John A. Lednicky

The Dynamics of Herpesvirus and Polyomavirus Reactivation and Shedding in Healthy Adults: A 14-Month Longitudinal Study

Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

Some Oral Poliovirus Vaccines Were Contaminated with Infectious SV40 after 1961

Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious SV40. It has been assumed that all polio vaccines were SV40 free in the United States after 1961 and in other countries after 1962. Following a WHO requirement that was prompted by the detection of SV40 in some human tumors, we conducted a multilaboratory study to test for SV40 polio vaccines prepared after 1961. Vaccine samples from 13 countries and the WHO seed were initially tested by PCR. The possible presence of intact and/or infectious SV40 DNA in PCR-positive samples was tested by transfection and infection of permissive CV-1 cells. All results were verified by immunohistochemistry, cloning, and sequencing. All the vaccines were SV40 free, except for vaccines from a major eastern European manufacturer that contained infectious SV40. We determined that the procedure used by this manufacturer to inactivate SV40 in oral poliovirus vaccine seed stocks based on heat inactivation in the presence of MgCl2 did not completely inactivate SV40. These SV40-contaminated vaccines were produced from early 1960s to about 1978 and were used throughout the world. Our findings underscore the potential risks of using primary monkey cells for preparing poliovirus vaccines, because of the possible contamination with SV40 or other monkey viruses, and emphasize the importance of using well-characterized cell substrates that are free from adventitious agents. Moreover, our results indicate possible geographic differences in SV40 exposure and offer a possible explanation for the different percentage of SV40-positive tumors detected in some laboratories.

SV40 DNA IN HUMAN OSTEOSARCOMAS SHOWS SEQUENCE VARIATION AMONG T-ANTIGEN GENES

Authentic simian virus 40 (SV40) has been detected in association with human choroid plexus and ependymoma tumors, and SV40‐like DNA sequences have been found in some human osteosarcomas. We report here an analysis of human osteosarcoma samples for the presence of SV40 DNA using PCR and primers directed at 4 distinct sites of the SV40 genome, coupled with sequence analysis. Authentic SV40 DNA sequences were detected in 5 of 10 osteosarcoma tumor samples. The SV40 regulatory region in each case was identical and of archetypal length (non‐duplicated enhancer), as is usually found in natural isolates of SV40 from monkeys and in human brain tumors. A section of the gene that encodes a viral late gene product (VP1) was detected in 5 of 10 tumors and had an exact match with the known sequence of SV40. Two separated segments of the large T‐antigen (T‐ag) gene were found in the same 5 tumors. Analysis of the DNA sequences encoding the T‐ag carboxy terminus revealed sequence variation among the tumors, as observed previously in viral DNA associated with human brain tumors. There does not appear to be a preferential association of a T‐ag variable domain sequence with a given tumor type. No sequences from the regulatory region of human polyomaviruses JCV and BKV were detected in the bone tumors. We also noted less efficient recovery of SV40 DNA from tumor samples fixed in paraffin as compared to frozen tumors. Our results confirm the presence of SV40 DNA in human bone tumors and, based on the sequence variation observed for the carboxy terminus of the T‐ag gene, suggest that there is not a specific SV40 strain associated with human osteosarcomas. Int. J. Cancer 72:791–800, 1997.

Hantaviruses A Short Review

OBJECTIVES Hantaviruses are rodent viruses that have been identified as etiologic agents of 2 diseases of humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). This article presents a concise review of hantavirus biology, the medical features of HFRS and HPS, and tests for the detection of hantavirus infections in humans. DATA SYNTHESIS Hemorrhagic fever with renal syndrome is a disease found outside the Americas and denotes a group of clinically similar illnesses that vary in severity relative to the causative agent. Hantavirus pulmonary syndrome is associated with higher mortality than HFRS, was first recognized as a hantavirus disease in 1993, and occurs within the American continents. Recent genetic studies show that both Old and New World hantavirus species coevolved with specific rodent hosts. The list of distinct hantaviruses associated with HPS is growing. The burgeoning human population is causing disruption of natural habitats as more and more land is cleared for commercial and residential purposes. Many rodents readily adapt to life in human settlements, where they generally benefit from reduced predation and where they sometimes proliferate to high numbers. CONCLUSIONS Although often referred to as emerging pathogens, HPS-associated hantaviruses emerge through increased exposure of humans to rodents and their excreta, not through genetic drift or reassortment of the viral genome. Based on current human population growth and development trends, hantavirus diseases will become more common in the near future unless public health measures are taken to curtail or eliminate rodents from human communities.

Sequence analyses of human tumor-associated SV40 DNAs and SV40 viral isolates from monkeys and humans

SV40 DNA has been found associated with several types of human tumors. We now report a sequence comparison of SV40 DNAs from pediatric brain tumors and from osteosarcomas with viral isolates from monkeys and from humans. We analyzed the entire genomic sequences of five isolates, Baylor and VA45-54 strains from monkeys and SVCPC, SVMEN, and SVPML-1 recovered from humans, and compared them to the reference virus SV40-776. The viral sequences were highly conserved, but isolates could be distinguished by variations in the structure of the viral regulatory region and in the nucleotide sequence of the variable domain at the C-terminus of the large T-antigen gene. We conclude that multiple strains of SV40 exist that can be identified on the basis of sequences in these regions of the viral genome. The isolates were more similar to each other and to the Baylor strain than to the reference strain SV40-776. Human isolates SVCPC and SVMEN were found to be identical. The DNAs present in some human brain and bone tumors were authentic SV40 sequences. Many of the C-terminal T-ag sequences associated with human tumors were unique, but some sequences were shared by independent sources. There was no compelling evidence for human-specific strains of SV40 or for tumor type-specific associations, suggesting that SV40 has a relatively broad host range. The source of the viral DNA found in human tumors remains unknown.

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

&NA; Systemic non‐Hodgkin lymphoma (S‐NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein‐Barr virus DNA is found in <40% of patients with AIDS‐related S‐NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS‐related S‐NHL samples, NHL samples from HIV‐negative patients, peripheral blood leukocytes from HIV‐infected and ‐uninfected patients without NHL, and lymph nodes without tumors from HIV‐infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N‐terminal region of the oncoprotein large tumor antigen (T‐ag) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T‐ag DNA sequences, proven to be SV40‐specific, were detected more frequently in AIDS‐related S‐NHL samples (6 of 26) than in peripheral blood leukocytes from HIV‐infected patients (6 of 26 vs. 0 of 69; p = .0001), NHL samples from HIV‐negative patients (6 of 26 vs. 0 of 10; p = .09), or lymph nodes (6 of 26 vs. 0 of 7; p = .16). Sequences of C‐terminal T‐ag DNA from SV40 were amplified from two AIDS‐related S‐NHL samples. Epstein‐Barr virus DNA sequences were detected in 38% (10 of 26) AIDS‐related S‐NHL samples, 50% (5 of 10) HIV‐negative S‐NHL samples, and 57% (4 of 7) lymph nodes. None of the S‐NHL samples were positive for both Epstein‐Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S‐NHL are warranted.

Zika Virus Outbreak in Haiti in 2014: Molecular and Clinical Data.

Background Zika virus (ZIKV), first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations) in the Caribbean. Methods From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV) and dengue (DENV) virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture. Findings Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas. Conclusions ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The virus appears to have been circulating in Haiti prior to the first reported cases in Brazil. Factors contributing to transmission and the possible linkage of this early Haitian outbreak with microcephaly remain to be determined.

Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)

BackgroundThere is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a animal model for humans in influenza pathogenicity and transmissibility studies. In this manuscript, a nose-only bioaerosol inhalation exposure system that was recently developed and validated was used in an inhalation exposure study of aerosolized A/Vietnam/1203/2004 (H5N1) virus in ferrets. The clinical spectrum of influenza resulting from exposure to A/Vietnam/1203/2004 (H5N1) through intranasal verses inhalation routes was analyzed.ResultsFerrets were successfully infected through intranasal instillation or through inhalation of small particle aerosols with four different doses of Influenza virus A/Vietnam/1203/2004 (H5N1). The animals developed severe influenza encephalomyelitis following intranasal or inhalation exposure to 101, 102, 103, or 104 infectious virus particles per ferret.ConclusionsAerosolized Influenza virus A/Vietnam/1203/2004 (H5N1) is highly infectious and lethal in ferrets. Clinical signs appeared earlier in animals infected through inhalation of aerosolized virus compared to those infected through intranasal instillation.

Phylogenetic Analysis of Polyomavirus Simian Virus 40 from Monkeys and Humans Reveals Genetic Variation

ABSTRACT A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This analysis included infectious isolates recovered between 1960 and 1999 from primary cultures of monkey kidney cells, from contaminated poliovaccines and an adenovirus seed stock, from human malignancies, and from transformed human cells. Maximum-parsimony and distance methods revealed distinct SV40 clades. However, no clear patterns of association between genotype and viral source were apparent. One clade (clade A) is derived from strain 776, the reference strain of SV40. Clade B contains isolates from poliovaccines (strains 777 and Baylor), from monkeys (strains N128, Rh911, and K661), and from human tumors (strains SVCPC and SVMEN). Thus, adaptation is not essential for SV40 survival in humans. The C terminus of the T-antigen (T-ag-C) gene contains the highest proportion of variable sites in the SV40 genome. An analysis based on just the T-ag-C region was highly congruent with the whole-genome analysis; hence, sequencing of just this one region is useful in strain identification. Analysis of an additional 16 strains for which only the T-ag-C gene was sequenced indicated that further SV40 genetic diversity is likely, resulting in a provisional clade (clade C) that currently contains strains associated with human tumors and human strain PML-1. Four other polymorphic regions in the genome were also identified. If these regions were analyzed in conjunction with the T-ag-C region, most of the phylogenetic signal could be captured without complete genome sequencing. This report represents the first whole-genome approach to establishing phylogenetic relatedness among different strains of SV40. It will be important in the future to develop a more complete catalog of SV40 variation in its natural monkey host, to determine if SV40 strains from different clades vary in biological or pathogenic properties, and to identify which SV40 strains are transmissible among humans.

Polyomavirus JCV excretion and genotype analysis in HIV-infected patients receiving highly active antiretroviral therapy.

Objective: To assess the frequency of shedding of polyomavirus JC virus (JCV) genotypes in urine of HIV-infected patients receiving highly active antiretroviral therapy (HAART). Methods: Single samples of urine and blood were collected prospectively from 70 adult HIV-infected patients and 68 uninfected volunteers. Inclusion criteria for HIV-infected patients included an HIV RNA viral load < 1000 copies, CD4 cell count of 200–700 × 106 cells/l, and stable HAART regimen. PCR assays and sequence analysis were carried out using JCV-specific primers against different regions of the virus genome. Results: JCV excretion in urine was more common in HIV-positive patients but not significantly different from that of the HIV-negative group [22/70 (31%) versus 13/68 (19%); P = 0.09]. HIV-positive patients lost the age-related pattern of JCV shedding (P = 0.13) displayed by uninfected subjects (P = 0.01). Among HIV-infected patients significant differences in JCV shedding were related to CD4 cell counts (P = 0.03). Sequence analysis of the JCV regulatory region from both HIV-infected patients and uninfected volunteers revealed all to be JCV archetypal strains. JCV genotypes 1 (36%) and 4 (36%) were the most common among HIV-infected patients, whereas type 2 (77%) was the most frequently detected among HIV-uninfected volunteers. Conclusion: These results suggest that JCV shedding is enhanced by modest depressions in immune function during HIV infection. JCV shedding occurred in younger HIV-positive persons than in the healthy controls. As the common types of JCV excreted varied among ethnic groups, JCV genotypes associated with progressive multifocal leukoencephalopathy may reflect demographics of those infected patient populations.