Genetic diversity of avian infectious bronchitis coronavirus strains isolated in China between 1995 and 2004

Summary. Twenty-six avian infectious bronchitis (IB) viruses (IBV) were isolated from outbreaks in chickens in China between 1995 and 2004. They were characterized by comparison with twenty-six Chinese reference strains and five other IBV strains. Chinese IBVs, which were mainly nephropathogenic, were placed into seven genotypes. Fourteen Chinese IBV isolates were placed in genotype I, having small evolutionary distances from each other. Genotype II included 6 strains that were isolated in the 1990s in China. Genotype III consisted of eight Chinese isolates that showed close relationship with Korean IBV isolates. Another eight IBV isolates clustered in genotype IV and showed larger evolutionary distances. The Massachusetts serotype was present in China in 1990s and was in a separate genotype. Two isolates, HN99 and CK/CH/LHN/00I, which might be a reisolation of vaccine strains, clustered into genotype VI. Four Chinese IBV isolates formed another genotype and showed larger evolutionary distances from other Chinese IBV genotypes (genotype VII). IBVs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%. The results showed that IBVs in China came from genetic changes both in IBV populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. IBVs showing various genetic differences are cocirculating in China.     

Molecular analysis of infectious bronchitis viruses isolated in Slovenia between 1990 and 2005: a retrospective study

Fifteen infectious bronchitis viruses (IBV) isolated from broiler and broiler breeder flocks in Slovenia between 1990 and 2005 were molecularly characterised. IBV strains were divided into four genotypes by the analysis of the S1 gene region. Four strains belonged to the Massachusetts genotype, one strain was placed into the QX genotype, one strain formed a cluster together with the B1648 strain and nine strains were classified into the 624/I genotype. Nine Slovenian strains of the 624/I genotype formed two subgroups independently of the time of isolation and the geographical origin. Phylogenetic analysis of the partial N gene sequences revealed lower sequence variability and different clustering of the Slovenian IBV. Fourteen strains were grouped together with the strains H120 and D1466. One strain formed a cluster with the strain 793/B.     

Genetic analysis revealed LX4 genotype strains of avian infectious bronchitis virus became predominant in recent years in Sichuan area,China

Between 2008 and 2010, 19 strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in Sichuan province, China. The S1 genes of the isolates were amplified and sequenced. Phylogenetic analysis revealed that the 19 isolates and 37 reference IBV strains can be grouped into eight genotypes. Although IBVs of Taiwan-I type, massachusetts type, and proventriculitis type were isolated, but most isolates were LX4 genotype. Homology analysis of the sequences of S1 genes of the 19 isolates and 37 reference IBV strains revealed that the identity of the nucleotides and amino acid sequences of the S1 genes between the 15 LX4-type isolates and other IBV strains were 71.9–99.3% and 72.1–99.1%, respectively, while those of the analyzed IBV of LX4 type were 96.0–99.9% and 94.3–99.8%, respectively. The results from this study and other published results in the GenBank database showed that isolates circulating in Sichuan province in recent years were mainly LX4 genotype, which is the predominant genotype circulated in China in recent years.     

fimA genotypes and multilocus sequence types of Porphyromonas gingivalis from patients with periodontitis

Fimbriae are important virulence factors of pathogenic bacteria, facilitating their attachment to host and bacterial cells. In the periodontal pathogen Porphyromonas gingivalis, the fimA gene is classified into six types (genotypes I, Ib, II, III, IV, and V) on the basis of different nucleotide sequences, with fimA genotypes II and IV being prevalent in isolates from patients with periodontitis. The aims of this study were to examine the distribution of fimA genotypes in a collection of 82 P. gingivalis isolates from adult periodontitis patients of worldwide origin and to investigate the relationship between the fimA genotypes and the sequence types (STs), as determined by multilocus sequence typing (MLST), of the isolates. The fimA gene was amplified by PCR with primer sets specific for each genotype. The STs of all strains were assigned according to the MLST database for P. gingivalis (www.pubmlst.org/pgingivalis). The 82 strains showed extensive genetic diversity and were assigned to 69 STs. Only isolates with closely related STs harbored the same fimA genotype. Twenty-eight (34.1%) strains harbored fimA genotype II, while only the reference strain for fimA genotype V reacted with the primers specific for this genotype. Twenty-one isolates (25.6%) were positive by more than one of the fimA PCR assays; the most frequent combinations were genotypes I, Ib, and II (eight isolates) and genotypes I and II (four isolates). Sequencing of the fimA gene from selected isolates did not support the observed specific fimA genotype combinations, suggesting that the genotyping method used for the major fimbriae in P. gingivalis should be reevaluated.     

Genotypes of Candida albicans involved in development of candidiasis and their distribution in oral cavity of non-candidiasis individuals

Genotype characteristics and distribution of commensal Candida albicans should be studied to predict the development of candidiasis, however, extensive genotype analysis of commensal C. albicans has not been made. In this study, 508 C. albicans isolates were collected from patients with/without candidiasis and divided into 4 isolate groups (SG-1, oral cavity of non-candidiasis patients; SG-2, patients with cutaneous candidiasis; SG-3, patients with vaginal candidiasis; SG-4, patients with candidemia). These isolates were characterized to study the relationship between genotypes and pathogenicity using microsatellite analysis. Using CDC3 and CAI, 5 genotypes (I, 111: 115/33: 41; II, 115: 119/23: 23; III, 115: 123/18: 27; IV, 115: 123/33: 40; and V, 123: 127/32: 41) were found in 4.2%, 8.9%, 7.1%, 2.2% and 3.1% of the isolates, respectively. Genotypes II and III were commonly found in all isolate groups. These genotypes were further divided into 28 types by additional HIS3 and CAIII microsatellite markers. In this analysis, C. albicans with type 6 and type 23 was widely distributed as a commensal species in the oral cavity of non-candidiasis patients and found to be related with candidiasis development. Additionally, genotypes I and IV were found in SG-2 and/or SG-4, suggesting that the fungus with those genotypes is also involved in this development. In contrast, genotype V was not identified in any infective isolates.     

Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene

Infectious bronchitis virus (IBV) isolates recovered in Russia, Ukraine, and Kazakhstan between 2007 and 2010 were subjected to molecular characterization and compared with those isolated a decade ago. The IBV genome was detected in 202 out of 605 field samples from chickens with various clinical signs. Partial sequencing of the S1 gene revealed 153 vaccine strains and 49 field isolates of several genetic groups. Massachusetts, 793/B and D274 remained the predominant IBV genotypes along with QX, whereas B1648, Italy-02, Arkansas and variants accounted for about 12% of the total number. Three IBVs contained recombinant S1 gene sequences comprising genome fragments of QX-type field isolates and vaccine strains H120 (UKR/02/2009) or 4/91 (RF/03/2010), and vaccine strains H120 and D274 (RF/01/2010). The results of the present study showed a significant decline in prevalence of variant IBVs and a further spread of QX-type isolates in commercial chicken flocks in Russia as compared with the 1998 to 2002 data.     

Molecular epidemiology of Newcastle disease viruses isolated in South Korea using sequencing of the fusion protein cleavage site region and phylogenetic relationships.

The study, using sequence analysis and the phylogenetic relationship of the fusion protein gene, divided the Korean epizootic isolates of Newcastle disease virus (NDV) into several lineages to determine the molecular epidemiology of the virus. A 695 base pair fragment was amplified by polymerase chain reaction between matrix protein gene and fusion protein gene of 30 Korean NDV isolates, which were isolated from field outbreaks of Newcastle disease between 1949 and 2002. All isolates showed the amino acid sequence 112R-R-Q/R-K-R116 at the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. These amino acid sequences were identical to a known virulent motif. The region of the F gene between nucleotides 47 and 435 was compared by phylogenetic analysis. Based on nucleotide sequence, the Korean NDV isolates belonged to genotype III, V, VI and VII corresponding to isolates in 1949, 1982 to 1984, 1988 to 1997, and 1995 to 2002, respectively. These data showed that genotypes of five Korean Newcastle disease epizootics had replaced each other serially (III, V, VI and VII) in chronological order. Further, the five Korean Newcastle disease epizootics were closely related with the Newcastle disease panzootics or Newcastle disease epizootics in other countries. Present study showed that the Korean genotype V isolated before 1984 was related with European Newcastle disease epizootics in the 1970s, whereas the Korean genotypes VI and VII isolated after 1988 were more closely related with Far East Newcastle disease epizootics, especially Newcastle disease epizootics in Japan, Taiwan and China. Since 1988, the genotypes VI and VII of Far East origin were dominant in South Korea. That might be due to the increased trade of agricultural products including poultry among Far East Asian countries.     

Newly isolated serotypes of infectious bronchitis virus: their role in disease

The pathogenicity of isolates of infectious bronchitis virus (IBV) made from broiler or breeder flocks in Britain between 1981 and 1983, together with three made from flocks of commercial laying hens during 1984, has been investigated. When 11-day-old IBV-free chicks were challenged with these IBV isolates, together with a pool of pathogenic strains of E. coli, some mortality was recorded, together with a high incidence of morbidity. Three IBV isolates, when given to groups of IBV-free brown-egg laying hens, caused only a minimal effect on egg production but markedly affected egg shell colour.     

Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea

Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57?% of chicken flocks and 28?% of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97?% nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.     

Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan

Summary In order to differentiate recent isolates of avian infectious bronchitis virus (IBV) in Taiwan, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and direct sequencing methods were used to type 25 IBV Taiwan isolates. Two conserved sequences that flank the hypervariable region I (HVR I) in the N-terminus of S1 protein gene were chosen as primers. Sequences of 228–231 base pairs (bp) were amplified by PCR from 25 Taiwan isolates and 4 reference strains (H120, Conn, JMK, Holte). PCR products were digested with 5 restriction endonucleases,BsoFI,DdeI,MboII,AluI,RsaI, and different IBV isolates were grouped according to their RFLP patterns. The RFLP patterns of the 4 reference strains in this study matched the published sequences in GenBank. Except 1 vaccine strain, the other 24 Taiwan isolates were different from these 4 and 18 other IBV strains whose sequences were published. The data from PCR-RFLP and sequencing of IBV genomes showed that the 24 Taiwan isolates can be divided into 2 distinct groups, I and II. Seven RFLP patterns are identified in group I and only 1 in group II.     

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.     

Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005�C2008

Seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons (Columba livia), geese and ostriches in Eastern China during 2005?C2008 were characterized biologically and phylogenetically. The results showed genetic diversity of these viruses: three class I viruses and one genotype I and 12 genotype II viruses of class II circulating in chickens were avirulent; four genotype VIb viruses isolated from pigeons were moderately virulent; and two genotype III viruses and 57 genotype VIId viruses were highly virulent. The three class I viruses were further classified as genotypes 2 and 3. The very high F protein sequence identity of one genotype I virus with strain Queensland V4 and 12 genotype II viruses with strain La Sota indicated that these viruses originated from the two vaccine strains. Two genotype III viruses shared greater than 99% sequence identity with the moderately virulent vaccine strain Mukteswar but exhibited significantly higher virulence, suggesting that they evolved from the vaccine virus and that the Mukteswar vaccine should be banned in China. Fifty-seven of the 63 virulent NDVs in this study belonged to genotype VIId, indicating its predominance in Eastern China. Genotype VIId viruses could be further classified into two subgroups. Four of the five NDVs isolated from pigeons belonged to genotype VIb, indicating its host-specific preference. Both the genotype VIb and VIId NDVs showed low amino acid similarity to the vaccine strains currently used in China, implying the urgent need to develop better vaccines against the most prevalent NDVs in China.     

First report of typical Brazilian Toxoplasma gondii genotypes from isolates of free-range chickens (Gallus gallus domesticus) circulating in the state of Paraíba,Northeast Brazil

This study evaluated, for the first time, the genetic diversity of Toxoplasma gondii isolates from free-range chickens from the state of Paraíba, Northeast Brazil. Tissue samples from 33 chickens from properties in five municipalities of Paraíba (Esperança, Olho d’Água, Malta, Monteiro, and Patos) were bioassayed in mice. The brains of mice infected with T. gondii cysts were used for DNA extraction and genotyping. Genotyping was performed using 11 PCR-RFLP markers and 15 microsatellite (MS) markers. Complete genotyping results were obtained for 29 isolates, with nine genotypes detected by RFLP and 15 genotypes identified by MS. Three genotypes (#273, #274, and #277) have only been recently identified from pigs in the region. Brazilian clonal types BrII and BrIII were identified from one isolate each. Clonal types I, II, and III were not detected by RFLP. Genotype #13 (Caribbean 1), detected in 48.3% (14/29) of isolates from four of the five municipalities investigated, was the most prevalent genotype in the state of Paraíba. However, the MS analysis showed that of these 14 isolates, only four were unique genotypes, and considering the distance between the municipalities from where they were collected, it is possible that only seven are independent isolates while the others are clones. The other genotypes were restricted to different microregions. The results indicate that the Caribbean 1 lineage of T. gondii is circulating widely in Northeast Brazil. The genotypic diversity of T. gondii in the state of Paraíba is high, and microsatellite analysis revealed this diversity with higher resolution than PCR-RFLP.

     

Continuous evolution of avian infectious bronchitis virus resulting in different variants co-circulating in southern China

Sixty field strains of avian infectious bronchitis virus (IBV) were isolated from chicken flocks in different regions of Guangxi from 1985 to 2012. Phylogenetic analysis of S1 subunit glycoprotein genes revealed that field isolates from 2009-2011 mostly belonged to the LX4 type, while those from 1985-2008 belonged to the HN08 type, and a few others belonged to the 4/91 type, the TW type and the Mass type. In addition, it is noteworthy that no obvious regional differences were found among these 60 strains isolated from six regions in Guangxi, while there was a high degree of sequence identity among the isolates in the same period of time.     

Molecular characterization of two genotypes of mumps virus circulated in Korea during 1998-2001

Sequence analyses of the entire small hydrophobic (SH) and hemagglutinin-neuraminidase (HN) genes of mumps viruses circulated in Korea from 1998 to 2001 showed that these isolates were grouped into two genotypes, H and I. While genotype I was predominant throughout the country during this period, genotype H was found in the restricted region, 1999. The nucleotide and deduced amino acid sequences of Korean isolates showed the type-specific changes including the signature motif at positions 28-30 in the SH gene and the neutralizing epitopes in the HN gene. Particularly, Asian strains including Korean isolates and European strains differed from 2.3 to 3.8% at the nucleotide sequence level in the SH gene although they belonged to the same genotype H. Furthermore, none of Korean isolates were genetically related to the vaccine strains used in Korea. The results provide important information to understand the epidemiology of mumps infection and to facilitate the development of more efficient vaccine program in Korea.