Archives of Virology

Nguyen, Van; Kim, Hye; Moon, Hyoung; Park, Seong; Keum, Hyun; Rho, Semi; Han, Jae; Park, Bong

doi: 10.1007/s00705-012-1234-xpmid: 22289963

This study was conducted to investigate the status and population dynamics of porcine circovirus type 2 (PCV2) in Korea and to assess the molecular evolutionary pattern of the two biologically important, overlapping open reading frames, the ORF1 and ORF3 genes. A wide range of PCV2 genomic sequences (entire genome, ORF1, ORF2 and ORF3) collected between 2001 and 2010 were analyzed using the Bayesian Markov chain Monte Carlo and maximum-likelihood approaches. These techniques identified the PCV2d genotype and the 2Ek cluster of PCV2a in Korea for the first time. Second, the genotypic shift of PCV2b dominating over PCV2a likely occurred between 2002 and 2004 due to a population expansion of PCV2b. In the context of positive Darwinian selection, the results uncovered independent evolutionary patterns in the ORF3 gene compared to the overlapping ORF1 gene and new sites in the viral ORFs/proteins that might relate to differences in the biological properties of the PCV2 groups.

Košík, Ivan; Krejnusová, Ingrid; Práznovská, Margaréta; Poláková, Katarína; Russ, Gustáv

doi: 10.1007/s00705-012-1238-6pmid: 22294447

Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD 50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.

Madani, Tariq; Abuelzein, El-Tayeb; Azhar, Esam; Kao, Moujahed; Al-Bar, Hussein; Abu-Araki, Huda; Ksiazek, Thomas

doi: 10.1007/s00705-012-1237-7pmid: 22294446

RT-PCR to detect Alkhumra virus (ALKV) RNA in plasma or serum has been the standard practice to confirm this infection in the first seven days of illness. In this study, RT-PCR detection of viral RNA from the plasma, serum, and buffy coat (BC) was compared to virus isolation. Plasma, serum, and BC were obtained from seven patients with clinically suspected ALKV infection in Najran, Saudi Arabia. Baby hamster kidney (BHK-21) and rhesus monkey kidney (LLC-MK2) cell culture monolayers were used for virus isolation. Real-time RT-PCR was used to confirm ALKV infection and to detect viral RNA directly from plasma, serum, and BC. ALKV was isolated from five of the seven patients. The virus was isolated from all three specimen types (plasma, serum, and BC) of the five confirmed patients. ALKV RNA was detected directly by RT-PCR in BC in all five (100%) culture-positive patients and in plasma or serum in only four (80%) of the five patients. Three of the five patients for whom ALKV RNA was detected in BC also had detectable viral RNA in plasma and serum. In the remaining two patients with detectable ALKV RNA in the BC, the plasma was positive but the serum was negative in one patient, whereas the serum was positive and the plasma was negative in the other patient. The use of real-time RT-PCR to detect ALKV RNA in the BC was superior to using plasma and serum and equivalent to virus isolation.

Tsakogiannis, D.; Ruether, I.; Kyriakopoulou, Z.; Pliaka, V.; Theoharopoulou, A.; Skordas, V.; Panotopoulou, E.; Nepka, C.; Markoulatos, P.

doi: 10.1007/s00705-012-1236-8pmid: 22294445

The E2 gene of human papilloma virus is expressed at the early stage of the viral life cycle, encoding the E2 transcription factor, and regulates the expression of E6 and E7 oncogenes. Disruption of E2 gene due to viral integration inhibits the transcriptional suppression of the HPV oncogenes, inducing cell proliferation. In the present study, a total of 22 HPV16-positive cytological specimens derived from high- and low-grade cervical intraepithelial lesions were investigated in order to identify sequence variations in the HPV16 E2 ORF. The E2 gene was amplified by PCR using external and internal overlapping sets of primers. Amplicons were cloned and sequenced. Disruption sites were detected in cervical samples diagnosed as high-grade cervical intraepithelial lesions. Moreover, sequence variations were identified in the E2 ORF and specific variations were associated with non-European variants such as African type I, African type II and Asian American. A total of three new sequence variations were identified at positions 2791, 2823 (transactivation domain) and 3361 (hinge region). Distinct phylogenetic branches were formed according to E2 analysis that characterized the different HPV16 variants. It was ascertained that non-European variants are circulating in the Greek population.

Yacoub, Alia; Leijon, Mikael; McMenamy, Michael; Ullman, Karin; McKillen, John; Allan, Gordon; Belák, Sándor

doi: 10.1007/s00705-012-1231-0pmid: 22302287

A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.

Wu, Guocai; Wang, Yun; Chao, Yan; Jia, Yuping; Zhao, Chengquan; Luo, Bing

doi: 10.1007/s00705-012-1241-ypmid: 22302288

Epstein-Barr virus nuclear antigen protein 3C (EBNA3C) is a 992-amino-acid protein that has been shown to play a complex regulatory role in the transcription of viral and cellular genes. In this study, we successfully amplified 26 Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs), 50 nasopharyngeal carcinomas (NPCs) and 27 throat washing (TW) samples from healthy donors. Based on a phylogenetic tree, the samples could be divided into three patterns. 3C-6 was the predominant subtype in northern China, and the variations between the strains sequenced in our study and those from southern China and Japan were similar, but differences were also identified. The distribution of EBNA3C subtypes among EBVaGCs, NPCs and healthy donors was not significantly different. These data suggest that EBNA3C gene variations are geographically restricted rather than tumor-specific polymorphisms.

Rajagopalan, Prem; Naik, Amruta; Katturi, Prashanth; Kurulekar, Meera; Kankanallu, Ravi; Anandalakshmi, Radhamani

doi: 10.1007/s00705-012-1225-ypmid: 22307170

Cotton leaf curl disease (CLCuD) is a major limitation to cotton production on the Indian subcontinent. A survey for viruses causing CLCuD was conducted during the 2009 and 2010 cropping seasons in the northwestern Indian cotton-growing belt in the states of Punjab, Haryana and Rajasthan. Partial sequences of 258 and full-length sequences of 22 virus genomes were determined. This study shows that the resistance-breaking cotton leaf curl Burewala virus (CLCuBuV) is now the dominant virus in many fields. The spread and establishment of the mutant CLCuBuV in northwestern India, the variation in its genomic sequence, its virulence and infectivity, and the implications for cotton breeding are discussed.

Meng, Chunchun; Qiu, Xvsheng; Jin, Shiqiang; Yu, Shengqing; Chen, Hongjun; Ding, Chan

doi: 10.1007/s00705-012-1248-4pmid: 22310996

A lentogenic Newcastle disease virus (NDV), Duck/JS/10 (JS10), was isolated from an unvaccinated duck in China. The complete genome of the virus contained 15,198 nucleotides. Based on length of the genome and a partial sequence of the F gene, the virus was classified as a class I genotype 4 NDV. The antigenicity of the virus was compared with that of NDV strain La Sota via hemagglutination inhibition (HI), virus neutralization (VN) assay and animal experiments. Our results show that JS10 generates higher HI and VN titers than La Sota against both class I and II virulent NDV strains. Experiments on animals demonstrate that virus shedding from chickens vaccinated with JS10 is significantly reduced when compared to those vaccinated with La Sota. Overall, this study strongly suggests that JS10 may qualify as a new vaccine candidate against Newcastle disease.

Ouyang, Yabo; Ma, Hui; Jin, Min; Wang, Xinwei; Wang, Jingfeng; Xu, Lu; Lin, Shuxiang; Shen, Zhiqiang; Chen, Zhaoli; Qiu, Zhigang; Gao, Zhixian; Peng, Lin; Li, Junwen

doi:

Zhou, Yumei; Chen, Xianfeng; Ushijima, Hiroshi; Frey, Teryl

doi: 10.1007/s00705-012-1243-9pmid: 22322905

Rubella virus (RUBV), a small, plus-strand RNA virus that is an important human pathogen, has the unique feature that the GC content of its genome (70%) is the highest (by 20%) among RNA viruses. To determine the effect of this GC content on genomic evolution, base and codon usage were analyzed across viruses from eight diverse genotypes of RUBV. Despite differences in frequency of codon use, the favored codons in the RUBV genome matched those in the human genome for 18 of the 20 amino acids, indicating adaptation to the host. Although usage patterns were conserved in corresponding genes in the diverse genotypes, within-genome comparison revealed that both base and codon usages varied regionally, particularly in the hypervariable region (HVR) of the P150 replicase gene. While directional mutation pressure was predominant in determining base and codon usage within most of the genome (with the strongest tendency being towards C’s at third codon positions), natural selection was predominant in the HVR region. The GC content of this region was the highest in the genome (>80%), and it was not clear if selection at the nucleotide level accompanied selection at the amino acid level. Dinucleotide frequency analysis of the RUBV genome revealed that TpA usage was lower than expected, similar to mammalian genes; however, CpG usage was not suppressed, and TpG usage was not enhanced, as is the case in mammalian genes.