Evidence of Human Exposure to Tamdy Virus, Northwest China.

We report the isolation of Tamdy virus from Hyalomma asiaticum ticks in northwest China and serologic evidence of human Tamdy virus infection in the same region. These findings highlight the need to further investigate a potential causal relationship between Tamdy virus and febrile illnesses of unknown etiology in that region.

Synthesis, antiviral activity, 3D-QSAR, and interaction mechanisms study of novel malonate derivatives containing quinazolin-4(3H)-one moiety.

A series of novel malonate derivatives containing quinazolin-4(3H)-one moiety were synthesized and evaluated for their antiviral activities against cucumber mosaic virus (CMV). Results indicated that the title compounds exhibited good antiviral activities. Notably, compounds g15, g16, g17, and g18 exhibited excellent curative activities in vivo against CMV, with 50% effective concentration (EC50) values of 208.36, 153.78, 181.47, and 164.72µg/mL, respectively, which were better than that of Ningnanmycin (256.35µg/mL) and Ribavirin (523.34µg/mL). Moreover, statistically valid three-dimensional quantitative structure-activity relationship (3D-QSAR) models with good correlation and predictive power were obtained with comparative molecular field analysis (CoMFA) steric and electrostatic fields (r(2)=0.990, q(2)=0.577) and comparative molecular similarity indices analysis (CoMSIA) with combined steric, electrostatic, hydrophobic and hydrogen bond acceptor fields (r(2)=0.977, q(2)=0.516), respectively. Based on those models, compound g25 was designed, synthesized, and showed better curative activity (146.30µg/mL) than that of compound g16. The interaction of between cucumber mosaic virus coat protein (CMV CP) and g25 with 1:1.83 ratio is typically spontaneous and exothermic with micromole binding affinity by isothermal titration calorimetry (ITC) and fluorescence spectroscopy investigation.

Molecular characterization of influenza B viruses isolated in east-central China in 2009-2010.

The current circulating influenza B viruses can be divided into two major phylogenetic lineages: the Victoria and Yamagata lineages. We conducted a survey of influenza B viruses in Hubei and Zhejiang provinces during 2009-2010. Out of 341 throat swabs, 18 influenza B viruses were isolated. Five isolates were selected for genetic and phylogenetic analysis. The molecular analyses revealed that all the isolates had similar antigenic characteristics to B/Brisbane/60/2008. However, in the three viruses isolated from Zhejiang, a single asparagine to aspartic acid substitution in position 197 was observed, thereby eliminating the glycosylation at that site and possibly causing an antigenic change. None of the viruses had amino acid mutations at positions 116, 149, 152, 198, 222, 250, 291, and 402 of the neuraminidase (NA) gene, predicting that the viruses would still be sensitive to NA inhibitors. Phylogenetic analyses revealed that all five isolates were closely related to B/Brisbane/60/2008-the 2010 vaccine strain-and contained Victoria-like hemagglutinin and Yamagata-like NA genes, suggesting that reassortment may had occurred. In addition, similar phylogenetic patterns among the acidic polymerase, nucleoprotein and matrix protein genes, as well as between the basic polymerase 1 and basic polymerase 2 genes, were observed, suggesting possible functional interactions among these proteins. All the results highlighted the importance of molecular monitoring of influenza B viruses for reassortment and antigenic drift.

Amino acids 473V and 598P of PB1 from an avian-origin influenza A virus contribute to polymerase activity, especially in mammalian cells.

It has been reported that the avian-origin influenza A virus PB1 protein (avian PB1) enhances influenza A virus polymerase activity in mammalian cells when it replaces the human-origin PB1 protein (human PB1). Characterization of the amino acid residues that contribute to this enhancement is needed. In this study, it was found that PB1 from an avian-origin influenza A virus [A/Cambodia/P0322095/2005, H5N1 (Cam)] could enhance the polymerase activity of an attenuated human isolated virus, A/WSN/33, carrying the PB2 K627E mutation (WSN627E) in vitro. Furthermore, 473V and 598P in the Cam PB1 were identified as the residues responsible for this enhanced activity. The results from recombinant virus experiments demonstrated the contribution of PB1 amino acids 473V and 598P to polymerase activity in mammalian cells and in mice. Interestingly, 473V is conserved in pH1N1 viruses from the 2009 pandemic. Substitution of 473V by leucine in pH1N1 PB1 led to a decreased viral polymerase activity and a lower growth rate in mammalian cells, suggesting that the PB1 473V also plays a role in maintaining efficient virus replication of the pH1N1 virus. Thus, it was concluded that two amino acids in avian-origin PB1, 473V and 598P, contribute to the polymerase activity of the H5N1 virus, especially in mammalian cells, and that 473V in PB1 also contributes to efficient replication of the pH1N1 strain.

Modification of nonstructural protein 1 of influenza A virus by SUMO1.

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.

MiRPara: a SVM-based software tool for prediction of most probable microRNA coding regions in genome scale sequences.

BACKGROUND: MicroRNAs are a family of ~22 nt small RNAs that can regulate gene expression at the post-transcriptional level. Identification of these molecules and their targets can aid understanding of regulatory processes. Recently, HTS has become a common identification method but there are two major limitations associated with the technique. Firstly, the method has low efficiency, with typically less than 1 in 10,000 sequences representing miRNA reads and secondly the method preferentially targets highly expressed miRNAs. If sequences are available, computational methods can provide a screening step to investigate the value of an HTS study and aid interpretation of results. However, current methods can only predict miRNAs for short fragments and have usually been trained against small datasets which don't always reflect the diversity of these molecules. RESULTS: We have developed a software tool, miRPara, that predicts most probable mature miRNA coding regions from genome scale sequences in a species specific manner. We classified sequences from miRBase into animal, plant and overall categories and used a support vector machine to train three models based on an initial set of 77 parameters related to the physical properties of the pre-miRNA and its miRNAs. By applying parameter filtering we found a subset of ~25 parameters produced higher prediction ability compared to the full set. Our software achieves an accuracy of up to 80% against experimentally verified mature miRNAs, making it one of the most accurate methods available. CONCLUSIONS: miRPara is an effective tool for locating miRNAs coding regions in genome sequences and can be used as a screening step prior to HTS experiments. It is available at http://www.whiov.ac.cn/bioinformatics/mirpara.

Influenza A virus replication induces cell cycle arrest in G0/G1 phase.

Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G(0)/G(1)-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G(0)/G(1) phase into S phase. Consistent with the G(0)/G(1)-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G(1) into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G(0)/G(1)-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G(0)/G(1) phase were observed compared to those in either unsynchronized cells or cells synchronized in the G(2)/M phase. G(0)/G(1)-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G(0)/G(1)-phase cell cycle arrest in infected cells.

Production and characterization of monoclonal antibodies against the hemagglutinin of H5N1 and antigenic investigation of avian influenza H5N1 viruses isolated from China.

Eight monoclonal antibodies against hemagglutinin of influenza A virus A/Chicken/Henan/01/2004(H5N1) were produced by a DNA prime and inactivated virions-boost immunization strategy. Among the monoclonal antibodies, 3 (H50, H56, and H57) exhibited hemagglutination inhibition activity. Western blot analyses revealed that all the monoclonal antibodies reacted to the prokaryotically expressed HA1 of A/Chicken/Henan/01/2004(H5N1). The monoclonal antibodies were then used to characterize 10 avian influenza H5N1 viruses isolated from China during 2004 to 2007, by using the hemagglutination inhibition test and the antigen-capture enzyme-linked immunosorbent assay. The isolates could be divided into 4 different antigenic groups according to their responses to the monoclonal antibodies. The antigenic grouping of these 10 H5N1 isolates, using these antibodies, did not completely match their phylogenetic classification based on the hemagglutinin sequences. The results showed there were antigenic variations within the subclade 2.3.4 of H5N1, which is predominant in China.

Migration of waterfowl in the East Asian flyway and spatial relationship to HPAI H5N1 outbreaks.

Poyang Lake is situated within the East Asian Flyway, a migratory corridor for waterfowl that also encompasses Guangdong Province, China, the epicenter of highly pathogenic avian influenza (HPAI) H5N1. The lake is the largest freshwater body in China and a significant congregation site for waterfowl; however, surrounding rice fields and poultry grazing have created an overlap with wild waterbirds, a situation conducive to avian influenza transmission. Reports of HPAI H5N1 in healthy wild ducks at Poyang Lake have raised concerns about the potential of resilient free-ranging birds to disseminate the virus. Yet the role wild ducks play in connecting regions of HPAI H5N1 outbreak in Asia is hindered by a lack of information about their migratory ecology. During 2007-08 we marked wild ducks at Poyang Lake with satellite transmitters to examine the location and timing of spring migration and identify any spatiotemporal relationship with HPAI H5N1 outbreaks. Species included the Eurasian wigeon (Anas penelope), northern pintail (Anas acuta), common teal (Anas crecca), falcated teal (Anas falcata), Baikal teal (Anas formosa), mallard (Anas platyrhynchos), garganey (Anas querquedula), and Chinese spotbill (Anas poecilohyncha). These wild ducks (excluding the resident mallard and Chinese spotbill ducks) followed the East Asian Flyway along the coast to breeding areas in northern China, eastern Mongolia, and eastern Russia. None migrated west toward Qinghai Lake (site of the largest wild bird epizootic), thus failing to demonstrate any migratory connection to the Central Asian Flyway. A newly developed Brownian bridge spatial analysis indicated that HPAI H5N1 outbreaks reported in the flyway were related to latitude and poultry density but not to the core migration corridor or to wetland habitats. Also, we found a temporal mismatch between timing of outbreaks and wild duck movements. These analyses depend on complete or representative reporting of outbreaks, but by documenting movements of wild waterfowl, we present ecological knowledge that better informs epidemiological investigations seeking to explain and predict the spread of avian influenza viruses.

Persistent circulation of highly pathogenic influenza H5N1 virus in Lake Qinghai area of China.

To trace the endemic situation of highly pathogenic influenza H5N1 virus in wildlife in the Lake Qinghai area of northwest China, a continuous surveillance program was implemented from September 2005 to September 2007. A total of 2699 field samples were collected, and 95 of the samples were positive by a reverse transcription-polymerase chain reaction (RT-PCR) test of subtype H5 influenza virus. To some extent, the detection rate correlated with the species, the location, and the seasons of collection. In 2007, two H5N1 isolates were identified from two species of migratory birds. The two 2007 isolates showed high similarities of the hemagglutinin and neuraminidase genes with those of the 2005 and 2006 Qinghai isolates (98.6-99.8% for HA and 98.7-98.9% for NA at the nucleotide level). Both isolates fell into clade 2.2.2 and were experimentally highly pathogenic to chickens and mice. According to our surveillance results, HPAI H5N1 viruses still exist in this region.

Molecular characterizations of avian polyomavirus isolated from budgerigar in China.

Budgerigar fledgling disease is an acute viral infectious disease caused by avian polyomavirus (APV). In this study, 34 liver tissue samples of young, dead budgerigar with typical symptoms were collected in 2004. All the samples had positive polymerase chain reaction (PCR) test based on the VP1 specific primers. VP1 genes of these samples were sequenced and had high similarities to each other (99%-100%). A strain (HBYM02) was isolated and sequenced. As shown in the phylogenetic tree, there are two branches. One branch was composed by strains isolated from Passeriformes, and the other was composed only by one strain isolated from Falconiformes. The genome similarities between our isolate and other reported isolates were very high (> 99%), and the evolution distances in the phylogenetic tree were very short (< 0.005), which suggests that APV in China has the same genotype as those in other regions. The results will be useful for the diagnoses of, and vaccine development for, APV.

Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006.

Avian influenza H5N1 viruses pose a significant threat to human health because of their ability to infect humans directly. In the paper, three highly pathogenic H5N1 influenza viruses were isolated from three species of migratory birds in Qinghai Province of China in 2006. The analysis of the genome sequences indicated that the three isolates shared high homology with each other (94% to 99%). Three isolates shared a common ancestor and were closest to strains isolated from Qinghai and Siberia in 2005, but distinct from poultry viruses found in Southeast Asia. In experimental infection, all three viruses were highly pathogenic to chickens and mice. The results suggest that highly pathogenic avian influenza H5N1 viruses still exist in the migratory birds and could spread to other regions with wild bird migration.

Design, synthesis, and antiviral activities of 1,5-benzothiazepine derivatives containing pyridine moiety.

In our previous work, a series of novel benzothiazepine derivatives containing pyridine moiety were successfully synthesized through chalcone 1,3-dipolar cycloaddition and determined their antiviral activity against tobacco mosaic virus (TMV). Bioassay results indicated that most of these target compounds exhibited improved curative, protection, and inactivation activity in vivo than the commercial agent ningnanmycin. Particularly, compound 3m exhibited marked curative activity against TMV, with an EC50 value of 352.2 µM, which was even better than that of ningnanmycin. The compound was identified as the most promising candidate for inhibiting plant virus and an excellent compound with antiviral activities against TMV. Structure-activity relationship experiment indicated that the 1,5-benzothiazepine moiety is crucial for potent anti-TMV activity.

Metagenomic profile of the viral communities in Rhipicephalus spp. ticks from Yunnan, China.

Besides mosquitoes, ticks are regarded as the primary source of vector-borne infectious diseases. Indeed, a wide variety of severe infectious human diseases, including those involving viruses, are transmitted by ticks in many parts of the world. To date, there are no published reports on the use of next-generation sequencing for studying viral diversity in ticks or discovering new viruses in these arthropods from China. Here, Ion-torrent sequencing was used to investigate the presence of viruses in three Rhipicephalus spp. tick pools (NY-11, NY-13, and MM-13) collected from the Menglian district of Yunnan, China. The sequencing run resulted in 3,641,088, 3,106,733, and 3,871,851 reads in each tick pool after trimming. Reads and assembled contiguous sequences (contigs) were subject to basic local alignment search tool analysis against the GenBank database. Large numbers of reads and contigs related to known viral sequences corresponding to a broad range of viral families were identified. Some of the sequences originated from viruses that have not been described previously in ticks. Our findings will facilitate better understanding of the tick virome, and add to our current knowledge of disease-causing viruses in ticks living under natural conditions.

Testing the Effect of Internal Genes Derived from a Wild-Bird-Origin H9N2 Influenza A Virus on the Pathogenicity of an A/H7N9 Virus.

Since 2013, avian influenza A(H7N9) viruses have diversified into multiple lineages by dynamically reassorting with other viruses, especially H9N2, in Chinese poultry. Despite concerns about the pandemic threat posed by H7N9 viruses, little is known about the biological properties of H7N9 viruses that may recruit internal genes from genetically distinct H9N2 viruses circulating among wild birds. Here, we generated 63 H7N9 reassortants derived from an avian H7N9 and a wild-bird-origin H9N2 virus. Compared with the wild-type parent, 25/63 reassortants had increased pathogenicity in mice. A reassortant containing PB1 of the H9N2 virus was highly lethal to mice and chickens but was not transmissible to guinea pigs by airborne routes; however, three substitutions associated with adaptation to mammals conferred airborne transmission to the virus. The emergence of the H7N9-pandemic reassortant virus highlights that continuous monitoring of H7N9 viruses is needed, especially at the domestic poultry/wild bird interface.

Improved methods for isolation of avian influenza virus.

Isolation of viruses using chick embryos is a classical virological method. Inoculation of the allantoic cavity and use of allantoic fluid is a common method of passaging isolated avian influenza viruses. In the present study, 2490 fresh fecal samples and 4967 old fecal samples were investigated and subjected to conventional passaging (allantoic fluid method). Two newly developed methods-the allantochorion and allantoic fluid mixed method and the chick embryo and allantoic fluid mixed method-were also examined. The rates of influenza virus isolation for these three methods were compared. There appeared to be little difference among these methods when fresh fecal samples were used. However, for the old fecal samples, isolation rates for influenza virus were significantly higher for the chick embryo and allantoic fluid mixed method compared with the conventional allantoic fluid method. All viruses isolated using the conventional allantoic fluid method were isolated successfully using the two newly developed methods. These results suggest that using chick embryos in conjunction with allantoic fluid is effective for early virus isolation, especially for fecal samples that are not fresh. Additionally, practical chick embryo passage methods are described that improve significantly the rate of isolation of influenza viruses from fecal samples of migratory birds in a complex wild ecological environment.

Freshwater cyanophages.

Cyanophages are double-stranded DNA viruses that infect cyanobacteria, and they can be found in both freshwater and marine environments. They have a complex pattern of host ranges and play important roles in controlling cyanobacteria population. Unlike marine cyanophages, for which there have been a number of recent investigations, very little attention has been paid to freshwater cyanophages. This review summarizes the taxonomy and morphology, host range, distribution, seasonal dynamics, and complete genomes of freshwater cyanophages, as well as diagnostic markers that can be used to identify them.

Mini-genome rescue of Crimean-Congo hemorrhagic fever virus and research into the evolutionary patterns of its untranslated regions.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of genus Nairovirus, family Bunyaviridae, which are distributed widely in Africa, Europe and Asia with several genotypes. As a BSL-4 level pathogen, the requirement of high-level biosafety facilities severely constrains researches on live virus manipulation. In this study, we developed a helper-virus-independent mini-genome rescue system for the Chinese YL04057 strain. Based on the enhanced green fluorescent protein (EGFP)-derived mini-genome plasmids, this polymerase I driven system permits easy observation and quantification. Unlike previous report, gradually reduced levels of activity of the CCHFV L, M and S untranslated regions (UTRs) were observed in our system. We also demonstrated that the UTRs at both ends were indispensable for mini-genome background expression. In addition, we phylogentically analyzed all six UTRs of CCHFV and showed that L-UTRs were clustered together approximately corresponding to their original geographical continents. The UTRs of M segment showed a similar branch structure to its open reading frames (ORFs), and nearly an identical tree was generated with 5' UTRs of S segment compared with its ORFs. However, the 3' UTRs of S segment formed new divergent groups. Compatibility tests of YL04057 strain nucleocapsid protein and L protein expression plasmids with Nigerian strain IbAr10200 mini-genomes revealed lower compatibility of L-UTRs without an obvious effect on M-UTRs. Moreover, we demonstrated that the L-UTRs could tolerate certain nucleotide mutations. This system may provide a foundation for future studies of the viral replication cycle, pathogenic mechanisms and evolutionary patterns of CCHFV.

Microbial transformation of curcumol by Aspergillus niger.

Curcumol is a representative index component for the quality control of the essential oil of Curcuma wenyujin Y.H. Chen et C. Ling, an antivirus and anticancer drug in China. Microbial transformation of curcumol (1) by Aspergillus niger AS 3.739 yielded two products. Their structures were elucidated as 3alpha-hydroxycurcumol (2) and 3alpha-(4'-methoxy-succinyloxy)-curcumol (3) by extensive spectroscopic methods including 2D-NMR and HRESI-MS. Among them, 3 is a new compound. Esterification of the substrate with succinic acid is a novel reaction in the field of microbial transformation of natural products. Compound 2, the major transformation product of 1, was a high regio- and stereo-specific hydroxylation product and showed significant antiviral effects.

An improved system for the evaluation of antiviral compounds against herpes simplex virus type 2.

Infection with herpes simplex virus type 2 (HSV-2) can result in lesions in reproductive organs, along with long-term latency. In this work, a non-lethal strain of HSV-2 which was isolated clinically was used to infect female mice intravaginally. Body weight, vulval lesions, histological examination of vaginal tissue, and viral load were monitored and used as indices for evaluating antiviral drugs against HSV-2 infection. The results indicated that mice infected with HSV-2 exhibited significant reduction in body weight, serious vulval lesions, massive lymphocyte invasion of vaginal tissue, and approximately 104 copies/µl of HSV-2 were found in vaginal and uterine tissues. Aciclovir (ACV) treatment inhibited loss in body weight, genital pathology and virus replication (reduced to 10°·³ copies/µl) effectively. The study provides a simple, reproducible and feasible animal model for anti-HSV-2 drugs evaluation and HSV-2 vaccine research.