome of V-105 shared only 76.8%-76.9% nucleotide sequence identities with rat HEV strains from Germany, which suggests that V-105 is a new genotype of rat HEV.

Characterization of self-assembled virus-like particles of ferret hepatitis E virus generated by recombinant baculoviruses.

Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in The Netherlands. Due to the lack of a cell-culture system for ferret HEV, the antigenicity, pathogenicity and epidemiology of this virus have remained unclear. In the present study, we used a recombinant baculovirus expression system to express the 112-N-terminus and 47-C-terminus-amino-acid-truncated ferret HEV ORF2 protein in insect Tn5 cells, and found that a large amount of a 53 kDa protein (F-p53) was expressed and efficiently released into the supernatant. Electron microscopic analysis revealed that F-p53 was self-assembled into virus-like particles (ferret HEV-LPs). These ferret HEV-LPs were estimated to be 24 nm in diameter, which is similar to the size of G1, G3, G4 and rat HEV-LPs derived from both the N-terminus- and C-terminus-truncated constructs. Antigenic analysis demonstrated that ferret HEV-LPs were cross-reactive with G1, G3, G4 and rat HEVs, and rat HEV and ferret HEV showed a stronger cross-reactivity to each other than either did to human HEV genotypes. However, the antibody against ferret HEV-LPs does not neutralize G3 HEV, suggesting that the serotypes of these two HEVs are different. An ELISA for detection of anti-ferret HEV IgG and IgM antibodies was established using ferret HEV-LPs as antigen, and this assay system will be useful for monitoring ferret HEV infection in ferrets as well as other animals. In addition, analysis of ferret HEV RNA detected in ferret sera collected from a breeding colony in the USA revealed the genetic diversity of ferret HEV.

Development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever in Africa, South Asia, and Southeast Asia. Because the mosquito vector Aedes albopictus is present in habitats across Europe, North America, and East Asia, CHIKV has become a serious worldwide public health concern. Infection with CHIKV typically causes fever, rash, myalgia, and arthralgia. One of the important questions yet to be answered is how the host immune system is involved in the development of this disease. In this study, we prepared a CHIKV-pseudotyped lentiviral vector for use in a safe and convenient neutralization (NT) assay and analyzed its efficacy. The CHIKV-pseudotyped lentiviral vector was prepared by cotransfection with plasmids encoding the CHIKV glycoproteins E3, E2, 6k, and E1, packaging elements, and a luciferase reporter. This alternative to native CHIKV can be safely handled in a biosafety level 2 facility. The NT assay was optimized using sera from CHIKV-immunized mice and then applied to human patient sera. The majority of the serum samples from patients with chikungunya in Thailand showed robust neutralization activities, with titers that were tightly correlated with those determined by a conventional NT assay. Moreover, there was a strong correlation with the CHIKV antibody titers as determined by enzyme-linked immunosorbent assay. Thus, the CHIKV-pseudotyped-lentiviral-vector-based NT assay system is a powerful tool for examining the neutralization activity of patient sera, which will lead to a better understanding of the immune responses involved in CHIKV infection.

Structural basis for the recognition of Lewis antigens by genogroup I norovirus.

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le(a)) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le(a), Le(b), H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le(a) antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le(a) antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le(a) antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le(a) antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.

Chikungunya virus induces a more moderate cytopathic effect in mosquito cells than in mammalian cells.

OBJECTIVES: Chikungunya virus (CHIKV) is an alphavirus belonging to the Togaviridae family. Alphaviruses cause a chronic non-cytopathic infection in mosquito cells, while they develop a highly cytopathic infection in cells originating from various vertebrates. In this study, we compared the cytopathic effect (CPE) induced by CHIKV in Vero cells and a mosquito cell line, C6/36 cells. METHODS: CPE and the virus titers were compared between the CHIKV-infected C6/36 and Vero cells. Apoptosis was measured by TUNEL assay, and the differences between the C6/36 and Vero cells were compared. RESULTS: CHIKV infection induced strong CPE and apoptosis in the Vero cells, but light CPE in the C6/36 cells. The virus titers produced in the C6/36 cells were much higher than those produced in the Vero cells. CONCLUSIONS: The reason CHIKV induced strong CPE is that this virus triggers strong apoptosis in Vero cells compared with C6/36 cells. CHIKV established a persistent infection in C6/36 cells after being passaged 20 times. CHIKV infection in mosquito cells was distinct from that in Vero cells. The cell and species specificity of CHIKV-induced cell death implies that the cellular and viral regulators involved in apoptosis may play an important role in determining the outcome of CHIKV infection.

Hepatitis E virus outbreak in monkey facility, Japan.

An outbreak of hepatitis E virus occurred in an outdoor monkey breeding facility in Japan during 2004-2006. Phylogenetic analysis indicated that this virus was genotype 3. This virus was experimentally transmitted to a cynomolgus monkey. Precautions should be taken by facility personnel who work with monkeys to prevent infection.