[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Sep 2014 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2013 · Virologica Sinica
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Here, we report the complete genome sequences of two Crimean-Congo hemorrhagic fever virus (CCHFV) strains, 79121M18 and YL04057,
isolated in Xinjiang, China. Sequence analysis showed that they represent a genotype of CCHFV that has not been reported before.
Full-text · Article · Jun 2013 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: 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, had 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 100.3 copies/μl) effectively. The study provides a simple, reproducible and feasible animal model for anti-HSV-2 drugs evaluation and HSV-2 vaccine research.
No preview · Article · Feb 2013 · Journal of virological methods
[Show abstract][Hide abstract] ABSTRACT: The embryonated chicken egg (ECE) provides a convenient, space-saving incubator for the cultivation of many kinds of animal viruses where the egg can be easily observed for viral replication throughout the development of the chicken embryo. Within the family Bunyaviridae, the embryonated egg has been used as a host system for many viruses such as Rift Valley fever virus and Akabane virus. The current study was conducted to determine the cultivation of Crimean-Congo hemorrhagic fever virus (CCHFV) in ECE. Four-day-old eggs were infected with CCHFV via the yolk sac route and harvested embryonic tissues and amino-allantoic fluid (AAF) that were used for virus passage and viral RNA (vRNA) detection. Quantification of vRNA copies was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our study indicated that CCHFV caused the death of the embryonated egg in a dose-dependent manner and the 50% egg infectious dose (EID(50)) was determined to be 6.47×10(5) copies/egg. CCHFV replicated and passaged well in the egg and high viral loads were detected both in embryonic tissue (10(9-10) copies/g) and AAF (10(7-9) copies/ml) of the embryonated egg. Thus, ECE could be used for viral cultivation and preservation, and as a potential host infection model for the study of the pathogenesis of CCHFV.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls. We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010. Out of 1036 cloacal swabs collected, three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards. All the isolates were genetically and phylogenetically characterized. The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics (QTRGL for H3N2 viruses, PEKASR for H4N6 virus) of lowly pathogenic avian influenza (LPAI) virus. The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses. The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses. In addition, co-infection of H3N2 and H4N6 in the same farm was observed. And interestingly, we isolated two subtypes viruses (H3N2 and H4N6) and their progeny virus (H3N2) with evidence of genome reassortment from the same farm, in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain. The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice, which was consistent with the results of genetic analysis.
Preview · Article · Sep 2012 · Chinese Science Bulletin
[Show abstract][Hide abstract] ABSTRACT: A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology (96%-99%) with known epidemic strains (A/California/04/2009(H1N1). Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences: A/Hubei/86/2009 PKVRDQEG → PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG → PKVRDQER, A/Hubei/75/2009 PKVRDQEG → PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein, 627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.
No preview · Article · Dec 2011 · Virologica Sinica
[Show abstract][Hide abstract] ABSTRACT: We aimed to determine the seroprevalence of Crimean-Congo hemorrhagic fever virus (CCHFV) infection in Yunnan Province, China.
One thousand six hundred and fifty-seven human serum samples and 1280 ticks (Hyalomma asiaticum) were collected from five counties (Menglian, Menghai, Lancang, Mengla, and Ximeng). Serum samples were analyzed independently by indirect immunofluorescence assay and Western blotting to detected CCHFV antibody. The ticks were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) to detect virus RNA.
The CCHFV IgG positivity was 3.4% (57/1657). A multivariate analysis was performed, and variables that increased the chance of infection were found to include history of tick bite or contact (odds ratio (OR) 16.6, 95% confidence interval (CI) 7.5-37.0) and age>30 years (OR 6.8, 95% CI 1.6-28.2). The RT-PCR positive rate for ticks was 14.3% (6/42).
The five counties (Menglian, Menghai, Lancang, Mengla, and Ximeng) in Yunnan are areas with the potential for CCHF outbreaks. Residents should protect themselves against tick bites and the surveillance of CCHFV in this region should be improved.
Full-text · Article · Jul 2011 · International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The molecular and pathogenic properties of avian influenza virus (A/duck/Hubei/216/1985/H7N8) isolated from Hubei Province
of China in 1985 were characterized. The hemagglutinin gene (HA) of Dk/Hb/216/85/H7N8 had the multiple amino acid sequences (-PEIPKGRG-) at the connecting peptide between HA1 and HA2, which
is considered to be a distinguishing molecular characteristic of low pathogenicity. The key sites of host markers among the
genes (M, NP, NS, PA and PB2) of Dk/Hb/216/85/H7N8 were similar to those of H5N1 viruses. Phylogenetic analysis showed that the genes (M, NS and PB2) of Dk/Hb/216/85/H7N8 clustered closely with those of Gs/Guangdong/1996/H5N1 (Gs/GD/96), implying that three ancient genes
of Gs/GD/96-like viruses had been circulating in Central China during the 1980s. In experimental infection, Dk/Hb/216/85/H7N8
was lowly pathogenic to chickens and mice, consistent with the molecular feature of HA gene.
Keywordsavian influenza virus-low pathogenicity-H7N8-H5N1
No preview · Article · Jun 2010 · Chinese Science Bulletin