Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton.

BACKGROUND: Cytoplasmic male sterility (CMS) is a maternally inherited trait failing to produce functional pollen. It plays a pivotal role in the exploitation of crop heterosis. The specific locus amplified fragment sequencing (SLAF-seq) as a high-resolution strategy for the identification of new SNPs on a large-scale is gradually applied for functional gene mining. The current study combined the bulked segregant analysis (BSA) with SLAF-seq to identify the candidate genes associated with fertility restorer gene (Rf) in CMS cotton. METHODS: Illumina sequencing systematically investigated the parents. A segregating population comprising of 30 + 30 F2 individuals was developed using 3096A (female parent) as sterile and 866R (male parent) as a restorer. The original data obtained by dual-index sequencing were analyzed to obtain the reads of each sample that were compared to the reference genome in order to identify the SLAF tag with a polymorphism in parent lines and the SNP with read-associated coverage. Based on SLAF tags, SNP-index analysis, Euclidean distance (ED) correlation analysis, and whole genome resequencing, the hot regions were annotated. RESULTS: A total of 165,007 high-quality SLAF tags, with an average depth of 47.90× in the parents and 50.78× in F2 individuals, were sequenced. In addition, a total of 137,741 SNPs were detected: 113,311 and 98,861 SNPs in the male and female parent, respectively. A correlation analysis by SNP-index and ED initially located the candidate gene on 1.35 Mb of chrD05, and 20 candidate genes were identified. These genes were involved in genetic variations, single base mutations, insertions, and deletions. Moreover, 42 InDel markers of the whole genome resequencing were also detected. CONCLUSIONS: In this study, associated markers identified by super-BSA could accelerate the study of CMS in cotton, and as well as in other crops. Some of the 20 genes' preliminary characteristics provided useful information for further studies on CMS crops.

Diversity and circulation of Jingmen tick virus in ticks and mammals.

Since its initial identification in ticks in 2010, Jingmen tick virus (JMTV) has been described in cattle, rodents, and primates. To better understand the diversity, evolution, and transmission of JMTV, we sampled 215 ticks, 104 cattle bloods, 216 bats, and 119 rodents in Wenzhou city, Zhejiang Province, China as well as 240 bats from Guizhou and Henan Provinces. JMTV was identified in 107 ticks (from two species), 54 bats (eleven species), 8 rodents (three species), and 10 cattle, with prevalence levels of 49.8, 11.8, 6.7, and 9.6 per cent, respectively, suggesting that bats may be a natural reservoir of JMTV. Phylogenetic analyses revealed that all the newly identified JMTVs were closely related to each other and to previously described viruses. Additionally, all tick and mammalian JMTV sampled in Wenzhou shared a consistent genomic structure, suggesting that the virus can cocirculate between ticks and mammals without observable variation in genome organization. All JMTVs sampled globally could be divided into two phylogenetic groups, Mantel tests suggested that geographic isolation, rather than host species, may be the main driver of JMTV diversity. However, the exact geographical origin of JMTV was difficult to determine, suggesting that this virus has a complex evolutionary history.

Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China.

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.

Investigation of the role of healthy dogs as potential carriers of rabies virus.

To investigate whether healthy animals are potential carriers of rabies virus in China, 153 domestic dogs were collected from a rabies enzootic area, Anlong county in Guizhou Province, and monitored for 6 months. Initially, findings of rabies virus antigen in the saliva of 15 dogs by an enzyme-linked immunosorbent assay (ELISA) test suggested they might be carriers. These 15 dogs were kept under observation for 6 months. None of the dogs showed any clinical signs of rabies during the observation period. Moreover, using the ELISA test alone, detection of rabies virus antigen in saliva of some animals was not consistent during the observation period. However, none of the saliva samples collected either at the time of acquisition or during the observation period was found to be positive for rabies virus RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, neither viral antigen nor viral RNA was detected in the brain samples collected at the time of euthanasia. These results do not provide support for the contention that healthy dogs act as carriers in rabies. Caution is urged when preliminary and nondefinitive tests, such as ELISA, are used to infer clinical status related to rabies.

Extensive diversity and evolution of hepadnaviruses in bats in China.

To better understand the evolution of hepadnaviruses, we sampled bats from Guizhou, Henan and Zhejiang provinces, China, and rodents from Zhejiang province. Genetically diverse hepadnaviruses were identified in a broad range of bat species, with an overall prevalence of 13.3%. In contrast, no rodent hepadnaviruses were identified. The newly discovered bat hepadnaviruses fell into two distinct phylogenetic groups. The viruses within the first group exhibited high diversity, with some closely related to viruses previously identified in Yunnan province. Strikingly, the newly discovered viruses sampled from Jiyuan city in the second phylogenetic group were most closely related to those found in bats from West Africa, suggestive of a long-term association between bats and hepadnaviruses. A co-phylogenetic analysis revealed frequent cross-species transmission among bats from different species, genera, and families. Overall, these data suggest that there are likely few barriers to the cross-species transmission of bat hepadnaviruses.

Extensive diversity of coronaviruses in bats from China.

To help reveal the diversity and evolution of bat coronaviruses we collected 1067 bats from 21 species in China. A total of 73 coronaviruses (32 alphacoronaviruses and 41 betacoronaviruses) were identified in these bats, with an overall prevalence of 6.84%. All newly-identified betacoronaviruses were SARS-related Rhinolophus bat coronaviruses (SARSr-Rh-BatCoV). Importantly, with the exception of the S gene, the genome sequences of the SARSr-Rh-BatCoVs sampled in Guizhou province were closely related to SARS-related human coronavirus. Additionally, the newly-identified alphacoronaviruses exhibited high genetic diversity and some may represent novel species. Our phylogenetic analyses also provided insights into the transmission of these viruses among bat species, revealing a general clustering by geographic location rather than by bat species. Inter-species transmission among bats from the same genus was also commonplace in both the alphacoronaviruses and betacoronaviruses. Overall, these data suggest that high contact rates among specific bat species enable the acquisition and spread of coronaviruses.

Genetic characterization of hantaviruses isolated from Guizhou, China: evidence for spillover and reassortment in nature.

To gain more insights into the epidemiology of hantaviruses in the Guizhou province, China, rodents were captured in Guizhou during the period from 2001 to 2003. In addition, serum sample was collected from one patient. Virus isolation was attempted from human serum and rodent samples. Four hantaviruses were isolated successfully in cell culture from one human, two A. agrarius, and one R. norvegicus. The nucleotide sequences for the entire S and M and partial L segment were determined from these four isolates as well as six viruses isolated in 1980s. Phylogenetic analysis revealed that the S segment from all isolates belong to the Hantaan virus (HTNV) clade, regardless of the sources from which they were derived. According to the S sequences, these viruses could be divided into three distinct phylogroups, showing geographical clustering. Analysis of the entire M and the partial L segment sequences demonstrated that 8 out of the 10 isolates belong to the HTNV clade. However, two isolates (CGRn8316 and CGRn9415) isolated from R. norvegicus belong to the Seoul virus (SEOV) clade. In addition, these two isolates were distinct from other known members of SEOV clade. Together, the data suggest that at least three groups of HTNV are co-circulating and one new variant of SEOV may be present in Guizhou. Our results also suggest that HTNV from A. agrarius spilled over to R. norvegicus and natural reassortment between HTNV and SEOV occurred during or after the spillover.

Isolation and characterization of the full coding sequence of a novel densovirus from the mosquito Culex pipiens pallens.

During an investigation of arboviruses in China, a novel densovirus (DNV) was isolated from the adult female Culex pipiens pallens. The virus, designated Culex pipiens pallens densovirus (CppDNV), caused cytopathic effect in C6/36 cells. The virus particles were icosahedral, non-enveloped and had a mean diameter of 24 nm. The complete coding region of CppDNV was found to be 3335 nt and it contained three open reading frames (ORFs). CppDNV shares 82-93 % identical nucleotides with isolates of the Aedes albopictus densovirus [isolates AalDNV-1, AalDNV-2 (C6/36 DNV) and AalDNV-3], Aedes aegypti densovirus (AaeDNV) and Haemagogus equines densovirus (HeDNV). The nucleotide sequence identity among CppDNV isolates exceeds 98 %. Phylogenetic trees based on non-structural (NS1 and NS2) and capsid (VP) genes show that CppDNV clustered with the species AaeDNV and represents a novel variant of this species within the genus Brevidensovirus.

Molecular diversity and phylogeny of Hantaan virus in Guizhou, China: evidence for Guizhou as a radiation center of the present Hantaan virus.

To gain further insight into the molecular epidemiology of Hantaan virus (HTNV) in Guizhou, China, rodents were captured in this region in 2004 and 2005. In addition, serum samples were collected from four patients. Ten hantaviruses were isolated successfully in cell culture from four humans, two Apodemus agrarius, three Rattus norvegicus and one Rattus nitidus. The nucleotide sequences for their small (S), medium (M) and partial large (L) segments were determined. Phylogenetic analysis of the S and M segment sequences revealed that all of these isolates belong to the species HTNV, suggesting a spillover of HTNV from A. agrarius to Rattus rats. All available isolates from Guizhou were divided into four distinct groups either in the S segment tree or in the M segment tree. The clustering pattern of these isolates in the S segment tree was not in agreement with that in the M or L segment tree, showing that genetic reassortment between HTNV had occurred naturally. Analysis of the S segment sequences from available HTNV strains indicated that they formed three clades. The first clade, which comprised only viruses from Guizhou, was the outgroup of clades II and III. The viruses in the second clade were found in Guizhou and mainly in the far-east Asian region, including China. However, the viruses in the third clade were found in most areas of China, including Guizhou, in which haemorrhagic fever with renal syndrome (HFRS) is endemic. Our results reveal that the highest genetic diversity of HTNV is in a limited geographical region of Guizhou, and suggest that Guizhou might be a radiation centre of the present form of HTNV.

[An epidemiological study of rabies virus in domestic dogs, cats and wildlife and the immunogenicity study for rabies vaccines derived from different cell cultured virus strains].

For epidemiological investigation of the rabies virus carrier rates of domestic dogs, cats and wild animals like rodent animals and bats,three kinds of regions where rabies had higher incidence (Hunan and Guizhou Provinces), lower incidence (Jiangsu Province, Wuhan City) and provisionally rabies-free (Shenyang City) were selected. Then the antigenic types, the genovariation of the isolaled viruses and the currently vaccine matching of the virus strains were analyzed. The results showed that in China the principal host of rabies is dog,the total virus carrier rate of the captured dogs was 2.56%, and the highest positive isolation rate was 20.0% in some monitoring site. However,there was no evidence about the rabies virus carrier rate in rodent animals,bats or other wild animals. The rabies vaccines which prepared from aG and CTN strains have already been produced successfully in China. The research showed that the nucleotide sequences of the newly isolated viruses were more similar with the glycoprotein gene of CTN strain. In order to evaluate the safety and the efficacy of the vaccines currently used, two groups (50 people each) were injected with vaccine of aG strain and CTN strain respectively in five surveillance points. The neutralizing antibody tested were 0.49 IU/mL-0.52 IU/mL and 6.7 IU/mL-7.53 IU/mL after the 7 and the 14 days of vaccine injection respectively. In addition, the rates of antibody positive seroconversion were 45.1%-47.9% and 100% respectively, and there was no moderate or severe adverse reactions observed. These data showed the vaccines have satisfactory effect on safety and protection.

[The survey of 21 cases of human rabies in Anlong county in Guizhou province].

OBJECTIVE: To analyze 21 cases of rabies from February 8 to May 1 in 2004 in Anlong county in Guizhou province, and to explore the possible factors causing the epidemics. METHODS: 21 cases of rabies were investigated. The canine brains were collected, and indirect immunofluorescence assay was used to detect rabies virus antigen in the brains. RESULTS: From February 8 to May 1st in this year, 21 cases of rabies were reported, with 5.12/100,000 of the prevalence rate. Dogs accounted for 20 cases, and cat for one case. Median incubation period was 36.52 days, but less than 15 days in 6 cases. Among 21 cases, 17 cases were not correctly treated, and 9 cases received vaccine, with 3 cases vaccinated in time. No cases received passive immunization of antirabies serum or human antirabies immune globulin. A total of 73 dogs' brains were examined for rabies viral antigen by IFA, and 9 (12.33%) were positive. CONCLUSION: The increasing number of dog, high rate of virus carrier in dogs, incorrect treatment of the wound, and as well as low inoculating rate of rabies vaccine might be responsible for the outbreak of rabies in Anlong county.

[Study on ribotyping of Lyme borreliosis spirochete in Guizhou province].

OBJECTIVE: To define the main genotypes in Guizhou agricultural areas by molecular epidemiologic investigation of 21 Borrelia burgdorferi sensu lato of Lyme disease spirochetes and to provide the scientific bases for formulating a preventive policy. METHODS: Polymerase chain reaction (PCR) technique was used to amplify the 23S(rrl)-5S(rrf) intergenic spacer, and amplified products were analyzed by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. RESULTS: There were two genospecies in the strains: 20 strains belong to Borrelia valaisiana, 1 strain is Borelia sp. CONCLUSION: Borrelia valaisiana was the main genotype in Guizhou agricultural areas. The harmness of B. valaisiana to human being has been confirmed. In order to efficiently prevent the harmness of agent to the people in Guizhou agriculture areas, we should study the risk further.

[Surveillance on hemorrhagic fever with renal syndrome in Guizhou during 1984-2000].

OBJECTIVE: To explore the relations between different areas on hemorrhagic fever with renal syndrome (HFRS) in Guizhou. METHODS: Various forms of infectious areas were surveyed continuously through epidemiologic surveillance system. RESULTS: In fixed areas under surveillance system, the mean positive rate of HFRSV among Apodemus agrarius was 3.39%, comparing with Rattus norvegicus 1.61% in Apodemus infectious areas of Zunyi county, 3.19% in Rattus norvegicus, but no HFRSV of Apodemus agrarius was identified in Rattus infectious area of Shiqian county. Both Apodemus and Rattus infectious areas were relatively stabilized. In both banks of Luowang river, Kaiyang county, which had been identified as areas of infections for Apodemus in the eastern part, Rattus infectious area in the west, slow change was noticed. In 1983 - 1984 was not found in Apodemus agrarius HFRSV, however the infectious rate of HFRSV in Apodemus agrarius was 13.85% (Ag 1/65, Ab 8/65) in the western part of the province in 1995 - 1998. CONCLUSION: Both Apodemus and Rattus infectious areas were stabilized but changed slowly. Mixed type and the result of mutual penetration were noticed.