Genetic characterization of Puumala hantavirus strains from Belgium: evidence for a distinct phylogenetic lineage

Puumala hantavirus (PUUV) sequences were recovered from red bank voles (Clethrionomys glareolus) trapped between 1996 and 1998 in four localities of southern Belgium: Thuin, Montbliart, Momignies and Couvin. In addition, three PUUV isolates originating from bank voles trapped in the 1980s in southern (Montbliart) and northern (Turnhout) Belgium were genetically characterized. Analysis of the complete S and partial M segment sequences showed that the Belgian PUUV strains constitute a genetic lineage, distinct from other known PUUV lineages from Europe and Japan. This lineage also includes a wild strain (Cg-Erft) originating from a neighbouring area of Germany. Within the Belgian lineage, geographical clustering of genetic variants was observed. In the Montbliart site, the range of diversity between the most temporally distant strains (from 1986 and 1996-1998) was higher than between those from 1996 and 1998, suggesting slight genetic drift via accumulation of neutral or quasi-neutral substitutions with time.     

Phylogenetic analysis of swine fever virus strains

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.     

A new Clethrionomys-derived hantavirus from Germany: evidence for distinct genetic sublineages of Puumala viruses in Western Europe.

Puumala (PUU) viruses are the predominant etiologic agents of hantavirus infections in Europe. The most important reservoir is the bank vole, Clethrionomys glareolus (Cg), belonging to the subfamily Arvicolinae of the Muridae family. Here we report on the molecular characterization of the first rodent-derived sequence (PUU/Cg-Erft) from Germany. Comparison of the S and M segment coding regions revealed 92.5 and 92.8% identity, respectively, with PUU/H-9013, a human isolate from France. However, only 83.1% identity was found with the S segment of a previously reported PUU sequence from a German HFRS case (PUU/H-Berkel) indicating the co-existence of two distinct sublineages in Germany. Phylogenetic and alignment analyses of S and M segment coding regions enabled us to assign PUU viruses/sequences to at least six distinct genetic sublineages. Membership was defined by nucleotide sequence differences of < 8%, whereas a diversity of > 14% clearly outgrouped a virus/sequence. Based on S segment sequences the sublineage represented by Clethrionomys rufocanus-derived viruses from Japan diverged at a well supported node from the clade harbouring all Clethrionomys glareolus-derived European PUU viruses. A correlation between genetic relationship and geographic origin of PUU viruses was observed which may support a co-evolution of PUU viruses with distinct subspecies of their reservoir host.     

Corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by Puumala virus infection

Reported here are two cases of hantavirus pulmonary syndrome caused by Puumala virus infection, which rapidly resolved after initiation of corticosteroid treatment combined with continuous veno-venous hemodiafiltration. These cases emphasize the role of the inflammatory response in the pathogenesis of hantavirus pulmonary syndrome.     

Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein

Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/H?lln?s. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.     

Phylogenetic analysis of avian infectious bronchitis virus strains isolated in Japan

Summary To define the origin and evolution of recent avian infectious bronchitis virus (IBV) in Japan, a genetic analysis was performed. By phylogenetic analysis based on the S1 gene including the sequence of the hypervariable regions, IBV isolates in Japan were classified into five genetic groups, which included two already-known groups (Mass and Gray). Among them, three major genetic groups were associated with the recent outbreaks of IB in Japan. One group is indigenous to Japan and could not be placed within the known existing groups in other countries. The remaining two groups, which have emerged recently, are related to isolates in China and Taiwan.     

Phylogenetic analysis of Ostreococcus virus sequences from the Patagonian Coast

A phylogenetic analysis of new Ostreococcus virus (OV) sequences from the Patagonian Coast, Argentina, and homologous sequences from public databases was performed. This analysis showed that the Patagonian sequences represented a divergent viral clade and that the rest of OV sequences analyzed here were clustered into six additional phylogenetic groups. Analyses of 18S gene libraries supported a close relationship of the Patagonian Ostreococcus host with clade A sequences described elsewhere, corroborating previous studies indicating that clade A strains are ubiquitous. Besides the Patagonian OV sequences, several phylogenetic groupings were linked to particular geographic locations, suggesting a role for allopatric cladogenesis in viral diversification. However, and in agreement with previous observations, other viral lineages included sequences with diverse geographic origins. These findings, together with analyses of ancestral trait trajectories performed here, are consistent with an evolutionary dynamics in which geographical isolation has a role in OV diversification but can be followed by rapid dispersion to remote places.     

Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by severe diarrhea, dehydration and high mortality in the affected pigs. Recently, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China. In this study, molecular detection of PEDV was conducted using RT-PCR (targeting the M gene) on samples collected from piglets with watery diarrhea from 15 pig farms, and phylogenetic analysis of PEDV field strains was carried out based on their M and S genes. In addition, the complete genome sequence of a PEDV field strain was determined. PEDV was detected in 92.7 % of the samples (267/288). The 15 M genes that were amplified shared 99.6-100 % nucleotide identity and 99.1-100 % amino acid similarity with each other. The 15 S genes exhibited 98.6-99.9 % homology, both at the nucleotide level and at the deduced amino acid level. Phylogenetic analysis showed that all of the amplified M genes grouped in cluster 3, together with some Chinese, Korean and Thai strains, while all of the amplified S genes were in cluster 3 and were closely related to Korean strains. Compared with previous PEDV strains, all of the S genes have common characteristics, namely, a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156, similar or identical to Korean KNU-serial strains reported in recent years. The genome of the sequenced PEDV field strain is 28,038 nucleotides in length, excluding the poly (A) tail. Our findings suggest that a novel PEDV with a characteristic variant S gene is responsible for recent outbreaks of clinical diarrhea in piglets in China.     

Immunogenicity of N-protein of Puumala hantavirus for noninbred mice in the intramuscular administration of its gene DNA

The outbreaks of hantavirus infections in some regions of the Russian Federation in some years involve considerable material and social losses. In this connection, the designing of the most effective types of vaccines is an urgent task. The authors have created plasmid constructions containing the gene of hemorrhagic fever with renal syndrome virus, with whose Intramuscular injection there is a specific immune response and plasmid DNA is detectable in the adjacent tissues within a month after injection.     

Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype

Summary.  Bluetongue virus (BTV) infection of ruminants is endemic throughout tropical and subtropical regions of the world. The S7 gene segments of prototype Chinese strains of BTV serotypes 1, 2, 3, 4, 12, 15, and 16 were sequenced and compared to the same genes of prototype strains of BTV from the US, Australia, and South Africa. The S7 genes and predicted VP7 proteins of the Chinese viruses were relatively conserved, with the notable exception of serotype 15. Furthermore, phylogenetic analysis of the S7 genes did not predict geographic origin of the various strains of BTV. Received August 28, 1999 Accepted December 7, 1999     

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.     

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype

Summary The hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3 484 coding for 1 148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18–20 and Sotkamo.The nucleotide sequence data reported in this paper have been deposited in Gen Bank under accession numbers U14137 and U14136.     

Application of a full-genome microarray-based assay for the study of genetic variability of West Nile virus

Viral adaptation through fixation of spontaneous mutations is an important factor potentially associated with reoccurrence of West Nile virus (WNV) outbreaks in the New World. The emergence of new genetic variants of WNV represents an important public health issue because it may affect the sensitivity of WNV screening and diagnostic assays, as well as the development and efficacy of WNV vaccines and anti-viral drugs. A microarray assay was developed and optimized to enable simple monitoring of WNV genetic variability and rapid detection of any nucleotide mutations within the entire viral genome. The assay was validated using 11 WNV isolates from the 2007 and 2009 U.S. epidemics. The developed microarray system can potentially serve as a high throughput, rapid, and effective approach for the detection of circulating WNV genetic variants.     

Resequencing of the Puumala virus strain Sotkamo from the WHO Arbovirus collection

RNA viruses exhibit a high mutation rate as the RNA-dependent RNA polymerase lacks proofreading and repair capabilities. It is known that serial passaging on cell culture leads to virus adaptation. Puumala virus (PUUV) strain Sotkamo is the prototype virus for the low-pathogenic hantavirus Puumala, family Bunyaviridae. A full-length sequence of the strain Sotkamos tripartite genome was made available more than 15 years ago, after at least 15 passages on Vero E6 cells. A distinct sample from the sequenced strain, with unknown passage history, was then included in the WHO Arbovirus collection. The genome sequence of this included sample was determined in this study and exhibited over 99 % identity in comparison to the previously published sequence. A total of 23 nucleotide changes across all genome segments were found. The small segment had the highest nucleotide variance without changes on the protein level. Within the extraviral domain of the glycoproteins, the majority of non-synonymous mutations were detected, whereas the large segment is most conserved on the nucleotide level. It seems possible that the PUUV strain Sotkamo adapted differently to serial passaging on cell culture in two different laboratories. In addition, a distinct passage number could exhibit itself within the nucleotide differences.     

Phylogenetic analysis of classical swine fever virus (CSFV) field isolates from outbreaks in South and Central America

To date, there is little information concerning the epidemiological situation of classical swine fever (CSF) in the Americas. Besides summarizing the available data, genotyping of isolates from outbreaks in domestic pigs in several countries of South and Central America was performed. For this, a 190 base fragment of the E2 envelope glycoprotein gene was used. European strains and isolates, and historical isolates from the United States (US) were included for comparison. In contrast to the situation in most parts of Europe, where group 2 isolates predominate, it was found that all the isolates from the American continent analyzed belonged to group 1 and were further resolved into three subgroups. The Cuban isolates clustered in subgroup 1.2, whereas the isolates from Honduras and Guatemala clustered in subgroup 1.3. The remaining isolates from Argentina, Brazil, Colombia and Mexico generated four poorly resolved clusters in subgroup 1.1, together with the vaccine strains, with historical European and US isolates, and with a recent Russian isolate. While the vaccine strains and the historical European isolates formed a relatively distinct cluster, one of the US isolates clustered together with the Mexican, and another one with Colombian isolates. Historically, CSF (hog cholera) was observed almost simultaneously in the US and in Europe in the first half of the 19th century, and its origin remains a matter of discussion. Our results showed that the US isolates are closely related to isolates from South America, while appearance of isolates in Cuba on one hand and in Honduras and Guatemala on the other hand, seems to have been due to unrelated events. This allows to speculate that at least in the American continent, CSF virus may have appeared independently in several regions, and spreading may have been a secondary effect.     

Characterization of hepatitis B virus strains from the Central African Republic: preliminary results

Objectives

Genotyping of Hepatitis B virus (HBV) strains from patients in Central African Republic and comparison with results obtained in other African countries.

Patients and methods

Sera were collected from patients admitted with symptoms of acute or chronic hepatitis to the “Hôpital de l’Amitié de Bangui”, Central African Republic (CAR). The complete sequence of preS2/S gene has been defined for determining genotypes.

Results

Hundred and ninety-six sera were collected from 112 men and 84 women. Ninety-two percent of patients had contact with HBV (anti-HBc postitive) and the HBsAg prevalence was about 62%. HBV DNA was detected in 66% of HBsAg positive sera. No HBV-DNA was evidenced among patients with negative HBsAg. Ninety-three percent of the HBV strains belonged to genotype E; one (3.4%) belonged to genotype A1, and one (3.4%) belonged to genotype D.

Conclusions

The high prevalence of HBV infection in the studied population is due to their recruitment. The genotype E is predominant in CAR and the intragroup variability of HBV genotype E reached only 1.8%. Genotypes A and D were less common in CAR their presence may be explained by importation.     

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan

Summary.  Feline immunodeficiency virus was isolated from four cats from Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C. Accepted February 3, 1997; Received November 20, 1996     

Phylogenetic analysis of previously nontypeable hepatitis C virus isolates from Argentina

Phylogenetic analysis of hepatitis C virus isolates from Argentina that were previously nontypeable by restriction fragment length polymorphism (RFLP) analysis revealed that they belong to genotype 1a. A substitution at position 107 (G-->A), which is the landmark of these strains, was shown to be distributed among isolates worldwide. The RFLP patterns obtained for these isolates should be added to the ones reported for genotype 1 isolates.