B Viruses Research Articles

Analysis of complete genomes of the rubella virus genotypes 1E and 2B which circulated in China, 2000-2013.

Rubella viruses of genotypes 1E and 2B are currently the most frequently detected wild-type viruses in the world. Genotype 1E viruses from China have been genetically distinct from genotype 1E viruses found elsewhere, while genotype 2B viruses found in China are not distinguishable from genotype 2B viruses from other areas. Genetic clusters of viruses of both genotypes were defined previously using sequences of the 739-nt genotyping window. Here we report phylogenic analysis using whole genomic sequences from seven genotype 1E and three genotype 2B viruses which were isolated in China between 2000 and 2013 and confirm the subgrouping of current circulating genotypes 1E and 2B viruses. In addition, the whole genomic characterization of Chinese rubella viruses was clarified. The results indicated that the Chinese rubella viruses were highly conserved at the genomic level, and no predicted amino acid variations were found at positions where functional domains of the proteins were identified. Therefore, it gives us the idea that the rubella control and elimination goal should be achieved if vaccine immunization coverage continues maintaining at the high level.

Motor coordination and balance measurements reveal differential pathogenicity of currently spreading enterovirus 71 strains in human SCARB2 transgenic mice.

Enterovirus 71 (EV71) has caused large-scale epidemics with neurological complications in the Asia-Pacific region. The C4a and B5 strains are the two major genotypes circulating in many countries recently. This study used a new protocol, a motor coordination task, to assess the differential pathogenicity of C4a and B5 strains in human SCARB2 transgenic mice. We found that the pathogenicity of C4a viruses was more severe than that of B5 viruses. Moreover, we discovered that an increased level of monocyte chemoattractant protein-1 was positively correlated with severely deficient motor function. This study provides a new method for evaluating EV71 infection in mice and distinguishing the severity of the symptoms caused by different clinical strains, which would contribute to studies of pathogenesis and development of vaccines and antivirals in EV71 infections.

Virological Perspectives and Characterization of Seasonal Influenza B Viruses in Mumbai

Acute respiratory infections caused by influenza viruses are major life-threatening infections affecting the global population. Information on influenza viruses in Mumbai region in India is limited. With an estimated population above one crore, it becomes imperative to identify the predominant circulating strains, seasonal evolution, epidemiology and impact of influenza virus infection on the public health of Mumbai. The present study is aimed to isolate and identify the prevalent influenza B virus strains circulating in the Mumbai region. Virological analysis of 482 throat and nasal samples from patients presenting with suspected influenza-like illness was conducted from January to December 2012 at a research institute in Mumbai. Isolation of positive influenza B samples was performed on Madin Darby Canine Kidney (MDCK) cell line. The isolates were further characterized by hemagglutination inhibition assay and one-step reverse transcriptase polymerase chain reaction. Among all the specimens tested, 71% were positive for influenza virus. The positivity for influenza B virus, influenza A (H1N1) pdm 09 and seasonal influenza A virus was 38%, 22% and 11% respectively. Of the 80 influenza B specimens cultured on MDCK, virus was isolated from 38 (48%) samples. Influenza B/Wisconsin/1/2010-like serotype belonging to the B/Yamagata/18/88 lineage was the predominant serotype identified. Influenza was a significant public health concern in the Mumbai region during the study period. Young children were the most affected age group. The study data generated will help understand the epidemiology of the disease in the Mumbai region as well as generate information for global influenza surveillance.

Antiviral effect of emodin from Rheum palmatum against coxsakievirus B5 and human respiratory syncytial virus in vitro.

Viral infections are the major causes of morbidity and mortality in elderly people and young children throughout the world. The most common pathogens include coxsackie virus (CV) and respiratory syncytial virus (RSV). However, no antiviral agents with low toxicity and drug resistance are currently available in clinic therapy. The present study aimed to examine the antiviral activities of emodin (an ingredient of Rheum palmatum) against CVB5 and RSV infections, in an attempt to discover new antiviral agents for virus infection. The monomer emodin was extracted and isolated from Rheum palmatum. The antiviral activities of emodin on HEp-2 cells were evaluated, including virus replication inhibition, virucidal and anti-absorption effects, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-razolium bromide (MTT) assay and plaque reduction assay (PRA). The kinetics of virus inhibition by emodin in a period of 14 h was further determined by plaque assay and quantitative real time PCR (qPCR). Cytokine (IFN-γ, TNF-α) mRNA expressions after emodin treatment (7.5, 15, 30 μmol/L) were also assessed by qPCR post-infection. The results showed that emodin had potent inhibitory activities against CVB5 and RSV, with the 50% effective concentration (EC50) ranging from 13.06 to 14.27 μmol/L and selectivity index (SI) being 5.38–6.41 μmol/L. However, emodin couldn’t directly inactivate the viruses or block their absorption to cells. It acted as a biological synthesis inhibitor against CVB4 and RSV in a concentration- and time-dependent manner, especially during the first 0–4 h post-infection. Moreover, emodin could decrease the mRNA expression of IFN-α but enhance TNF-γ expression significantly compared to the viral controls in vitro. Our results provide a molecular basis for development of emodin as a novel and safe antiviral agent for human enterovirus and respiratory virus infection in the clinical therapy.

A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.

Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

BackgroundCCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates.MethodsDual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing.ResultsIn the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells.ConclusionsIn the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12981-015-0066-7) contains supplementary material, which is available to authorized users.

Phylodynamics of Enterovirus A71-Associated Hand, Foot, and Mouth Disease in Viet Nam.

ABSTRACTEnterovirus A71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011 and 2013 and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time frame of sampling, whereas that of B5 increased and remained >1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated area of endemicity. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia.IMPORTANCE EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

Design, antiviral and cytostatic properties of isoxazolidine-containing amonafide analogues

A novel series of 5-arylcarbamoyl- and 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesized via cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone with N-substituted naphthalimide acrylamides and N-allylnaphthalimides. All cis- and trans-isoxazolidine phosphonates obtained herein were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines trans-9d and trans-9f exhibited the highest activity (EC50=8.9μM) toward cytomegalovirus. Compounds cis- and trans-9d as well as cis- and trans-9f were found potent against HSV and Vaccinia viruses (EC50 in the 45–58μM range), whereas isoxazolidines 10a and 10d suppressed replication of Coxsackie B4 and Punta Toro viruses (EC50 in the 45–73μM range). Antiproliferative evaluation of all obtained isoxazolidines revealed the promising activity of cis-9b, cis-9d, trans-9d, cis-9e, trans-9e, cis-9f and trans-9f toward tested cancer cell lines with IC50 in the 1.1–19μM range.

Reemergence of enterovirus 71 epidemic in northern Taiwan, 2012.

BackgroundEnterovirus 71 (EV71) belongs to picornavirus family and could be classified phylogenetically into three major genogroups (A, B and C) including 11 genotypes (A, B1-B5 and C1-C5). Since 1997, EV71 has caused large-scale of epidemics with neurological complications in Asian children. In Taiwan, nationwide EV71 epidemics with different predominant genotypes have occurred cyclically since 1998. A nationwide EV71 epidemic occurred again in 2012. We conducted genetic and antigenic characterizations of the 2012 epidemic.MethodsChang Gung Memorial Hospital (CGMH) is a medical center in northern Taiwan. In CGMH, specimens were collected from pediatric inpatients with suspected enterovirus infections for virus isolation. Enterovirus isolates were serotyped and genotyped and sera from EV71 inpatients were collected for measuring neutralizing antibody titers.ResultsThere were 10, 16 and 99 EV71 inpatients identified in 2010, 2011 and 2012, respectively. There were 82 EV71 isolates genotyped, which identified 17 genotype C4a viruses and 65 genotype B5 viruses. The genotype B5 viruses were not detected until November 2011 and caused epidemics in 2012. Interestingly, the B5-2011 viruses were genetically distinguishable from the B5 viruses causing the 2008 epidemic and are likely introduced from China or Southeastern Asia. Based on antigenic analysis, minor antigenic variations were detected among the B5-2008, B5-2011, C4a-2008 and C4a-2012 viruses but these viruses antigenically differed from genotype A.ConclusionsGenotype B5 and C4a viruses antigenically differ from genotype A viruses which have disappeared globally for 30 years but have been detected in China since 2008. Enterovirus surveillance should monitor genetic and antigenic variations of EV71.

Evolutionary analysis of rubella viruses in mainland China during 2010-2012: endemic circulation of genotype 1E and introductions of genotype 2B.

Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010–2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010–2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.

Adaptive evolution in the S gene of hepatitis B virus from patients with occult HBV infection

Objective To detect the positively selected sites in the surface （ S） gene of hepatitis B viruses （ HBVs） from patients with occult HBV infection and to study the molecular mechanism of occult HBV infection.Methods The sequence of S gene from patients with occult HBV infection and reference strains of eight HBV genotypes （ A through H） were downloaded from GenBank and then alignment analysis were performed by using Clustal W software .Phylogenetic trees were constructed by using MEGA 5.05 soft-ware package.PAML4.7 was used to analyze positively selected sites .Results A total of 1286 HBV se-quences from patients with occult infection were searched in GenBank .One hundred and seventy-four com-plete gene sequences encoding surface S protein were screened after alignment analysis and confirmation , of which 13 sequences with nonsense mutation were removed .The likelihood ratio test showed that for both the 161 remained sequences and the 31 reference sequences , the selection models of M2, M3 and M8 were sig-nificantly better than the neutral models of M0, M1 and M7 （2△lnL〈55.12, P〈0.001）.By using Bayes Empirical Bayes （BEB） analysis, 14 positively selected sites （including codon 3, 8, 40, 45, 46, 47, 49, 68, 126, 127, 164, 184, 207 and 210） were detected in the surface gene of HBVs from patients with occult HBV infection, eight of which were located at the immune epitope of HBsAg .However, only 2 positively se-lected sites were identified in reference sequences .Conclusion The long-lasting persistence of HBV in pa-tients with occult HBV infection might be caused by the adaptive evolution of their surface gene in a form of escape mutant under immune suppressive condition . Key words: Hepatitis B virus; Occult infection; Adaptive evolution

Investigation of A Severe Enteroviral Encephalitis and Circulating Genotypes during Hand, Foot and Mouth Disease Surge in Nakhon Ratchasima Province, Thailand, August 2011

On 7 Aug 2011, a 1-month-old baby with encephalitis caused by unspecified enterovirus was reported during the period of widespread hand, foot and mouth disease (HFMD) outbreaks in Nakhon Ratchasima Province. An investigation was carried out to confirm the diagnosis, identify etiology of all severe enteroviral infection cases, determine magnitude of HFMD and enterovirus infection, including asymptomatic infections, and recommend prevention and control measures. A descriptive study was conducted by interviewing family members of the index case. Throat swab and stool specimens were collected to identify enterovirus. Survey on asymptomatic infection was done in schools attended by the index case’s siblings. Stool culture and isolation for enterovirus were also performed. Coxsackie B5 virus was isolated from fresh stool specimens of the index case. Among total 244 students screened for HFMD symptoms in four schools, only seven (3%) met the suspect case definition. During HFMD outbreaks caused by EV-71 B5, coxsackie A16 and coxsackie B5, proportion of asymptomatic infection among students was 22%. Risk factors such as no soap in toilets and misuse of alcohol gel to clean hands in schools were observed during the environmental survey.

Neurosteroids and Host Immune Resistance

Androstene steroids up-regulate immunity to increase host resistance against lethal infection by viruses, bacteria, and parasites. They also repair injury after lethal radiation exposure by mediating a rapid recovery of hematopoietic precursor cells. Androstenes increase the levels of the TH1 cytokines, IL-2, IL-3, IFN and counteract hydrocortisone mediated immune suppression. Increased host resistance results in protection from lethal infection by DNA or RNA viruses such as herpes virus, coxsackievirus B4, influenza, and arthropod borne viruses. They provide similar protection against lethal Gram positive and negative bacteria as well as parasitic infections. During shock response, the hypothalamic-pituitary-adrenal axis (HPA) causes unchecked inflammation and sepsis. Androstenetriol treatment reduces Th2 and increases in Th1 cytokines, and significantly increases survival after severe trauma hemorrhage and shock by modulating the HPA response. In contrast to its epimer, 17 androstenediol inhibits proliferation and mediates apoptosis in tumor cells of murine and human origin and autophagy in human glioblastoma. The anti-proliferative functions or the immune up regulation by these neurosteroids are independent of either the estrogen or androgen receptors for activity. Results demonstrate that endoplasmic reticulum (ER) stress is linked to 17 androstenediol induced autophagy by PERK/eIF2 signaling in human malignant glioma cells and transformed fibroblast. The androstene hormones increase host resistance to infections by enhancing cell mediated immunity while androstene exerts an anti- tumor effect by apoptosis, autophagy and increase ER stress.

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors

Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment.

Temperature Effects for High-Pressure Processing of Picornaviruses

Investigation of the effects of pre-pressurization temperature on the high-pressure inactivation for single strains of aichivirus (AiV), coxsackievirus A9 (CAV9) and B5 (CBV5) viruses, as well as human parechovirus-1 (HPeV) was performed. For CAV9, an average 1.99 log10 greater inactivation was observed at 4°C after a 400-MPa-5-min treatments compared to 20°C treatments. For CBV5, an average of 2.54 log10 greater inactivation was noted after 600-MPa-10-min treatments at 4°C in comparison to 20°C treatments. In contrast, inactivation was reduced by an average of 1.59 log10 at 4°C for HPeV. AiV was resistant to pressure treatments of 600MPa for as long as 15min at 4, 20, and 30°C temperatures. Thus, different pre-pressurization temperatures result in different inactivation effects for picornaviruses.

Microbiologic Findings in Acute Facial Palsy in Children

Microbiologic causes of facial palsy in children were investigated. Prospective clinical study. Tertiary referral center. Forty-six children aged 0 to 16 years with peripheral facial palsy. Paired serum samples and cerebrospinal fluid were tested to find indications of microbes associated with facial palsy. The microbes tested were herpes simplex virus 1 and 2, varicella-zoster virus, human herpesvirus-6, Mycoplasma pneumoniae, Borrelia burgdorferi, influenza A and B virus, picorna, cytomegalovirus, parainfluenza virus, respiratory syncytial virus, coxsackie B5 virus, adenovirus, and enterovirus, Chlamydia psittaci, and Toxoplasma gondii. Besides the routine tests in clinical practice, serum and cerebrospinal fluid samples were tested with a highly sensitive microarray assay for DNA of herpes simplex virus 1 and 2; human herpes virus 6A, 6B, and 7; Epstein-Barr virus, cytomegalovirus, and varicella zoster virus. Incidence for facial palsy was 8.6/100,000/children/year. Cause was highly plausible in 67% and probable in an additional 11% of cases. Borrelia burgdorferi caused facial palsy in 14 patients (30%), varicella zoster virus in 5 (11%) (one with concomitant adenovirus), influenza A in 3 (6%), herpes simplex virus 1 in 2 (4%) (one with concomitant enterovirus), otitis media in 2 (4%), and human herpesvirus 6 in 2 (4%). Mycoplasma pneumoniae, neurofibromatosis, and neonatal age facial palsy affected 1 child (2%) each. Microbiologic etiology association of pediatric facial palsy could frequently be confirmed. Borreliosis was the single most common cause; hence, cerebrospinal fluid sampling is recommended for all pediatric cases in endemic areas. Varicella zoster virus accounted for 11% of the cases, being the second most common factor.

Molecular surveillance of rubella viruses in Taiwan from 2005 to 2011

Rubella has been listed as a mandatory notifiable disease in Taiwan since 1988. Because of high coverage rates with an effective vaccine, rubella cases have decreased dramatically in Taiwan since 1994. However, rubella outbreaks still occur due to imported transmission. Five large clusters were detected in Taiwan from 2007 to 2011. In 2007, one cluster was caused by rubella genotype 1E viruses that were imported from Vietnam, whereas another cluster was caused by genotype 2B viruses and was untraceable. In 2008, two clusters were caused by different lineages of genotype 1E viruses that were imported from Malaysia. In 2009, a cluster that was caused by genotype 2B viruses was associated with imported cases from Vietnam. The rubella viruses from 124 confirmed cases from 2005 to 2011 were characterized, and the data revealed that these viruses were distributed in the following four genotypes: 1E (n = 56), 1h (n = 1), 1j (n = 4), and 2B (n = 63). Of these viruses, 93 (75%) were associated with imported cases, and 43 of 56 genotype 1E viruses were associated with imported cases from China, Vietnam, Malaysia, and Indonesia. One genotype 1h virus was imported from Belarus, and three of four genotype 1j viruses were imported from the Philippines. Of 63 rubella genotype 2B viruses, 46 were imported from Vietnam, Thailand, Malaysia, China, Germany, and South Africa. Molecular surveillance allows for the differentiation of circulating rubella viruses and can be used to investigate transmission pathways, which are important to identify the interruption of endemic virus transmission.

Detection and significance of gene variation in hepatitis B virus S region

To explore the variations of gene S in hepatitis B viruses of hepatitis B patients and provide experimental evidences for the mutation analysis of viral gene. The virus DNA load in hepatitis B patient donors was detected by real-time polymerase chain reaction (PCR) and gene sequence analysis. And a comparison was made with standard strain by the software DNAstar. (1) Gene S was successfully amplified and sequenced in 15 hepatitis B patients. Three samples had I→T mutation at residue 126 in HBsAg "a" antigenic determinant. (2) Sixteen hepatitis B patients had 67 nucleotide mutations, including 14 residues in PreS1 and 6 residues in PreS2. Mutations nt 3036 T→C, nt 3039 T→G, nt 3066 C→T and L88V existed in PreS1 gene in all samples. HBV genome is susceptible to nucleotide mutations. Some residues have geographically restricted mutations in gene S region. And understanding the significance of these mutations may help clarify the pathogenesis of hepatitis B and provide new experimental evidence for its gene diagnosis and prevention.

Increased Interaction between Vaccinia Virus Proteins A33 and B5 Is Detrimental to Infectious Extracellular Enveloped Virion Production

Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion to this paper (W. M. Chan and B. M. Ward, J. Virol. 86:8210-8220, 2012), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding. Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this interaction needs to be regulated for the production of fully infectious extracellular virions.

Production of Infectious Chimeric Hepatitis C Virus Genotype 2b Harboring Minimal Regions of JFH-1

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.