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Fulltext Viral Protein Accumulation of Zika Virus Variants Links with Regulation of Innate Immunity for Differential Control of Viral Replication, Spread, and Response to Interferon

Lu AY, Gustin A, Newhouse D, Gale M

J Virol, 2023 May 31;97(5):e0198222.
PMID: 37162358 DOI: 10.1128/jvi.01982-22

Asian lineage Zika virus (ZIKV) strains emerged globally, causing outbreaks linked with critical clinical disease outcomes unless the virus is effectively restricted by host immunity. We have previously shown that retinoic acid-inducible gene-I (RIG-I) senses ZIKV to trigger innate immunity to direct interferon (IFN) production and antiviral responses that can control ZIKV infection. However, ZIKV proteins have been demonstrated to antagonize IFN. Here, we conducted in vitro analyses to assess how divergent prototypic ZIKV variants differ in virologic properties, innate immune regulation, and infection outcome. We comparatively assessed African lineage ZIKV/Dakar/1984/ArD41519 (ZIKV/Dakar) and Asian lineage ZIKV/Malaysia/1966/P6740 (ZIKV/Malaysia) in a human epithelial cell infection model. De novo viral sequence determination identified amino acid changes within the ZIKV/Dakar genome compared to ZIKV/Malaysia. Viral growth analyses revealed that ZIKV/Malaysia accumulated viral proteins and genome copies earlier and to higher levels than ZIKV/Dakar. Both ZIKV strains activated RIG-I/IFN regulatory factor (IRF3) and NF-κB pathways to induce inflammatory cytokine expression and types I and III IFNs. However, ZIKV/Malaysia, but not ZIKV/Dakar, potently blocked downstream IFN signaling. Remarkably, ZIKV/Dakar protein accumulation and genome replication were rescued in RIG-I knockout (KO) cells late in acute infection, resulting in ZIKV/Dakar-mediated blockade of IFN signaling. We found that RIG-I signaling specifically restricts viral protein accumulation late in acute infection where early accumulation of viral proteins in infected cells confers enhanced ability to limit IFN signaling, promoting viral replication and spread. Our results demonstrate that RIG-I-mediated innate immune signaling imparts restriction of ZIKV protein accumulation, which permits IFN signaling and antiviral actions controlling ZIKV infection. IMPORTANCE ZIKV isolates are classified under African or Asian lineages. Infection with emerging Asian lineage-derived ZIKV strains is associated with increased incidence of neurological symptoms that were not previously reported during infection with African or preemergent Asian lineage viruses. In this study, we utilized in vitro models to compare the virologic properties of and innate immune responses to two prototypic ZIKV strains from distinct lineages: African lineage ZIKV/Dakar and Asian lineage ZIKV/Malaysia. Compared to ZIKV/Dakar, ZIKV/Malaysia accumulates viral proteins earlier, replicates to higher levels, and robustly blocks IFN signaling during acute infection. Early accumulation of ZIKV/Malaysia NS5 protein confers enhanced ability to antagonize IFN signaling, dampening innate immune responses to promote viral spread. Our data identify the kinetics of viral protein accumulation as a major regulator of host innate immunity, influencing host-mediated control of ZIKV replication and spread. Importantly, these findings provide a novel framework for evaluating the virulence of emerging variants.
Tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues

Yaiw KC, Bingham J, Crameri G, Mungall B, Hyatt A, Yu M, et al.

J Virol, 2008 Jan;82(1):565-8.
PMID: 17913804

Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission.
Fulltext The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis

Yunus MA, Lin X, Bailey D, Karakasiliotis I, Chaudhry Y, Vashist S, et al.

J Virol, 2015 Jan 15;89(2):1218-29.
PMID: 25392209 DOI: 10.1128/JVI.02432-14

All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.
IMPORTANCE: Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.
Fulltext The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response

Prow NA, Setoh YX, Biron RM, Sester DP, Kim KS, Hobson-Peters J, et al.

J Virol, 2014 Sep 1;88(17):9947-62.
PMID: 24942584 DOI: 10.1128/JVI.01304-14

The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II.
Recombinant nipah virus vaccines protect pigs against challenge

Weingartl HM, Berhane Y, Caswell JL, Loosmore S, Audonnet JC, Roth JA, et al.

J Virol, 2006 Aug;80(16):7929-38.
PMID: 16873250

Nipah virus (NiV), of the family Paramyxoviridae, was isolated in 1999 in Malaysia from a human fatality in an outbreak of severe human encephalitis, when human infections were linked to transmission of the virus from pigs. Consequently, a swine vaccine able to abolish virus shedding is of veterinary and human health interest. Canarypox virus-based vaccine vectors carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F) were used to intramuscularly immunize four pigs per group, either with 10(8) PFU each or in combination. Pigs were boosted 14 days postvaccination and challenged with 2.5 x 10(5) PFU of NiV two weeks later. The combined ALVAC-F/G vaccine induced the highest levels of neutralization antibodies (2,560); despite the low neutralizing antibody levels in the F vaccinees (160), all vaccinated animals appeared to be protected against challenge. Virus was not isolated from the tissues of any of the vaccinated pigs postchallenge, and a real-time reverse transcription (RT)-PCR assay detected only small amounts of viral RNA in several samples. In challenge control pigs, virus was isolated from a number of tissues (10(4.4) PFU/g) or detected by real-time RT-PCR. Vaccination of the ALVAC-F/G vaccinees appeared to stimulate both type 1 and type 2 cytokine responses. Histopathological findings indicated that there was no enhancement of lesions in the vaccinees. No virus shedding was detected in vaccinated animals, in contrast to challenge control pigs, from which virus was isolated from the throat and nose (10(2.9) PFU/ml). Based on the data presented, the combined ALVAC-F/G vaccine appears to be a very promising vaccine candidate for swine.
Fulltext RSAD2 and AIM2 Modulate Coxsackievirus A16 and Enterovirus A71 Replication in Neuronal Cells in Different Ways That May Be Associated with Their 5' Nontranslated Regions

Yogarajah T, Ong KC, Perera D, Wong KT

J Virol, 2018 03 15;92(6).
PMID: 29263272 DOI: 10.1128/JVI.01914-17

Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot, and mouth disease (HFMD), but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and real-time quantitative PCR (RT-qPCR) in CV-A16-infected neuroblastoma cells (SK-N-SH), and the results showed that the radical S-adenosylmethionine domain containing 2 (RSAD2) was the highest upregulated gene in the antimicrobial pathway. Increased RSAD2 expression was correlated with reduced viral replication, while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2), which is associated with pyroptotic cell death, was upregulated in EV-A71-infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5' nontranslated regions (5' NTRs) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5' NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5' NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16-associated neurovirulence in HFMD outbreaks compared to EV-A71 infection.IMPORTANCE Although coxsackievirus A16 (CV-A16) and enterovirus A17 (EV-A71) both cause hand, foot, and mouth disease, EV-A71 has emerged as a leading cause of nonpolio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact viral neuropathogenesis. We further showed that viral 5' NTRs may play significant roles in eliciting different host response mechanisms.
Fulltext Puzzling Origins of the Ebola Outbreak in the Democratic Republic of the Congo, 2014

Lam TT, Zhu H, Chong YL, Holmes EC, Guan Y

J Virol, 2015 Oct;89(19):10130-2.
PMID: 26202242 DOI: 10.1128/JVI.01226-15
Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia

McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa MJ

J Virol, 2001 Aug;75(16):7732-8.
PMID: 11462047

Enterovirus 71 (EV71) is a frequent cause of hand, foot, and mouth disease (HFMD) epidemics associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case fatality rates. In this study, we show that four genetic lineages of EV71 have been prevalent in the Asia-Pacific region since 1997, including two previously undescribed genogroups (B3 and B4). Furthermore, we show that viruses belonging to genogroups B3 and B4 have circulated endemically in Southeast Asia during this period and have been the primary cause of several large HFMD or encephalitis epidemics in Malaysia, Singapore, and Western Australia.
Fulltext Phylodynamics of Enterovirus A71-Associated Hand, Foot, and Mouth Disease in Viet Nam

Geoghegan JL, Tan le V, Kühnert D, Halpin RA, Lin X, Simenauer A, et al.

J Virol, 2015 Sep;89(17):8871-9.
PMID: 26085170 DOI: 10.1128/JVI.00706-15

Enterovirus A71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011 and 2013 and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time frame of sampling, whereas that of B5 increased and remained >1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated area of endemicity. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia.
IMPORTANCE: EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.
Origin and evolution of Japanese encephalitis virus in southeast Asia

Solomon T, Ni H, Beasley DW, Ekkelenkamp M, Cardosa MJ, Barrett AD

J Virol, 2003 Mar;77(5):3091-8.
PMID: 12584335

Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.
Fulltext Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability

Dietzel E, Kolesnikova L, Sawatsky B, Heiner A, Weis M, Kobinger GP, et al.

J Virol, 2016 Mar;90(5):2514-22.
PMID: 26676785 DOI: 10.1128/JVI.02920-15

Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles.
Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998

Brown BA, Oberste MS, Alexander JP, Kennett ML, Pallansch MA

J Virol, 1999 Dec;73(12):9969-75.
PMID: 10559310

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.
Fulltext Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes

Sun W, McCrory TS, Khaw WY, Petzing S, Myers T, Schmitt AP

J Virol, 2014 Nov;88(22):13099-110.
PMID: 25210190 DOI: 10.1128/JVI.02103-14

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections.
Fulltext Identification of the progenitors of Indonesian and Vietnamese avian influenza A (H5N1) viruses from southern China

Wang J, Vijaykrishna D, Duan L, Bahl J, Zhang JX, Webster RG, et al.

J Virol, 2008 Apr;82(7):3405-14.
PMID: 18216109 DOI: 10.1128/JVI.02468-07

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.
Fulltext Henipavirus pathogenesis in human respiratory epithelial cells

Escaffre O, Borisevich V, Carmical JR, Prusak D, Prescott J, Feldmann H, et al.

J Virol, 2013 Mar;87(6):3284-94.
PMID: 23302882 DOI: 10.1128/JVI.02576-12

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.
Fulltext Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences

Jelen MM, Chen Z, Kocjan BJ, Burt FJ, Chan PK, Chouhy D, et al.

J Virol, 2014 Jul;88(13):7307-16.
PMID: 24741079 DOI: 10.1128/JVI.00621-14

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution.
IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.
Fulltext Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences

Jelen MM, Chen Z, Kocjan BJ, Hošnjak L, Burt FJ, Chan PKS, et al.

J Virol, 2016 Jun 01;90(11):5503-5513.
PMID: 27030261 DOI: 10.1128/JVI.03149-15

Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B.
IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.
Genome sequences of a novel HIV-1 CRF53_01B identified in Malaysia

Chow WZ, Al-Darraji H, Lee YM, Takebe Y, Kamarulzaman A, Tee KK

J Virol, 2012 Oct;86(20):11398-9.
PMID: 22997419

A novel HIV-1 genotype designated CRF53_01B was recently characterized from three epidemiologically unrelated persons in Malaysia. Here we announced three recently isolated full-length genomes of CRF53_01B, which is likely to be phylogenetically linked to CRF33_01B, circulating widely in Southeast Asia. The genome sequences may contribute to HIV-1 molecular surveillance and future vaccine development in the region.
Genome sequence of a novel HIV-1 circulating recombinant form 54_01B from Malaysia

Ng KT, Ong LY, Takebe Y, Kamarulzaman A, Tee KK

J Virol, 2012 Oct;86(20):11405-6.
PMID: 22997423

We report here the first novel HIV-1 circulating recombinant form (CRF) 54_01B (CRF54_01B) isolated from three epidemiologically unlinked subjects of different risk groups in Malaysia. These recently sampled recombinants showed a complex genome organization composed of parental subtype B' and CRF01_AE, with identical recombination breakpoints observed in the gag, pol, and vif genes. Such a discovery highlights the ongoing active generation and spread of intersubtype recombinants involving the subtype B' and CRF01_AE lineages and indicates the potential of the new CRF in bridging HIV-1 transmission among different risk groups in Southeast Asia.
Fulltext Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease

Ong KC, Devi S, Cardosa MJ, Wong KT

J Virol, 2010 Jan;84(1):661-5.
PMID: 19864378 DOI: 10.1128/JVI.00999-09

Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

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