A strain‐specific multiplex RT‐PCR for Australian rabbit haemorrhagic disease viruses uncovers a new recombinant virus variant in rabbits and hares

Rabbit haemorrhagic disease virus (RHDV , or GI .1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus ) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI .1a‐Aus, previously called RHDV a‐Aus, is a GI .1a virus detected in January 2014, and the novel lagovirus GI .2 (previously known as RHDV 2). Furthermore, an additional GI .1a strain, GI .1a‐K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain‐specific multiplex RT ‐PCR assay was developed, which allows fast, cost‐effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT ‐qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non‐structural genes of GI .1a‐Aus and the structural genes of GI .2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus ). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI .2, requires further studies.     

Worldwide rapid spread of the novel rabbit haemorrhagic disease virus (GI.2/RHDV2/b)

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so‐called ‘novel’ rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two‐step in‐depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future.     

Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden

Incursion of rabbit haemorrhagic disease virus (RHDV ) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus ) populations. Rabbit haemorrhagic disease virus 2 (RHDV 2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV 2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD ) do not distinguish between types of virus causing disease. Using RHDV 2‐specific RT ‐qPCR , sequencing of the VP 60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA ) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV 2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV 2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP 60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV 2, suggesting that RHDV 2 may be replacing RHDV as the predominant cause of RHD in Sweden.     

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free‐living wild European rabbits in Portugal

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV 2 or RHDV b (Lagovirus europaeus /GI .2, henceforth GI .2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit‐sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI .2 in free‐living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV , henceforth GI .1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR‐positive to GI .2, whereas GI .1 strains were not detected in any of the samples. The outbreaks of GI .2 revealed a marked seasonality, with peaks during the breeding season (November‐May). We also found that approximately, one‐third of free‐ranging European rabbits in Portugal have seroconverted to GI .2. We demonstrate that the GI .2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI .2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.     

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full‐length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016–2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild‐type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.     

Isolation and characterization of novel goose parvovirus‐related virus reveal the evolution of waterfowl parvovirus

Short beak and dwarfism syndrome (SBDS ) has been constantly breaking out in China since 2015. It is caused by a novel goose parvovirus‐related virus (NGPV ) and can severely restrict the growth of ducks. In this study, seven NGPV stains were isolated from different regions in China between 2015 and 2016. To better understand the correlation between NGPV and goose parvovirus (GPV ), we conducted complete genome sequencing and a comprehensive analysis of the NGPV genome. The phylogenetic and alignment analysis showed that NGPV is a branch of GPV , sharing 92.2%–97.1% nucleotide identity with GPV . Compared with classical GPV , five consensus nucleotide mutations in all the seven NGPV isolates and two 14‐nucleotide‐pair deletions in six NGPV isolates were found in the inverted terminal repeats, twelve and eight synchronous amino acid changes were found in the replication protein and capsid protein of NGPV , respectively, which might be important for viral gene regulation, humoral immune responses, and host transfer. Notably, SDLY 1602 was demonstrated a recombinant strain, with the potential major parent GPV vaccine strain 82‐0321v and the minor parent GPV wild strain GD aGPV . This is the first report showing that the recombination between two classical GPV strains generated a NGPV strain circulating in nature. This study will advance our understanding of NGPV molecular biology and facilitate to elucidate the evolutionary characteristics of GPV .     

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome‐Like Disease in Italy and Spain

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.     

Emergence and expansion of novel pathogenic reassortant strains of infectious bursal disease virus causing acute outbreaks of the disease in Europe

Infectious bursal disease virus (IBDV) is the aetiological agent of a highly contagious chicken immunodeficiency disorder known as Gumboro disease, which cause severe economic loses to the poultry worldwide. The emergence of very virulent IBDV strains (vvIBDV) during the late 80s resulted in drastic changes to the epidemiology of IBDV with a dramatic increase in the mortality of the animals affected. Molecular studies determined that the emergence of the vvIBDV was a consequence of a genomic reorganization of IBDV known as reassortant event by which the virus combined two emergent genetic background vvIBDV for segment A and vvIBDV for segment B. In the current study, a retrospective analysis was conducted, and samples collected during acute outbreaks of Gumboro disease in Poland during 1992–2015 were submitted to sequencing and further molecular and phylogenetic analyses. The results obtained not only revealed a high genetic diversity for Polish IBDV strains but a new population of IBDV was identified. These novel reassortant strains with a unique genetic background contain the segment A from very virulent strains and segment B from an unidentified source, phylogenetically segregated and classified as ‘transitional lineage’. The results obtained also showed the presence of this new lineage in Finland, evidencing the expansion of this new genomic reorganized viral strain in Europe representing an additional threat to the global situation of IBDV.     

Experimental infection with high‐ and low‐virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free‐ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high‐virulence (Cadí‐6) and low‐virulence (Freser‐5) BDV strains was performed. Pregnant and non‐pregnant animals with and without antibodies against BDV were included in each group. Cadí‐6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser‐5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí‐6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí‐6 group. Chamois from both groups presented lesions in brain but the ones infected with the low‐virulence Freser‐5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low‐virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross‐protection in chamois against high‐virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.     

Origin and global spreading of an ancestral lineage of the infectious bursal disease virus

Infectious bursal disease virus (IBDV) is an economically relevant and widespread pathogen that produces immunosuppression in young chickens. IBDV is genetically classified into seven genogroups (G1–G7), where the traditional classic, variant and very virulent strains correspond to G1, G2 and G3, respectively. The G4 strains, also known as ‘distinct’ (dIBDV), have recently acquired increased relevance because of their prevalence and notorious impair to the poultry industry in South America. Here, worldwide dIBDV strains were studied using phylogenetic and phylodynamic approaches. The phylogenetic analyses performed using partial and complete sequences of both viral segments (A and B) consistently clustered the dIBDV strains in a monophyletic group. The analyses of the VP5, polyprotein and VP1 coding regions identified amino acid residues that act as markers for the identification of the entire dIBDV group or different sub‐populations. The phylodynamic analyses performed using the hypervariable region of VP2 indicated that the dIBDV strains emerged in the early 1930s in Eastern Europe, shortly after the emergence of classic strains (1927) and before variant (1949) and very virulent strains (1967). The analysis of the migration routes indicated that after its emergence, the dIBDV strains spread to Eastern Asia around 1959, to Brazil around 1963, and to Argentina around 1990. These inter‐continental migrations resulted in three sub‐populations that are currently represented by strains from (a) Brazil, (b) Eastern Asia and Canada, and (c) Eastern Europe, Argentina and Uruguay. Taken together, our results highlight the complex evolutionary history of IBDV and the importance of new phylodynamic data to unravel and nearly follow the different evolutionary pathways taken by this important poultry pathogen.     

A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.     

Factors associated with the development of post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) - seronegative adult liver transplant recipients

Abstract Epstein-Barr virus (EBV) infection is recognized as the principal aetiological factor in the pathogenesis of post-transplant lymphoproliferative disease (PTLD), particularly when primary EBV infection occurs after transplantation. We analysed, using a time-dependent proportional hazards model, the factors associated with development of PTLD in 40 adult liver transplant recipients who were seronegative for EBV prior to transplantation. Of 40 patients, 13 (33%) had a tissue diagnosis of PTLD at a median time of 126 days after transplantation. The multivariate analysis showed that prior CMV disease, the number of steroid boluses given and the number of units of RBC and FFP transfused were significant risk factors for development of PTLD.     

Calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of Hirschsprung's disease

Background/Purpose

Identifying ganglion cells by rectal suction biopsy is a basic diagnostic tool for the diagnosis of Hirschsprung's disease (HD). However, the difficult interpretation of conventionally processed slides often necessitates ancillary staining methods. The aim of this study was to evaluate the usefulness of calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of HD.

Methods

We analyzed 52 rectal suction biopsy specimens (37 from 15 HD patients and 15 from 7 non-HD patients) for ganglion cells with calretinin and MAP-2 immunohistochemistry. We also analyzed full-thickness, frozen biopsy samples obtained from 15 HD patients who underwent surgery utilizing calretinin and MAP-2 immunohistochemistry.

Results

Both calretinin and MAP-2 positively stained ganglion cells in the submucosal plexus of the ganglionic bowel but not aganglionic bowel. Calretinin usually stained ganglion cell cytoplasm and nuclei more intensely than MAP-2, which only stained cytoplasm. No nerve fiber staining in the submucosal layer was observed for either antibody. In 21.1% (11/52) of samples, calretinin and MAP-2 staining found ganglion cells which were reported not to have ganglion cells in the original surgical pathology reports. Immunohistochemical staining for calretinin using paraffin-embedded tissue sections after cryostat sections clearly demonstrated decreased staining intensity compared to MAP-2.

Conclusion

Calretinin and MAP-2 are useful diagnostic markers for diagnosing HD in rectal suction biopsies. These complementary methods could ameliorate the diagnostic difficulties associated with HD.     

Generation of monoclonal antibodies against foot‐and‐mouth disease virus SAT 2 and the development of a lateral flow strip test for virus detection

Foot‐and‐mouth disease (FMD) remains a major economic concern for the livestock productivity in many developing countries and a continued threat to countries that are disease free because of its potential devastating impact on agricultural, food chain and tourism sectors. FMD virus (FMDV) is recognized as having seven serotypes: O, A, C, Asia 1, South African Territories (SAT) 1, 2, 3 and multiple subtypes within each serotype. FMD outbreaks due to SAT 2 have been reported in many African countries. The development of a rapid and easily performed test for FMD detection is critical for controlling FMD outbreaks and containing its spread. The present project developed a lateral flow immunochromatographic (LFI) strip test for the rapid detection of FMDV SAT 2. A panel of monoclonal antibodies (mAbs) against FMDV serotype SAT 2 was produced and characterized. One mAb (#10) was selected as the capture mAb because it reacted to all 23 SAT 2 isolates archived at the National Center for Foreign Animal Disease. The LFI strip test was developed using biotin‐conjugated mAb #10, and the colloid gold‐conjugated FMDV serotype‐independent mAb as the detection mAb. A generic Rapid Assay Device (gRAD) with one test line and a control line was used for the test. The LFI strip test detected all 23 tested SAT 2 isolates and recent outbreak strains. The results indicated that the diagnostic specificity and sensitivity of the LFI strip test were greater than the double antibody sandwich (DAS) DAS ELISA. The ability of the LFI strip test to produce rapid diagnostic results will be useful for early on‐site diagnosis during FMD outbreaks.     

Isolation,identification and retrospective study of foot‐and‐mouth disease virus from affected Mithun (Bos frontalis) in north‐eastern India

Foot‐and‐mouth disease (FMD ) is a contagious disease of cloven‐hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a “carrier state” among all cloven‐hoofed animals. The Mithun (Bos frontalis ), popularly called the “Cattle of Mountain,” is found in the geographically isolated, hilly region of north‐east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA , multiplex RT ‐PCR (MRT ‐PCR ) and liquid‐phase blocking enzyme‐linked immunosorbent assay and isolated in the BHK ‐21 cell line. The results indicate the presence of FMDV serotype “O.” The sequencing and molecular phylogenies have revealed close relationships in the lineage of type “O” isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population.     

新型冠状病毒肺炎疫情下髋/膝关节置换术互联网+延续管理模式的构建及应用效果分析

目的探讨新型冠状病毒肺炎疫情下髋/膝关节置换术互联网+延续管理模式的构建与应用效果。方法选择疫情爆发后2020年1月20日至1月31日在华西医院骨科行髋/膝关节置换手术的34例病人纳入观察组,采用互联网+延续管理模式;同时将2019年12月1日至12月20日行髋/膝关节置换手术的34例病人纳入对照组,采用传统延续管理模式。出院当天、出院后2周和出院后1个月,采用Harris髋关节功能评分和美国特种外科医院(Hospital for Special Surgery,HSS)膝关节功能评分评价两组病人髋/膝关节恢复情况,华西心晴指数量表(HEI)评价病人情绪障碍程度;记录术后并发症和病人延续护理满意度。结果观察组病人在互联网+延续管理模式下,未出现新型冠状病毒肺炎确诊病例,情绪亦未受疫情影响。出院后2周和1个月,观察组的髋/膝关节功能评分均高于对照组,差异均有统计学意义(P均<0.05)。两组病人均未发生与髋/膝关节置换术相关的并发症。观察组病人延续护理满意度为100%,优于对照组的85.29%,差异有统计学意义(χ~2=5.397,P=0.020)。结论新型冠状病毒肺炎疫情下,髋/膝关节置换术病人采用互联网+延续管理模式,有效避免了新型冠状病毒肺炎交叉感染,促进了关节置换术后关节功能恢复,且病人心理状况未受疫情影响,无相关并发症发生,保障了病人的医疗安全,提高了延续管理满意度。     

先天性巨结肠症和肛门直肠畸形中甲基化CpG结合蛋白2基因第3外显子突变分析

目的探讨甲基化CpG结合蛋白2（MeCP2）基因第3外显子突变与先天性巨结肠症（HSCR）和先天性肛门直肠畸形（ARM）的关系。方法采用PCR和DNA直接测序的方法．检测120例HSCR、50例ARM患儿和120名健康儿童外周血MeCP2基因第3外显子（MeCP2．E3）的突变情况。结果MeCP2．E3测序结果显示,120例HSCR患儿的碱基置换突变有45例（37．5％）．其中12例（10．0％）为突变型纯合子;50例ARM患儿的碱基置换突变有14例（28．0％）,其中4例（8％）为突变型纯合子：而健康对照儿童均未发现突变（P〈0．05）。结论HSCR和ARM患儿外周血MeCP2．E3存在突变．可能与疾病的发生有关．     

Emergence of novel lineage of foot‐and‐mouth disease virus serotype Asia1 BD‐18 (G‐IX) in Bangladesh

Foot‐and‐mouth disease virus (FMDV) is a highly evolutionary divergent pathogen causing great economic havoc in many countries. Among its seven existing serotypes, Asia1 is the least divergent with a single topotype both genetically and antigenically. It is reported sporadically in Indian subcontinent and was classified under lineage G‐VIII. In 2018, serotype Asia1 re‐emerged in Bangladesh after 2013, along with circulation of a novel serotype Asia1 BD‐18 (G‐IX) lineage. VP1 phylogeny and sequence variation clearly demonstrated the novel strains which was estimated to have at least >5% nucleotide divergence with distinct clade formation. Also, the Bayesian phylogeographic inferences traced back to the origin time of lineage G‐IX in early 2017 and a possible origin in Bangladesh. Mutational analysis considering established eight lineages revealed that the virus strains belonged to lineage G‐IX contained a unique mutation at 44 position in the B‐C loop region of VP1. Inappropriate vaccination and inefficient outbreak surveillance possibly contributed to the current episode of emergence. Therefore, active surveillance and continued vigilance are essential to assess and timely detect the occurrence, extent and distribution of this novel Asia1 strains in Bangladesh and the neighbouring countries.     

Emergence of two novel sublineages Ind2001BD1 and Ind2001BD2 of foot‐and‐mouth disease virus serotype O in Bangladesh

Foot‐and‐mouth disease (FMD ) is endemic in Bangladesh, and the implementation of a control programme for this disease is at an early stage, according to the FAO ‐ and OIE ‐proposed Progressive Control Pathway for FMD (PCP ‐FMD ) Roadmap. To develop an effective control programme, understanding of foot‐and‐mouth disease virus (FMDV ) serotypes, even subtypes within the serotypes is essential. The present investigation aims at viral VP 1 coding region sequence‐based analysis of FMD samples collected from 34 FMD outbreaks during 2012–2016 in Bangladesh. Foot‐and‐mouth disease virus (FMDV ) serotype O was responsible for 82% of the outbreaks in Bangladesh, showing its dominance over serotype A and Asia1. The VP 1 phylogeny revealed the emergence of two novel sublineages of serotype O, named as Ind2001BD 1 and Ind2001BD 2, within the Ind2001 lineage along with the circulation of Ind2001d sublineage in Bangladesh, which was further supported by the multidimensional scaling with distinct clusters for each sublineage. The novel sublineages had evident genetic variability with other established sublineages within Ind2001 lineage. Ten mutations with three or more amino acid variations were detected within B‐C loop, G‐H loop and C‐terminal region of the VP 1 protein of FMDV serotype O viruses isolated exclusively from Bangladesh. Furthermore, two amino acid substitutions at positions 197 and 198 within the VP 1 C‐terminal region are unique to the novel sublineages. The existence of widespread genetic variations among circulatory FMDV serotype O viruses makes the FMD control programme complex in Bangladesh. Adequate epidemiological data, disease reporting, animal movement control, appropriate vaccination and above all stringent policies of the government are necessary to combat FMD in Bangladesh.     

肿瘤浸润淋巴细胞、重组白细胞介素2治疗对手术后原发性肝癌病人细胞免疫功能的影响

对５例经手术切除的原发性肝癌病人用肿瘤浸润淋巴细胞（ＴＩＬ）和重组白细胞介素２（ｒＩＬ－２）治疗。治疗前１周及治疗后１个月检测细胞免疫功能，治疗后外周血淋巴细胞的ＮＫ活性、白细胞介素２产生及活性均明显升高；植物血凝素（ＰＨＡ）皮试阴性转阳性。提示；ｒＩＬ－２和ＴＩＬ应用可明显提高原发性肝癌病人的细胞免疫功能，对杀灭残留癌细胞，抗肿瘤复发、转移，提高远期疗效有一定作用。可能成为肝癌术后抗肿瘤复发，转移的重要疗法。