Sequence analysis of F, SH, and HN genes among mumps virus strains in Japan

Summary.  The fusion (F), small hydrophobic (SH), and hemagglutinin-neuram- inidase (HN) regions of 15 mumps virus (MuV) strains were sequenced to demon- strate the genetic variability since 1976 in Japan. The MuV strains were classified into 2 major genotypes, A and B, and genotype A was subdivided into three subtypes (A1, A2, and A3). A1 and A2 strains were mainly isolated in 1977 and 1980. A3 strains were isolated in 1985 and 1989 and genotype B strains in 1993 and 1994. Genotypes A1, A2, and A3 were closely related indigenous lineages in Japan but genotype B was in the similar cluster in Europe and North America. Received August 12, 1998 Accepted October 16, 1998     

Genetic analysis of human immunodeficiency virus type 1 nef in Portugal: subtyping, identification of mosaic genes, and amino acid sequence variability

Extending our previous genetic characterization of human immunodeficiency virus type 1 (HIV-1) strains circulating in Portugal, we here report the first phylogenetic and putative amino acid sequence variability analyses of nef accessory gene. Viral sequences (n = 53) were amplified by nested PCR from proviral DNA purified from peripheral blood mononuclear cells of HIV-1 infected individuals (n = 49). Phylogenetic inference analysis demonstrated a distribution of the viral sequences between subtypes A (sub-subtype A1), B, D, F (sub-subtype F1), G, H, and J, with subtypes G and B accounting altogether for more than half of the genotypes found. A significant number of the proviral DNA sequences analyzed (18.4%) were shown to correspond to intragenic nef recombinants, with the majority having the typical CRF02_AG nef structure. In addition, three novel intragenic recombinant structures were found (B/G/B, CRF02_AG/H, and D/G). From phylogenetic analysis, it was concluded that part of the non-recombinant nef genes might have actually been amplified from mosaic viruses: CRF06_cpx, CRF14_BG, and a new envA/nefJ recombinant. While comparing all the putative Nef sequences, significant amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed and numerous amino acid changes fell outside these regions. The results presented unambiguously endorse the high level of complexity of HIV-1 epidemics in Portugal.     

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV.     

Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.     

Hepatitis B virus in Mar del Plata,Argentina: Genomic characterization and evolutionary analysis of subgenotype F1b

The aim is to describe the molecular epidemiology and perform a genomic characterization of hepatitis B virus (HBV) circulating in Mar del Plata and to identify the origin and diversification patterns of the most prevalent genotype. The S gene and the region encompassing the X gene, basal core promoter (BCP), and precore (preC) was analyzed in 56 samples. They were genotyped as: 80% F1b, 9% A2, 7% D3, and 2% D1. A recombinant F4/D2 genome was detected. The double substitution G1764A/A1762T at the BCP (reduced HBeAg expression) was found in 20% F1b, 2% A2, 2% D1, and 2% D3 samples. A unique D3 presented the G1896A substitution at the preC (HBeAg negative phenotype). A 13% of the samples showed mutations at the HBsAg “a” immunodeterminant (escape from neutralizing antibodies). Mutations at the polymerase (antiviral resistance) were found in 52% of the samples. Coalescent analysis of subgenotype F1b, the most prevalent in the city, showed that viral diversification in Mar del Plata started by year 2000. F1b was the most prevalent genotype detected, being a characteristic of actual HBV infections in Mar del Plata. Local HBV exhibit clinically relevant mutations, but a minority of them was shown to be associated to potential vaccination escape or antiviral resistance. Nevertheless, further studies are needed to determine whether any of these mutants could pose a threat to prevention, diagnosis, or treatment.     

Epidemiological and molecular characterization of rubella virus isolated in São Paulo,Brazil during 1997–2004

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997–2004 were isolated in cell culture and genotyped. Twenty‐eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil. J. Med. Virol. 84:1831–1838, 2012. © 2012 Wiley Periodicals, Inc.     

Molecular analysis of measles virus genome derived from SSPE and acute measles patients in Papua,New Guinea

A very high annual incidence of 56 per million population below the age of 20 years for subacute sclerosing panencephalitis (SSPE) has been reported from Papua New Guinea (PNG). In a more recent study, we have confirmed this unusual high incidence for Eastern Highlands Province (EHP) of PNG. In the study, it was observed that the vaccination rate among SSPE patients registered at Goroka Base General Hospital (GBGH) in EHP was higher than that of other infants in the province in recent years. To identify the measles virus (MV) responsible for SSPE in EHP, sequence analysis of hypervariable region of the N gene was performed from 13 MV genomes: 2 amplified from clinical specimens of SSPE patients and 11 from acute measles patients. In 2 cases among the 11 with acute measles, nucleotide sequence of the entire H gene derived from isolated viruses was determined. Both nucleotide sequence and phylogenetic tree analyses showed that the amplified MV cDNAs were closely related to one another and belonged to the D3 genotype though they were different from any previously reported MV sequences. No genome sequences of vaccine strains were detected. These findings suggest that the MV strains prevailing in the highlands of PNG belong to genotype D3 of the MV and this wild-type MV rather than the vaccine strains was likely to be responsible for SSPE in these patients.     

Fusion (F) protein gene of newcastle disease virus: Sequence and hydrophobicity comparative analysis between virulent and avirulent strains

The nucleotide and predicted amino acid sequences have been obtained for the fusion (F) protein gene of the avirulent strain La Sota of Newcastle disease virus (NDV). The F₁ N-terminus begins with the tripeptide Leu-Ileu-Gly instead of Phe-X-Gly as usually observed in fusion peptide. It was found that the cleavage-activation domain of the avirulent La Sota strain contained single (but no pairs of) basic residues in the sequence Gly-Arg-Gln-Gly-Arg. Hydrophobicity analysis suggested that the cleavage-activation domain became more hydrophobic and could be less accessible for host-specific protease(s); dibasic residues next to the F₁ N-terminus were shown to be important for keeping the cleavage-activation site in exposed positioning, suitable for F protein activation. Comparative sequence analysis of the NDV F proteins revealed a striking homology between lentogenic La Sota and mesogenic Beaudette C strains. Furthermore, 58 variable positions were recorded in the NDV F protein, excluding signal sequence; some of these mutations, in the cysteine-clustered region, were surmised to alter virulence.Requests for reprints should be addressed to Arsene Burny, Laboratory of Biological Chemistry, Department of Molecular Biology, Free University of Brussels, ULB, Rhode-St-Genèse, Belgium.     

Molecular characterization,isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.     

Genetic analysis of hepatitis A virus outbreak in France confirms the co-circulation of subgenotypes Ia, Ib and reveals a new genetic lineage

Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.     

Identification of three lineages of wild measles virus by nucleotide sequence analysis of N,P, M,F, and L genes in Japan

The nucleotide sequences of nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), and large protein (L) genes were partly determined for 19 wild strains of measles virus (MV) isolated over the past 10 years in Japan (nucleotide position N: 1301–1700, P: 1751–2190, M: 3571–4057, F: 6621–7210, L: 10381–11133) and also for a MV strain obtained from a patient with subacute sclerosing panencephalitis (SSPE) who had natural measles in 1980. The phylogenetic trees of these strains drawn for respective genes were very similar to each other and revealed that all the wild strains were classified chronologically into 3 subgroups, those isolated in 1984, 1984–1989, and 1990–1994. The SSPE strain was classified into the subgroup of 1984. Phylogenetic tree analyses including other strains in the world revealed that Japanese strains in 1984 were classified into a distinct lineage which might correlate with the European strains from late 1970s to mid 1980s. Japanese strains from 1984 to 1989 were almost identical to those of the United States isolated from 1989 to 1992, and Japanese strains in 1990s were related closely to some of the MV strains isolated in 1994 in the United States. Genetic recombination among the MV genes seemed not to have occurred. J. Med. Virol. 52:113–120, 1997. © 1997 Wiley-Liss, Inc.     

Serologic and genetic characterization analyses of a highly pathogenic influenza virus (H5N1) isolated from an infected man in Shenzhen

Highly pathogenic avian influenza (HPAI) H5N1 virus caused a wave of outbreaks in China during 2005--2006, resulting in a total of 20 cases of human infection in 14 provinces of China. On June16, 2006, a case of H5N1 human infection was confirmed in Shenzhen. The virus isolated from the patient, A/Guangdong/2/06, was characterized genetically and the relationship between the tracheal virus load and the antibody titer of the infected man was analyzed. Serological analysis confirmed that the patient's neutralizating antibodies had been generated 2 weeks after the onset of symptoms. The patient's serum antibodies could efficiently neutralize A/Guandong/2/06 infectivity in vitro. Phylogenetic analysis showed that the H5N1 virus of Shenzhen belonged to subclade 2.3.4, which contained viruses that were mainly responsible for the outbreaks in domestic poultry and in the cases of human infection in southern China. Homology and molecular characterization analysis revealed that all the segments of Shenzhen H5N1 virus still belonged to avian segments. Several specific amino acid residue mutations were detected.     

Identification and characterization of a Hantavirus strain of unknown origin by nucleotide sequence analysis of the cDNA derived from the viral S RNA segment

The genetic characterization of a serologically Hantaan-like virus but of unknown origin (termedDX) was carried out by molecular cloning and nucleotide sequencing of the corresponding cDNA of the viral S RNA segment. The S RNA was found to be 1765 nucleotides long with 3 and 5 termini being complementary for 24 bases. The virus messenger-sense RNA contains one major open reading frame (ORF) encoding 428 amino acids or a 50 kD polypeptide. A comparison of the DX S RNA segment to those of Sapporo rat, Hantaan, Puumala/Hällnäs B1, and Prospect Hill viruses reveals 95.4, 71.3, 55.3, and 60.9% homology at the nucleotide sequence level, and 94.7, 80.1, 58.4, and 59.8% at the deduced amino acid sequence level. Thus Hantavirus strain DX is very closely related to Sapporo rat virus. We also analyzed the S RNA segments of these Hantaviruses for the presence of a second ORF encoding a potential nonstructural NSs protein. All potential second ORFs detected in the different S RNA segments differ substantially in length and position among the viruses, despite the high conservation of the nucleotide sequences and the overall structure of the nucleocapsid proteins. This suggests that the nucleocapsid protein is the only polypeptide encoded by Hanta-virus S RNA segments, setting them apart from the other members of the Bunyaviridae family.