Consensus PCR Primers Research Articles

Equus caballus papillomavirus Type 7 is a rare cause of equine penile squamous cell carcinomas

Penile squamous cell carcinomas (SCCs) are common, potentially life-threatening neoplasms of horses. They are well-recognized to be caused by Equus caballus papillomavirus (EcPV) type 2, although EcPV2 cannot be detected in all cases. A 23-year-old standardbred gelding developed multiple penile in situ and invasive SCCs that contained histological evidence of PV infection. By using both consensus and specific PCR primers, these lesions were found to contain EcPV7 DNA, but not DNA from EcPV2 or any other PV type. To determine how frequently EcPV7 is present in equine penile SCCs, specific primers were used to detect EcPV2 and EcPV7 in a series of 20 archived samples. EcPV7 was the only PV detected in one, both EcPV2 and 7 were detected in five, and only EcPV2 was detected in 14 SCCs. EcPV7 DNA was also detected in three of 10 archived oropharyngeal SCCs, although only as a co- infection with EcPV2. This is the first report of EcPV7 causing disease in horses. These results suggest EcPV7 could cause a subset of equine penile SCCs, and this is the first evidence that PV types other than EcPV2 can cause these neoplasms. The detection of EcPV7 in the oropharyngeal SCCs suggests a potential role of this PV type in the development of these SCCs. There were no clinical or histological features that differentiated lesions containing EcPV7 DNA from those containing EcPV2 DNA. If EcPV7 causes a proportion of equine penile SCCs, vaccines to prevent EcPV2 infection may not prevent all equine penile SCCs.

Genomic Characterisation of Canis Familiaris Papillomavirus Type 24, a Novel Papillomavirus Associated with Extensive Pigmented Plaque Formation in a Pug Dog.

Numerous large dark plaques developed over the ventrum, legs and head of a 9-year-old pug dog over a 4-year-period. Histology confirmed a diagnosis of viral pigmented plaque and a short section of a novel papillomavirus (PV) type was amplified using consensus PCR primers. Taking advantage of the circular nature of PV DNA, 'outward facing' PCR primers allowed amplification of the full sequence. As this is the 24th PV known to infect dogs, the novel PV was designated canine papillomavirus (CPV) type 24. The CPV24 genome contained putative coding regions for 5 early proteins and 2 late ones. The CPV24 open reading frame L1 showed the highest (78.2%) similarity to CPV4 and phylogenetic analysis showed that CPV24 clustered with CPV4 and CPV16 suggesting CPV24 is the third species 2 Chipapillomavirus type identified in dogs. This is the third report of extensive pigmented plaques covering a high proportion of the skin. Both previous cases were caused CPV4 and, considering the high genetic similarity between CPV4 and CP24, infection by these CPV types may predispose to more severe clinical disease. In addition, as plaques caused by CPV16 appear more likely to progress to neoplasia, the detection of a species 2 Chipapillomavirus within a pigmented plaque may indicate the potential for more severe disease.

Detection of Alpha- and Betacoronaviruses in Frugivorous and Insectivorous Bats in Nigeria

The rise of bat-associated zoonotic viruses necessitates a close monitoring of their natural hosts. Since the detection of severe acute respiratory syndrome coronavirus (SARS-CoV), it is evident that bats are vital reservoirs of coronaviruses (CoVs). In this study, we investigated the presence of CoVs in multiple bat species in Nigeria to identify viruses in bats at high-risk human contact interfaces. Four hundred and nine bats comprising four bat species close to human habitats were individually sampled from five states in Nigeria between 2019 and 2021. Coronavirus detection was done using broadly reactive consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene of CoVs. Coronavirus RNA was detected in 39 samples (9.5%, CI 95%: [7.0, 12.8]), of which 29 were successfully sequenced. The identified CoVs in Nigerian bats were from the unclassified African alphacoronavirus lineage and betacoronavirus lineage D (Nobecovirus), with one sample from Hipposideros ruber coinfected with alphacoronavirus and betacoronavirus. Different bat species roosting in similar or other places had CoVs from the same genetic lineage. The phylogenetic and evolutionary dynamics data indicated a high CoV diversity in Nigeria, while host switching may have contributed to CoV evolution. Robust sentinel surveillance is recommended to enhance our knowledge of emerging and re-emerging coronaviruses.

Evidence from a Series of 104 Equine Sarcoids Suggests That Most Sarcoids in New Zealand Are Caused by Bovine Papillomavirus Type 2, although Both BPV1 and BPV2 DNA Are Detectable in around 10% of Sarcoids.

Simple SummaryEquine sarcoids are common cancers of horses that are caused by bovine papillomaviruses (BPVs). Previous studies have suggested that most sarcoids are caused by either BPV1 or BPV2, with the proportion caused by each BPV type dependent on the country in which the horse lived. Additionally, some studies also suggest that other papillomavirus (PV) types could cause equine sarcoids. The study was comprised of 104 sarcoids from New Zealand horses and used both specific and consensus PCR primer sets. Overall, PV DNA was amplified from 90% of the sarcoids. Of the ones that contained BPV DNA, 88% contained only BPV2 DNA, 10% BPV1 and BPV2 DNA, and 2% only BPV1 DNA. Only the primers specific for BPV1 or specific for BPV2 amplified DNA and no other PV types were detected. There was little variability in the rates of detection between different regions of New Zealand and rates were consistent when two distinct time periods were compared. These results suggest that sarcoids from New Zealand horses are consistently most often caused by BPV2 and thus if vaccination is used to prevent these cancers, it will be important to use a vaccine that provides good protection against this BPV type. Equine sarcoids are common mesenchymal neoplasms of horses that are caused by cross-species infection by deltapapillomaviruses. While bovine papillomavirus (BPV) 1 and 2 are the most common causes, there are differences between countries regarding which of these BPV types cause the majority of sarcoids. Additionally, no causative PV can be detected in a subset of sarcoids, suggesting that other PV types could be rarer causes of these neoplasms. In the present study, consensus PCR primers and PCR primers specific for the five deltapapillomavirus types currently known to cause mesenchymal neoplasia (BPV1, BPV2, BPV13, BPV14, and Ovis aries PV2 DNA) were used to investigate the presence of PV DNA in 104 sarcoids from three defined regions in New Zealand and from two distinct time periods separated by 15 years. PV DNA was detected in 94 (90.4%) sarcoids. Of the sarcoids containing PV DNA, 83 (88.3%) contained only BPV2 DNA, 9 (9.6%) BPV1 and BPV2 DNA, and 2 (2.1%) only BPV1 DNA. No other PV types were detected. The predominance of BPV2 is consistent with studies of sarcoids from North America but dissimilar to studies of sarcoids from Europe and Australia. Detection rates of BPV1 and BPV2 were similar in sarcoids from different regions of New Zealand and in sarcoids from different time periods. These results suggest that most equine sarcoids in New Zealand are caused by BPV2 and thus if vaccines are developed to prevent sarcoids, vaccines that provide good protection against BPV2 will be required in this country.

Detection of Ovis aries papillomavirus type 2 DNA sequences in a sarcoid-like mass in the mouth of a pig

An oral mass was surgically excised from the gingiva of a pig. As the mass had a similar histological appearance to an equine sarcoid, DNA was extracted and consensus PCR primers used to amplify papillomavirus (PV) DNA. DNA sequences from Ovis aries papillomavirus (OaPV) type 2 were amplified both from a section of the entire mass as well as an area deeper within the mass away from the surface of the lesion. No other PV types were detected within the oral lesion. Ovis aries PV2 is a Delta PV that is closely related to the bovine Delta PVs that cause sarcoids in horses and cats. These results suggest that OaPV2 may be able to infect pigs and this virus could have caused the mesenchymal neoplasm in the mouth of this pig. This is the first evidence that a non-bovine PV can infect a non-host species and the first report of a sarcoid-like mass in pigs. These observations add to the range of species in which PV-associated neoplasia has been reported and suggest that cross-species infection by other Delta PV types may be possible.

Papillomavirus DNA is not Amplifiable from Bladder, Lung, or Mammary Gland Cancers in Dogs or Cats.

Simple SummaryPapillomaviruses (PVs) are well established to cause genital and oral cancer in humans. Additionally, some evidence suggests that these viruses may also have a role in the development of human lung, breast, and bladder cancer. Recent studies have revealed that PVs can influence the development of some cancers in cats and, less frequently, in dogs. In the present study, 47 lung, mammary gland, and bladder cancers in dogs and 25 cancers in cats were evaluated for evidence of any role PVs may have in cancer development. Histology did not reveal any lesions suggestive of PV infection, and no PV DNA was amplified from any cancer. Therefore, these findings suggest that PVs do not frequently infect these tissues and are therefore unlikely to be significant factors in the development of lung, mammary gland, or bladder cancer in either dogs or cats.Papillomaviruses (PVs) cause around 5% of all human cancers, including most cervical cancers and around a quarter of all oral cancers. Additionally, some studies have suggested that PVs could cause a proportion of human lung, breast, and bladder cancers. As PVs have been associated with skin cancer in cats and, more rarely, dogs, it was hypothesized that these viruses could also contribute to epithelial cancers of the lung, mammary gland, and bladder of dogs and cats. Formalin-fixed paraffin-embedded samples of 47 canine and 25 feline cancers were examined histologically for evidence of PV infection. Additionally, three sets of consensus PCR primers were used to amplify PV DNA from the samples. No histological evidence of PV infection was visible in any of the cancers. DNA from a bovine PV type was amplified from one sample, while two different samples were found to contain human PV DNA. However, these were considered to be contaminants, and no canine or feline PV types were amplified from any of the cancers. These results suggest that PVs do not frequently infect the lung, mammary gland, or bladder of dogs and cats and therefore are unlikely to be significant factors in the development of cancers in these tissues.

Genomic characterisation of Felis catus papillomavirus type 5 with proposed classification within a new papillomavirus genus

Four Felis catus papillomavirus (FcaPV) types have been fully sequenced from domestic cats. Of these, FcaPV-2 and -3 are thought to cause feline viral plaques and Bowenoid in situ carcinomas. Two short sequences of DNA from a previously unreported PV type were amplified from a feline viral plaque using consensus PCR primers. DNA was then extracted from a swab of the lesion and two sets of ‘outward facing’ primers were designed using the short sequences to amplify the entire 7600bp genome of the novel PV. The PV was designated FcaPV-5 and contained putative coding regions that were predicted to produce five early proteins and two late ones. The ORF L1 showed over 65% similarity to that of FcaPV-3 and -4. Assignment to a genus was difficult as the PV was over 60% similar to PV types from 4 different genera. However, due to the ORF L1 similarity of FcaPV-3, -4, and -5, the shared host species of all three PVs, and the similar lesions associated with FcaPV-3 and -5, it is proposed all three PVs are classified within a new genus. FcaPV-5 is the third PV type to be associated with feline viral plaques. The plaque that contained FcaPV-5 showed unusual histological features including hyperplasia and PV-induced cell changes in sebaceous glands and deep within hair follicles. While additional study of further lesions of this type is required, it is possible that FcaPV-5 may be able to infect a broader range of cells than other PV types.

Virus Hunting: Discovery of New Episomal Circular Viruses by Rolling Circle Techniques.

Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the sequence-independent amplification of circular viral genomes that is based on the rolling-circle mechanism used by certain viruses in their natural replication cycle. The amplification of the virus of interest is coupled to the isolation of the viral genome by gel extraction following a restriction digestion. Once isolated, the sequence of the viral genome can be determined by nanopore sequencing, a rapid and inexpensive next-generation sequencing technology that generates long reads in real time. The method described in this unit was originally developed for the discovery of papillomaviruses, but can be used for the identification of all types of circular DNA viruses. © 2017 by John Wiley & Sons, Inc.

Prevalence of Oxacillinase Groups I, II and III in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Genotyping by ERIC-PCR Methods

Background: Pseudomonas aeruginosa is main bacterial pathogen accountable for nosocomial infections. Furthermore, it could potentially become resistant to β-lactams, aminoglycosides and fluoroquinolones antibiotics. Objectives: The purpose of this study was to determine the antibiotic susceptibility pattern and genotyping of P. aeruginosa in hospitals of Tabriz (Iran) and investigate the prevalence of OXA producer isolates. Methods: Overall, 151 non-replicated isolates of P. aeruginosa were collected from October 2013 until September 2014. Antibiotic susceptibility pattern was determined by disk diffusion (Kirby Bauer) method, according to the clinical laboratory standards institute (CLSI) guideline. Genes encoding OXA (Ambler class D) β-lactamase were detected by PCR for all isolates. Polymerase chain reaction with Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) primers was used to establish the clonal relationship between the different isolates. Results: The frequencies of resistance to antibiotics were as follows: gentamicin: 68%, ceftazidime: 67%, piperacillin: 66%, cefepime: 64%, ciprofloxacin: 62%, tobramycin: 61%, amikacin: 60%, imipenem: 52%, gatifloxacin: 28%, polymyxin B: 2 % and colistin: 2%. The OXA group I genes was identified in 82 (56%), the OXA group II gene in 26 (18%), OXA group III in 14 (9%), OXA-1 in 22 (15%) and OXA-4 in 3 (2%) isolates. The ERIC-PCR indicated high genetic diversity among P. aeruginosa isolates. Conclusions: The high prevalence of OXA β-lactamase and high genetic diversity of P. aeruginosa indicated that the resistance of P. aeruginosa might be expanding in our studied hospitals. Keywords: Antibiotic; Pseudomonas aeruginosa; Genotyping; Beta-Lactamase OXA; Polymerase Chain Reaction

Abstract B23: Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC)

Abstract Background: HPV is an emerging risk factor for HNSCC, however, molecular mechanisms in HPV mediated HNSCC remains poorly delineated. Recent studies suggest that non-coding RNAs may play an important role in the aetiology of HNSCC. In the present study, we examined microRNAs involved in HPV-16 induced HNSCC. Material and Method: Tissue samples from HNSCC patients (n=300) were screened for HPV-16 using PCR based approach with consensus PCR primers (MY09/MY11, Gp5+/Gp6+), HPV16-specific PCR, HPV-16 E7 mRNA, immunohistochemistry (p16, p53 and pRB) and viral-load determination. In-silico approach was used to identify host miRNAs with HPV-16 mRNAs as putative target. Differential expression of identified miRNAs was determined by qRT-PCR in HPV-16 positive (UPCI:SCC090, CaSki, SiHa), HPV negative (UPCI:SCC116, HaCat) and transfected with HPV 16 full genome plasmid cell lines, and, HPV 16-positive/HPV-negative tissue samples. Putative targets of identified miRNAs were identified by bioinformatic approach and their mRNA levels were determined by qRT-PCR in HPV positive and negative cell lines, and tissue samples. Over-expression and inhibition studies with miRNA mimic and inhibitor were done to identify affected viral and host oncogenic pathways. Results: Total 32 HNSCC patients were found to be HPV-16 positive. We identified 10 miRNAs with HPV-16 as putative target. Among them hsa-miR-139-3p showed significant down-regulation in HPV-16 positive samples and cell lines compared to HPV negative controls. Using bioinformatics tools, hsa-miR-139-3p was found to target viral HPV-16 E1 and host HOXB6 gene. Transfection with hsa-miR-139-3p mimic resulted in significant down-regulation of HPV 16 early mRNA as well as its targeted genes in luciferase-3'UTR assay. Moreover, mimic transfected HPV-positive cell lines showed dysregulated MEK-ERK signaling pathway. Conclusion: Our results suggest hsa-miR-139-3p may be involved in patho-physiology of HPV-16 positive HNSCC as it deregulates expression of HPV oncogenic proteins and cell proliferation by targeting MEK-ERK signaling pathway. Citation Format: Malay Kumar Sannigrahi, Varinder Singh, Rajni Sharma, Naresh K. Panda, Madhu Khullar. Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B23.

Distribución de los genotipos del virus del papiloma humano en mujeres de Bogotá con anomalías en la citología cervicouterina

ObjectiveTo determine the distribution of HPV genotypes in women from Bogotá with abnormal cervical smear. MethodsCervical samples were collected from 191 women with ASCUS, 236 with low-grade (LSIL) and 116 with high-grade squamous intraepithelial lesions (HSIL). HPV typing was performed using consensus PCR primers GP5+/GP6 + and reverse line blot. ResultsThe prevalence of HPV was 76.1%. Unique infections were observed in 41.4% of the participants and co-infections in 34.6%. The frequency of HPV according to diagnosis was: 60.2%, 84.7% and 84.5% in ASCUS, LSIL and HSIL respectively. HPV16 and 58 were the more frequent types in the three groups, HPV16 frequencies were 20.4%, 33.9% and 38.8%, and HPV58 frequencies were 7.3%, 13.6%, and 18,1% for ASCUS, LSIL and HSIL respectively. The third type in frequency in ASCUS and LSIL was HPV56 (6.8% and 11.0%), while in HSIL was HPV18 (10.3%). Infections with probably oncogenic types or with low risk types were much less frequent and generally were observed as co-infections with high-risk types. ConclusionsInfections with high risk HPV types were the most frequent in all studied lesions, VPH-58 ranked second in frequency as has been reported in Mexico and in general in South America. The change to new prophylactic vaccines including more viral types as HPV58 could have a higher impact in reducing the incidence of pre-neoplastic lesions in our region.

Diversity of coronavirus in bats from Eastern Thailand.

BackgroundBats are reservoirs for a diverse range of coronaviruses (CoVs), including those closely related to human pathogens such as Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome CoV. There are approximately 139 bat species reported to date in Thailand, of which two are endemic species. Due to the zoonotic potential of CoVs, standardized surveillance efforts to characterize viral diversity in wildlife are imperative.FindingsA total of 626 bats from 19 different bat species were individually sampled from 5 provinces in Eastern Thailand between 2008 and 2013 (84 fecal and 542 rectal swabs). Samples collected (either fresh feces or rectal swabs) were placed directly into RNA stabilization reagent, transported on ice within 24 hours and preserved at −80°C until further analysis. CoV RNA was detected in 47 specimens (7.6%), from 13 different bat species, using broadly reactive consensus PCR primers targeting the RNA-Dependent RNA Polymerase gene designed to detect all CoVs. Thirty seven alphacoronaviruses, nine lineage D betacoronaviruses, and one lineage B betacoronavirus (SARS-CoV related) were identified. Six new bat CoV reservoirs were identified in our study, namely Cynopterus sphinx, Taphozous melanopogon, Hipposideros lekaguli, Rhinolophus shameli, Scotophilus heathii and Megaderma lyra.ConclusionsCoVs from the same genetic lineage were found in different bat species roosting in similar or different locations. These data suggest that bat CoV lineages are not strictly concordant with their hosts. Our phylogenetic data indicates high diversity and a complex ecology of CoVs in bats sampled from specific areas in eastern regions of Thailand. Further characterization of additional CoV genes may be useful to better describe the CoV divergence.

Papillomaviral DNA sequences are not amplifiable from canine subungual squamous cell carcinomas

AIM: To determine if papillomaviral DNA is more frequently present within canine subungual squamous cell carcinomas (SCCs) than in non-SCC digit lesions. METHODS: Total DNA was extracted from 23 canine subungual SCCs and 23 non-SCC digit lesions. The presence of amplifiable DNA within each sample was confirmed by amplifying a section of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene. Two different consensus PCR primer sets were used to amplify papillomaviral DNA from the samples. RESULTS: The consensus primers only amplified papillomaviral DNA from the positive control samples. None of the 46 canine digit samples contained DNA that was amplifiable by the consensus PCR primers. CONCLUSION: Papillomaviruses are unlikely to be a significant cause of canine subungual SCCs. CLINICAL RELEVANCE: While circumstantial evidence suggests that canine subungual SCCs could develop due to papillomaviral infection, this study did not reveal any evidence to support papillomaviral aetiology of these neoplasms.

Analysis of the expression of partial self-incompatibility in almond (Prunus dulcis)

SummarySeveral pollination studies carried out on different, self-incompatible almond cultivars and seedlings have shown the presence of variable levels of fruit set following self-pollination that could be attributed to partial self-incompatibility (PSI). PSI is an intermediate reproductive behaviour, described in some angiosperm species, which indicates that self-incompatibility is a quantitative and plastic trait. The present study was performed to substantiate the occurrence of PSI in almond by studying ten cultivars that are traditionally considered to be self-incompatible (four of which had previously shown fruit set after self-pollination). These cultivars were analysed by microscopic observations of the progression of pollen tubes through the pistil following controlled self-pollination, determinations of fruit set after bagging flower buds, and molecular identification of the parentage of the fruits obtained using consensus PCR-primers for Prunus S-RNase alleles. The results showed that, in nearly all cases, the pollen tubes did not enter the ovary and, from a total of 5,349 bagged flower buds, only 17 fruits were obtained. In all cases, PCR analysis of the plants obtained after germination of these seeds showed a band corresponding to an S-RNase allele not present in the maternal progenitor, clearly indicating that they could only have arisen from cross-pollination.Therefore, the low fruit set values observed in some of the cultivars studied were not due to a breakdown in the self-incompatibility response that confers PSI, but to very low rates of contamination with foreign pollen. These results corroborate the self-incompatibility phenotype of the cultivars studied here, and highlight the importance of ascertaining the identity of the parentage in any progeny obtained after bagging.

Amplification of feline sarcoid‐associated papillomavirus DNA sequences from bovine skin

Feline sarcoids are uncommon dermal neoplasms that are thought to be caused by papillomaviral (PV) infection. Feline sarcoid-associated PV (FeSarPV) has been consistently detected in sarcoids from North American and New Zealand cats but has not been detected within any other feline sample. This suggests that feline sarcoids may develop due to cross-species infection by a PV from an unidentified reservoir host. While there is some epidemiological evidence to suggest that cattle are the reservoir host of FeSarPV, this PV has never been identified within any bovine sample. In this study both consensus PCR primers and primers specific to FeSarPV were used to investigate the presence of PV DNA within five fibropapillomas and 18 samples of inflammatory skin disease from cattle. Consensus primers amplified bovine PV-2 DNA from four fibropapillomas, but none of the dermatitis samples. However, specific primers amplified FeSarPV DNA from four fibropapillomas and five inflammatory skin lesions. To the best of our knowledge this is the first time that FeSarPV has been detected within any sample other than a feline sarcoid. The ability of FeSarPV to asymptomatically infect bovine skin suggests that cattle are the reservoir host of this PV and feline sarcoids could be the result of cross-species infection of a dead-end host by a bovine PV.

Validation of human papillomavirus genotyping by signature DNA sequence analysis

BackgroundScreening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists.MethodsThe HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction. The nested PCR products were used for direct automated DNA sequencing. A 34- to 50-base sequence including the GP5+ priming site was selected as the signature sequence for routine genotyping by online BLAST sequence alignment algorithms.ResultsOf 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype. The most common genotype was HPV-16 (68 isolates), followed by HPV-52 (25 isolates). More than half (53.7%) of the total number of HPV isolates relied on a nested PCR for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel variants of HPV with less than "100% identities" signature sequence match with those stored in the Genbank database were also detected by signature DNA sequencing in this rural and suburban population of the United States.ConclusionLaboratory staff must be familiar with the limitations of the consensus PCR primers, the locations of the signature sequence in the L1 gene for some HPV genotypes, and HPV genotype sequence variants in order to perform accurate HPV genotyping.

HPV genotypes in women with squamous intraepithelial lesions and normal cervixes participating in a community-based microbicide study in Pretoria, South Africa

Background Little is known regarding the human papillomaviruses (HPV) genotypes prevalent in women in South Africa, a country with a high incidence of cervical cancer. Objective To determine the prevalence and HPV genotypes in women with squamous abnormalities and normal cervixes participating in a community-based microbicide study. Study design A total of 159 cervical specimens, including 56 specimens from women with abnormal cytology (cases) and 103 randomly selected specimens from women with normal cytology (controls), were collected. HPV was detected by consensus PCR primers and HPV genotypes were determined by Roche Linear Array ® HPV genotyping assay. Results HPV genotypes were found in 91% of cases and 40% of controls ( p < 0.005). High-risk HPV was detected in all high-grade squamous intraepithelial lesions (HSILs), 69% of low-grade squamous intraepithelial lesions (LSILs), 57% of atypical squamous cells of undetermined significance (ASCUS), and 86% of ASCUS in which HSIL could not be excluded (ASCUS-H), and 73% of HPV positive controls. HPV-35 was the predominant genotype in HSILs; HPV-18 in ASCUS; HPV-58 in ASCUS-H and HPV-16 in LSILs and controls. Conclusion High-risk HPV prevalence was high in both cases and controls. HPV genotype distribution in HSILs was different from that reported worldwide and from other studies in South Africa.

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S C ) and seven alleles for self-incompatibility (S1–S7) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S8–S16) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification.

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

Gene Expression Profiling in Ecotoxicology

Gene expression profiling is a powerful new end point for ecotoxicology and a means for bringing the genomics revolution to this field. We review the usefulness of gene expression profiling as an end point in ecotoxicology and describe methods for applying this approach to non-model organisms. Since genomes contain thousands of genes representing hundreds of pathways, it is possible to identify toxicant-specific responses from this wide array of possibilities. Stressor-specific signatures in gene expression profiles can be used to diagnose which stressors are impacting populations in the field. Screening for stress-induced genes requires special techniques in organisms without sequenced genomes. These techniques include differential display polymerase chain reaction (DD PCR), suppressive subtractive hybridization PCR (SSH PCR), and representational difference analysis. Gene expression profiling in model organisms like yeast has identified hundreds of genes that are up-regulated in response to various stressors, including several that are well characterized (e.g., hsp78, metallothionein, superoxide dismutase). Using consensus PCR primers from several animal sequences, it is possible to amplify some of these well characterized stress-induced genes from organisms of interest in ecotoxicology. We describe how several stress-induced genes can be grouped into cDNA arrays for rapidly screening samples.